Numerous studies have been performed
in mammals on the correlation between
the ability of cells to repair DNA and
the life span of the species from which
the cells were taken. The life spans of
the species varied from 1.5 years for the
shrew to 95 years for man. Almost all of
the studies showed a positive correlation
betweenDNArepair capacity and life span.
Many experiments have been performed
on the effect of adding antioxidants to the
diets of organisms upon the organism’s
life span. Although the results of such
experiments are not entirely consistent,
certain antioxidants have been found to
generally increase life span. Vitamin E,
for example, has been found to increase
the life span of rat, insects, rotifers,
nematodes, and paramecium.
More than 50 studies have been performed
to examine the possible experimental
acceleration of aging by externally
applied DNA-damaging agents. Overall,
it has been found that sublethal doses
of ionizing radiation or DNA-damaging
chemicals in the diet shorten life span,
but many specific aspects of normal aging
are not accelerated. Several authors
have noted that the distribution (over time
and in different tissues) of DNA damages
induced by external agents does not
closely mimic that of natural damages.
This difference could explain why the
life-shortening effects induced by external
agents do not closely conform to natural
aging. In particular, natural damages probably
accumulate gradually, so they would
tend to build up in nondividing cells, while
they would be diluted out in dividing cells.
Exposure to an external agent over a brief
period, on the other hand, could cause
equally large numbers of damages to nondividing
and rapidly dividing cells. The
effect on rapidly dividing cells could be
very large by interfering with DNA replication.
In addition, if oxidative damages
are important in normal aging, then brain
cells, which have a high level of oxidative
metabolism should have more damages
than most other cell types. Externally applied
damages would not be expected to
produce this particular type of bias. Thus,
the general finding that sublethal exposure
to DNA-damaging agents shortens
life span, while not uniformly accelerating
the natural aging process, is consistent
with the DNA damage theory of aging.
Показват се публикациите с етикет Molecular medicine. Показване на всички публикации
Показват се публикациите с етикет Molecular medicine. Показване на всички публикации
неделя, 21 юни 2009 г.
Molecular medicine-Negative Correlation between Mitochondrial ROS Production and Life Span
IfDNAdamage is the major cause of aging,
ROS are a major source of DNA damage,
and mitochondria are a major source of
ROS, then animals with mitochondria
producing higher levels of ROS may
have a shorter life span, other factors
being equal. Comparisons were made
between long-lived birds and short-lived
mammals. Pigeons, with a maximum life
span of 35 years were compared with rats
(of similar body size) with a maximum
life span of 4 years, and parakeets and
canaries (maximum life spans of 21 and
24 years, respectively) were compared with
mice (maximum life span of 3.5 years).
Mitochondrial ROS production was lower
in the longer-lived avian species. However,
in addition, pigeons were shown to have
higher levels of SOD in brain, heart,
and kidney than the levels shown by
rats, so there was also higher antioxidant
enzymatic protection in the longer-lived
pigeon. This indicates that antioxidant
enzymes, which confer resistance to an
externally added source of ROS may be
of comparable importance to longevity as
endogenous rates of ROS production. The
values of one measured DNA-damaged
base, 8-oxodeoxyguanine, were lower in
canary brain and parakeet heart nuclear
DNA than in the comparison tissues of the
mouse, while in the other comparisons,
the level of this one damaged base was
not significantly different in nuclear DNA.
In another experiment quoted by Herrero
and Barja, other workers showed that
nuclei of starlings (another long-lived bird,
maximum life span of 20 years) have less
DNAbreaks and abasic sites after exposure
to H2O2 than those of mice.
Mice heterozygous for a MnSOD defect
(MnSOD+/− mice) have higher levels
of oxidative damage to DNA, protein,
and lipids in their mitochondria, but
no increased damage to nuclear DNA
or cytoplasmic proteins. The MnSOD+/−
mice live as long as wild-type mice,
showing that mitochondrial DNA damage
(as distinct from nuclear DNA damage)
may not be central to longevity. This may
be because there are on the order of 1,000
mitochondria per cell, and mitochondria
with excess damage may be replaced by
replication of less damaged mitochondria.
In Section 1.3.2, we mentioned that the
long-lived strains of fruit flies have higher
levels of antioxidant defense enzymes. The
long-lived flies also had mitochondria that
had lower levels of ROS leakage. These
less leaky mitochondria, when transferred
to short-lived flies through maternal inheritance
(only maternal mitochondria are
passed on to and maintained in progeny
66 Aging and Sex, DNA Repair in
Tab. 4 Normal aging: alterations in genes controlling DNA repair or oxidant status but which do not alter life span.
Organism Genetic
alteration
Pathway Aging
phenotype
Fertility Spontaneous
cancer
Effect On
Cellular
ROS
Induced
DNA
damage
Spont.
mutation
Induced
apoptosis
Mouse XPA defect NER Unchangeda Unchanged Increased
liver
cancer
n.t. n.t. Increased in
liver, no
change in
brain
n.t.
Mouse XPC defect GGR (NER) Unchangeda Unchanged Unchanged
(when not
exposed to
UV light)
n.t. n.t. Increased n.t.
Fruit fly Excess SOD Removes ROS Unchanged Unchanged n.t. n.t. n.t. n.t. n.t.
Notes: ROS: reactive oxygen species; Spont.: spontaneous; n.t.: not tested.
aBased on observations during the first half of life span.
Aging and Sex, DNA Repair in 67
flies), also transferred the ability to live
about 25% longer.
ROS are a major source of DNA damage,
and mitochondria are a major source of
ROS, then animals with mitochondria
producing higher levels of ROS may
have a shorter life span, other factors
being equal. Comparisons were made
between long-lived birds and short-lived
mammals. Pigeons, with a maximum life
span of 35 years were compared with rats
(of similar body size) with a maximum
life span of 4 years, and parakeets and
canaries (maximum life spans of 21 and
24 years, respectively) were compared with
mice (maximum life span of 3.5 years).
Mitochondrial ROS production was lower
in the longer-lived avian species. However,
in addition, pigeons were shown to have
higher levels of SOD in brain, heart,
and kidney than the levels shown by
rats, so there was also higher antioxidant
enzymatic protection in the longer-lived
pigeon. This indicates that antioxidant
enzymes, which confer resistance to an
externally added source of ROS may be
of comparable importance to longevity as
endogenous rates of ROS production. The
values of one measured DNA-damaged
base, 8-oxodeoxyguanine, were lower in
canary brain and parakeet heart nuclear
DNA than in the comparison tissues of the
mouse, while in the other comparisons,
the level of this one damaged base was
not significantly different in nuclear DNA.
In another experiment quoted by Herrero
and Barja, other workers showed that
nuclei of starlings (another long-lived bird,
maximum life span of 20 years) have less
DNAbreaks and abasic sites after exposure
to H2O2 than those of mice.
Mice heterozygous for a MnSOD defect
(MnSOD+/− mice) have higher levels
of oxidative damage to DNA, protein,
and lipids in their mitochondria, but
no increased damage to nuclear DNA
or cytoplasmic proteins. The MnSOD+/−
mice live as long as wild-type mice,
showing that mitochondrial DNA damage
(as distinct from nuclear DNA damage)
may not be central to longevity. This may
be because there are on the order of 1,000
mitochondria per cell, and mitochondria
with excess damage may be replaced by
replication of less damaged mitochondria.
In Section 1.3.2, we mentioned that the
long-lived strains of fruit flies have higher
levels of antioxidant defense enzymes. The
long-lived flies also had mitochondria that
had lower levels of ROS leakage. These
less leaky mitochondria, when transferred
to short-lived flies through maternal inheritance
(only maternal mitochondria are
passed on to and maintained in progeny
66 Aging and Sex, DNA Repair in
Tab. 4 Normal aging: alterations in genes controlling DNA repair or oxidant status but which do not alter life span.
Organism Genetic
alteration
Pathway Aging
phenotype
Fertility Spontaneous
cancer
Effect On
Cellular
ROS
Induced
DNA
damage
Spont.
mutation
Induced
apoptosis
Mouse XPA defect NER Unchangeda Unchanged Increased
liver
cancer
n.t. n.t. Increased in
liver, no
change in
brain
n.t.
Mouse XPC defect GGR (NER) Unchangeda Unchanged Unchanged
(when not
exposed to
UV light)
n.t. n.t. Increased n.t.
Fruit fly Excess SOD Removes ROS Unchanged Unchanged n.t. n.t. n.t. n.t. n.t.
Notes: ROS: reactive oxygen species; Spont.: spontaneous; n.t.: not tested.
aBased on observations during the first half of life span.
Aging and Sex, DNA Repair in 67
flies), also transferred the ability to live
about 25% longer.
Molecular medicine-Normal Aging in spite of Certain Defects in DNA Repair or Increases in Antioxidant Enzyme Production
If a DNA repair pathway lacks an enzyme,
but the missing enzyme is partially
compensated for by a similar enzyme,
then repair may be sufficient to allow
survival, growth, and normal aging. While
such fairly good compensation may allow
normal aging, the repair of DNA damages
would still be less than if the repair
pathway were intact, and that could lead to
Aging and Sex, DNA Repair in 65
increased carcinogenesis. Such fairly good
compensation for DNA repair enzyme
defects may be the basis for normal aging,
but increased carcinogenesis, shown by
mice with the DNA repair mutations listed
in Table 4. As shown in Fig. 1, XPA and
XPC proteins each occur as one member
of a pair of complexed proteins whose
function is recognition of DNA damage
to be repaired by an NER pathway. It is
possible that the other complexes that can
recognize DNA damage can compensate,
at some level, when one of the recognition
complexes is absent.
Further, in some instances, genetic
alterations caused increased SOD production
in fruit flies but did not affect aging.
However, the inserted SOD gene may have
been turned on under the control of promoters
expressing in tissues where it may
not have been useful, or at rather low levels.
but the missing enzyme is partially
compensated for by a similar enzyme,
then repair may be sufficient to allow
survival, growth, and normal aging. While
such fairly good compensation may allow
normal aging, the repair of DNA damages
would still be less than if the repair
pathway were intact, and that could lead to
Aging and Sex, DNA Repair in 65
increased carcinogenesis. Such fairly good
compensation for DNA repair enzyme
defects may be the basis for normal aging,
but increased carcinogenesis, shown by
mice with the DNA repair mutations listed
in Table 4. As shown in Fig. 1, XPA and
XPC proteins each occur as one member
of a pair of complexed proteins whose
function is recognition of DNA damage
to be repaired by an NER pathway. It is
possible that the other complexes that can
recognize DNA damage can compensate,
at some level, when one of the recognition
complexes is absent.
Further, in some instances, genetic
alterations caused increased SOD production
in fruit flies but did not affect aging.
However, the inserted SOD gene may have
been turned on under the control of promoters
expressing in tissues where it may
not have been useful, or at rather low levels.
Molecular medicine-Premature Aging Associated with Defects in DNA Repair or Increased Oxidant Status
DNA damages are so frequent (Table 1)
that total absence of DNA repair of a
common damage is likely to be incompatible
with life. If a DNA repair pathway
lacks an essential enzyme, but the missing
enzyme can be, at least, partially compensated
for by a similar enzyme, then
repairmay be adequate to allow sufficient
survival and growth to show premature
aging. This will also occur if a particular
DNA damage is preferentially repaired
by one pathway, but another repair pathway,
with less efficiency, also repairs
that damage.
1. Helicase. There are at least 31 human
enzymes that are helicases or contain
helicase-motif domains. Helicases are
enzymes that unwind and separate the
strands of DNA, usually using the hydrolysis
of ATP to provide the necessary energy.
Some enzymes with multiple helicasemotif
domains only act as ATPases, providing
energy to DNA-related processes.
Helicases or enzymes with helicase-motif
domains participate in DNA repair, DNA
replication, and DNA recombination. Usually,
the helicase activity is specific for a
particular DNA configuration. Some helicases
involved in particular DNA-repair
pathways may be partially replaceable, at
least at a low level, by other helicases. That
may be why five genes, which code for
enzymes with helicase functions, or helicase
motifs plus an ATPase function, and
which are required in different DNA repair
pathways, when genetically defective,
cause syndromes characterized by early
aging in humans (Table 3). These syndromes
areWerner syndrome, Bloom syndrome,
Rothmund–Thomson syndrome,
Trichothiodystrophy and Cockayne syndrome
(Table 3). Similarly, in the mouse,
a defect in the Ku-80 gene, which normally
activates the Ku-70 helicase function, results
in an early aging phenotype (Table 3).
The different helicases listed in Table 3
have specificities for HRR, NHEJ, NER,
TCR or BER, so that defects in each of
these DNA repair pathways may allow
accumulation of different types of DNA
damage, each type being able to contribute
to premature aging.
2. Topoisomerase. Topoisomerases interact
with helicases in DNA repair, recombination,
and replication. When a helicase
64 Aging and Sex, DNA Repair in
unwinds the two DNA strands of the
double helix, this introduces supercoiling
of the associated DNA. Topoisomerases
introduce controlled breaks plus reattachments
in DNA to relieve supercoiling.
There are a number of topoisomerases
in mouse and human cells. The different
topoisomerases interact specifically with
different helicases. However, some topoisomerases
may be partially replaceable by
another topoisomerase at a low level. In the
mouse, a mutant lacking topoisomerase
IIIβ develops to maturity but shows
early aging (Table 3). Topoisomerase IIIβ
interactswith humanRecQ5β helicase and
is thought to act inDNArepair, replication,
or recombination (Table 3).
3. ERCC1. Excision Repair Cross Complementing
1 (ERCC1), when defective, is
another gene whose absence or truncation
causes an early aging phenotype in the
mouse (Table 3). ERCC1 functions in both
NER and interstrand cross-link repair (in
a step prior to HRR). ERCC1 has homology
with an endonuclease active in NER
in yeast, and that yeast endonuclease can
compensate for the loss of a topoisomerase
or a helicase. Thus, ERCC1 may have some
functional similarity to topoisomerase or
helicase in DNA repair. Conversely, loss
of ERCC1 may be partially compensated
for by a helicase or topoisomerase, or by
another endonuclease in mouse, so that a
defect in ERCC1 is not lethal but causes
early aging. ERCC1 primarily functions in
NER as an endonuclease as illustrated in
Fig. 1.
4. p53. Similar to helicase and topoisomerase,
p53 occurs as one of a family of
enzymes, p53, p63, and p73 (and both p73
and p63 have multiple isoforms), which
share significant homology and have similar
functions. In particular, p73 has a role
in activating DNA repair enzymes and in
carrying out apoptosis in the face of excess
DNA damage (see below in Fig. 3).
Thus, loss of p53 may, in part, be compensated
for by functions of p73 and/or p63.
In Section 1.3.4, we briefly discussed an
overactive form of p53 that causes early aging.
This mutant form of p53 has its effect
in the presence of a wild-type p53 (a heterozygous
situation) where it may increase
some functions detrimental to the cell. Although
it is not known which functions
it increases, an increase of p66Shc under
p53 control could reasonably be expected
to cause early aging, since it would increase
DNA damage through increases in
ROS. In addition, a p53 knockout mouse,
lacking all functions of p53, including its
functionality in three DNA repair pathways
(NER, BER, and HRR), is also viable
but ages prematurely (Table 3).
5. MsrA. As discussed in Section 1.3.3,
if cellular genes, which code for activity
in the replacement of damaged proteins,
are themselves damaged, then damaged
proteins may not turn over as rapidly, and
protein damages may become important
as they accumulate with age. Added activity
of MsrA in the fruit fly gave greater
longevity. Defective MsrA in the mouse
caused early aging ).
that total absence of DNA repair of a
common damage is likely to be incompatible
with life. If a DNA repair pathway
lacks an essential enzyme, but the missing
enzyme can be, at least, partially compensated
for by a similar enzyme, then
repairmay be adequate to allow sufficient
survival and growth to show premature
aging. This will also occur if a particular
DNA damage is preferentially repaired
by one pathway, but another repair pathway,
with less efficiency, also repairs
that damage.
1. Helicase. There are at least 31 human
enzymes that are helicases or contain
helicase-motif domains. Helicases are
enzymes that unwind and separate the
strands of DNA, usually using the hydrolysis
of ATP to provide the necessary energy.
Some enzymes with multiple helicasemotif
domains only act as ATPases, providing
energy to DNA-related processes.
Helicases or enzymes with helicase-motif
domains participate in DNA repair, DNA
replication, and DNA recombination. Usually,
the helicase activity is specific for a
particular DNA configuration. Some helicases
involved in particular DNA-repair
pathways may be partially replaceable, at
least at a low level, by other helicases. That
may be why five genes, which code for
enzymes with helicase functions, or helicase
motifs plus an ATPase function, and
which are required in different DNA repair
pathways, when genetically defective,
cause syndromes characterized by early
aging in humans (Table 3). These syndromes
areWerner syndrome, Bloom syndrome,
Rothmund–Thomson syndrome,
Trichothiodystrophy and Cockayne syndrome
(Table 3). Similarly, in the mouse,
a defect in the Ku-80 gene, which normally
activates the Ku-70 helicase function, results
in an early aging phenotype (Table 3).
The different helicases listed in Table 3
have specificities for HRR, NHEJ, NER,
TCR or BER, so that defects in each of
these DNA repair pathways may allow
accumulation of different types of DNA
damage, each type being able to contribute
to premature aging.
2. Topoisomerase. Topoisomerases interact
with helicases in DNA repair, recombination,
and replication. When a helicase
64 Aging and Sex, DNA Repair in
unwinds the two DNA strands of the
double helix, this introduces supercoiling
of the associated DNA. Topoisomerases
introduce controlled breaks plus reattachments
in DNA to relieve supercoiling.
There are a number of topoisomerases
in mouse and human cells. The different
topoisomerases interact specifically with
different helicases. However, some topoisomerases
may be partially replaceable by
another topoisomerase at a low level. In the
mouse, a mutant lacking topoisomerase
IIIβ develops to maturity but shows
early aging (Table 3). Topoisomerase IIIβ
interactswith humanRecQ5β helicase and
is thought to act inDNArepair, replication,
or recombination (Table 3).
3. ERCC1. Excision Repair Cross Complementing
1 (ERCC1), when defective, is
another gene whose absence or truncation
causes an early aging phenotype in the
mouse (Table 3). ERCC1 functions in both
NER and interstrand cross-link repair (in
a step prior to HRR). ERCC1 has homology
with an endonuclease active in NER
in yeast, and that yeast endonuclease can
compensate for the loss of a topoisomerase
or a helicase. Thus, ERCC1 may have some
functional similarity to topoisomerase or
helicase in DNA repair. Conversely, loss
of ERCC1 may be partially compensated
for by a helicase or topoisomerase, or by
another endonuclease in mouse, so that a
defect in ERCC1 is not lethal but causes
early aging. ERCC1 primarily functions in
NER as an endonuclease as illustrated in
Fig. 1.
4. p53. Similar to helicase and topoisomerase,
p53 occurs as one of a family of
enzymes, p53, p63, and p73 (and both p73
and p63 have multiple isoforms), which
share significant homology and have similar
functions. In particular, p73 has a role
in activating DNA repair enzymes and in
carrying out apoptosis in the face of excess
DNA damage (see below in Fig. 3).
Thus, loss of p53 may, in part, be compensated
for by functions of p73 and/or p63.
In Section 1.3.4, we briefly discussed an
overactive form of p53 that causes early aging.
This mutant form of p53 has its effect
in the presence of a wild-type p53 (a heterozygous
situation) where it may increase
some functions detrimental to the cell. Although
it is not known which functions
it increases, an increase of p66Shc under
p53 control could reasonably be expected
to cause early aging, since it would increase
DNA damage through increases in
ROS. In addition, a p53 knockout mouse,
lacking all functions of p53, including its
functionality in three DNA repair pathways
(NER, BER, and HRR), is also viable
but ages prematurely (Table 3).
5. MsrA. As discussed in Section 1.3.3,
if cellular genes, which code for activity
in the replacement of damaged proteins,
are themselves damaged, then damaged
proteins may not turn over as rapidly, and
protein damages may become important
as they accumulate with age. Added activity
of MsrA in the fruit fly gave greater
longevity. Defective MsrA in the mouse
caused early aging ).
Molecular medicine-Life Span Extension by Genetic Alterations that Increase DNA Repair, Reduce Oxidative Damage, or Reduce Cell Suicide (Apoptosis) due to DNA Damage
Table 2 lists alterations in genes controlling
DNA repair, oxidant status, or
apoptosis that result in increased life span.
The increases in life span found with
the genetic alterations in Table 2 are usually
an increase in the maximum life
span (not just the mean life span) by
about 30 to 40%. Mean life span can
be extended by reductions in tumorigenesis
or acute and sporadic diseases, not
generally regarded as a cause of aging.
The organisms with increased maximum
life span reported here showed longer
spans of normal vigorous activity (not
merely slowed metabolism, which can
also extend life span). The cellular roles
58 Aging and Sex, DNA Repair in
Tab. 2 Life span extension: increased life span from alterations in genes controlling DNA repair, apoptosis, or oxidant status.
Organism Genetic Pathway Aging Fertility Spontaneous Effect on
alteration phenotype phenotype cancer
phenotype Cellular Induced Spont. Induced
ROS DNA mutation apoptosis
damage
Mouse 100-fold excess
copies MGMT
gene
O6-meG DNA
repair
Life span
extension
n.t. Reduced n.t. Reduced
O6-meG
No effect at
H-ras
locus
n.t.
Fruit fly Excess SOD in
neurons or all
tissues or with
catalase in all
tissues
Removes ROS Life span
extension
No change n.t. Reduced
occurrence of
oxidized
proteins
n.t. n.t. n.t.
Fruit fly MsrA excess Methionine
sulfoxide
reductase
Life span
extension
Extended n.t. Repaired protein
oxidation
n.t. n.t. n.t.
Mouse p66shc defect Blocks oxidant
and apoptosis
parts of p53
pathways
Life span
extension
n.t. n.t. Reduced Reduced
oxidative
damage
n.t. Reduced
Human Higher specific
activity of PARP
BER Life span
extension
n.t. n.t. n.t. n.t. n.t. n.t.
Notes: ROS: reactive oxygen species; Spont.: spontaneous; MGMT: O6-methylguanine-DNA methyltransferase; n.t.: not tested; SOD: superoxide
dismutase.
Aging and Sex, DNA Repair in 59
of these genetic alterations are described
below.
1. MGMT. One frequent type of DNA
damage (see Table 1) isO6-methylguanine,
caused by low levels of alkylating agents
present in food, water, air, and tobacco
smoke, and formed by normal processes in
the body mediated by gastric bacteria and
macrophages.O6-methylguanine is specifically
repaired by a DNA repair enzyme
called O6-methylguanine-DNA methyltransferase
(MGMT). MGMT transfers the extra
methyl group from guanine in DNA to
a particular amino acid within itself and
becomes ‘‘used up’’ after the transfer occurs.
The MGMT gene codes for one of
the five DNA repair mechanisms listed
in Section 1.1. As indicated in Table 2,
when 100 copies of the MGMT gene were
inserted into the mouse genome, these
mice (under the usual conditions of mouse
maintenance) had their life span extended
and died at a considerably slower rate than
wild-type mice.
2. SOD. Another important type of
metabolically caused DNA damage is oxidative
damage, themost frequent damage
identified (Table 1). An apparently unavoidable
by-product of normal respiratory
metabolism is the production of reactive
oxygen species (ROS) from molecular
oxygen, and ROS cause oxidative damage.
ROS include free radicals (where
the symbol • indicates an unpaired electron):
the superoxide radical (O2
•−) and
the hydroxyl radical (OH•). Another oxygen
respiration by-product is hydrogen
peroxide (H2O2). H2O2, if not removed,
it diffuses fairly easily through the cell,
and when it encounters Fe2+ (the ferrous
ion), it can undergo the Fenton reaction
and produce OH• and other ROS.
ROS produce a number of lesions in
DNA, including base lesions, sugar lesions
(the deoxyribose sugar is in the
backbone of DNA), DNA–protein crosslinks,
single-strand breaks, double-strand
breaks, and abasic sites.
The major ROS produced by the cell
is O2
•−, formed in the mitochondria (the
energy-producing organelles of the cell).
Superoxide dismutase (SOD) occurs in
two forms, manganese SOD (MnSOD)
and copper/zinc SOD (Cu/ZnSOD). Both
forms of SODconvertO2
•− to the less damaging
H2O2, and then another enzyme,
catalase, converts H2O2 to molecular oxygen
and water. MnSOD occurs in the mitochondria
and Cu/ZnSOD occurs in the
cytoplasm.As shown in Table 2, a number
of investigators have found that inserting
genes producing higher than normal
levels of superoxide dismutase into the
fruit fly (Drosophila melanogaster) genome
results in life span extension. Insertion
of genes producing either MnSOD or
Cu/ZnSOD caused life span extension, although
the artificially inserted Cu/ZnSOD
only produced life span extension when
its expression was restricted to the motor
neurons, or solely to the adult phase of the
fruit fly life cycle.
Aging has been found to correlate with
increased levels of oxidative products, such
as protein carbonyls and 8-oxo-guanine in
DNA, and fruit flies lacking either catalase
or Cu/ZnSOD have a reduced life span.
Further, selection of a population of fruit
flies for increased life span correlates
with strongly increased expression of
both MnSOD and Cu/ZnSOD. Reverse
selection of these long life span flies to
a shorter life span resulted in reduced
expression of Cu/ZnSOD.
3. MsrA. In addition to DNA damage,
free radicals damage proteins, lipids,
and carbohydrates. Most proteins have
a short half-life (averaging about three
days in mouse liver). Oxidatively damaged
proteins and lipids are subject to both
60 Aging and Sex, DNA Repair in
degradation and some repair reactions.
If cellular genes that code for enzymes
involved in the replacement of damaged
proteins are themselves damaged, then
damaged proteins may not turn over
as rapidly, and protein damages may
become important as they accumulate with
age. Table 2 shows that insertion of an
extra gene encoding bovine methionine
sulfoxide reductase (MsrA) in the fruit fly
genome, which helps repair oxidatively
damaged proteins, leads to life span
extension. Consistent with this, MsrA,
when defective in the mouse, results in
early aging (Table 3).
4. p66Shc. The p53 gene has a central
role in response to DNA damage. The
p53 protein is directly active in three
forms of DNA repair (NER, BER, and
HRR).When there is no externally induced
DNA damage, p53 has a half-life of
only 5 to 40 minutes since specific
enzymes target p53 for degradation. Thus,
p53 is kept at a low level when there
is no DNA damage. However, upon
exposure of a cell to DNA-damaging
agents, p53 becomes metabolically stable
and, in addition, more copies of it are
produced in the cell. In the presence
of various types of DNA damage, p53
undergoes modifications at some of the
18 different sites within the protein. Some
of these modifications [phosphorylations,
acetylations, poly(ADP-ribosyl)ations, or
sumoylations (covalent attachments of
small ubiquitin-like proteins) allow the
p53 protein to act as a regulatory agent,
activating numerous other genes, carrying
out different responses to different kinds
or levels of DNA damage. The p53 protein
can regulate or act in at least four major
types of responses to DNA damage (acting
as a ‘‘master switch’’), and which action or
transactivation (regulating the induction
of other genes) it performs depends on
the level and type of DNA damage. p53
can (1) send the cell into cell cycle arrest
(to allow extra time for repair of DNA
damage); (2) act directly in DNA repair
(see Fig. 1 for where p53 acts in NER);
(3) cause the cell to switch into a cell
suicide mode (apoptosis); or (4) cause the
cell to produce higher levels of ROS
(apparently as a preliminary to entering
the cell suicide mode of apoptosis). When
acting to increase the internal level of ROS
and entry into apoptosis, p53 acts through
another gene it controls, p66Shc.
When a mouse embryo is produced
with both copies of its p66Shc gene
inactive (a p66Shc ‘‘knockout’’), mouse
embryo fibroblast cells derived from it
have intracellular levels of ROS reduced by
about 40%. Consistent with this reduction
in ROS, there is also greatly reduced
oxidative damage accumulation in both
nuclear and mitochondrial DNA of these
cells. A similar reduction in nuclear and
mitochondrial DNA damage is seen in
vivo in the tissues of lung, spleen, liver,
and skin in 3- and 24-month-old p66Shc
knockout mice, although there is no
reduction in the brain, where p66Shc is
not normally expressed. Cells of thesemice
are inhibited from undergoing apoptosis
after cellular oxidative damage (when
challenged with externally applied H2O2).
Knockout mice without p66Shc show
life span extension without any notable
increase in cancer or other pathological
defects (Table 2). Mice with a type of
overactive p53 (an increase in some
p53 functions) and intact p66Shc show
early aging (Table 3). On the other hand,
removal of all p53 functions (some of
which are protective in DNA repair) also
results in early aging (Table 3).
5. PARP. DNA damages caused by
alkylating agents (such as those that
methylate guanine, discussed above),
Aging and Sex, DNA Repair in 61
Tab. 3 Early aging: decreased life span from alterations in genes controlling DNA repair or protein oxidation.
Organism Genetic Pathway Aging Fertility Spontaneous Effect on
alteration phenotype cancer
Cellular Induced Spont. Induced
ROS DNA mutation apoptosis
damage
Human RECQ3 helicase and
exonuclease defect
(Werner syndrome)
HRR and NHEJ Early aging Reduced Increased n.t. Increased Increased n.t.
Human RECQ2 helicase defect
(Bloom syndrome)
HRR and NHEJ Early aging Reduced Increased n.t. Increased Increased n.t.
Human RECQ4 helicase defect
(Rothmund–Thomson
syndrome)
DNA repair
pathway,
unknown type
Early aging Reduced Increased n.t. n.t. Increased n.t.
Human and
Mouse
XPD helicase defective at
certain sites
(Trichothiodystrophy)
NER, also alters
transcription
initiation
Early aging Reduced No change n.t. n.t. n.t. Increased
Human CSB defect at 2 helicase
motifs or ATPase motif
(Cockayne syndrome)
BER if defective at
helicase motif V
or VI, TCR if
defective in
ATPase function
Early aging n.t. No change n.t. Increased n.t. Increased
Mouse Ku-80 (activator of Ku-70
helicase) defect
NHEJ Early aging n.t. Increased n.t. n.t. n.t.
Mouse Topoisomerase IIIβ
defect
Unknown, but
probably DNA
repair,
replication, or
recombination
Early aging n.t. n.t. n.t. n.t. n.t.
(continued overleaf )
62 Aging and Sex, DNA Repair in
Tab. 3 (continued)
Organism Genetic Pathway Aging Fertility Spontaneous Effect on
alteration phenotype cancer
Cellular Induced Spont. Induced
ROS DNA mutation apoptosis
damage
Mouse ERCC1 defect NER and interstrand
cross-link repair
(HRR)
Early aging Infertile No change n.t. Increased Increased
Mouse p53 overactivated Increases some p53
functions
Early aging n.t. Reduced n.t. n.t. n.t.
Mouse p53 defect Blocks all p53
functions,
including NER,
BER and HRR
Early aging Reduced Increased n.t. Increased Increased
Mouse MsrA Defect Methionine
sulfoxide
reductase
Early aging n.t. n.t. Increased
protein
oxidation
n.t. n.t.
Notes: ROS: reactive oxygen species; Spont.: spontaneous; n.t.: not tested.
Aging and Sex, DNA Repair in 63
ionizing radiation (which produces DNA
single- and double-strand breaks and
oxidative damages), and ROS result in
rapid activation of an enzyme called
poly(ADP-ribose) polymerase, or PARP.
PARP, similar to p53 discussed above,
has a role as a ‘‘master switch’’. PARP
can (1) act directly in one form of DNA
repair, BER, (2) control the function of
many other proteins by catalyzing the addition
of ADP-ribose branched polymers
onto them (either activating or repressing
their function), and (3) trigger apoptosis
(cell suicide). In addition, PARP controls
new transcription or activities of a number
of genes affecting survival or apoptosis,
including p53. It was found that centenarians
(humans who have lived for more
than 100 years) have a modified form
of PARP, which is more efficiently activated
than the PARP of noncentenarians
(Table 2), thereby apparently causing life
span extension. In addition, the maximal
poly(ADP-ribosyl)ation capacity (efficiency
of activation of PARP) in leukocytes of
13 mammalian species of different life
span was measured. There was a strong
correlation of PARP efficiency of activation
with species-specific life span.
DNA repair, oxidant status, or
apoptosis that result in increased life span.
The increases in life span found with
the genetic alterations in Table 2 are usually
an increase in the maximum life
span (not just the mean life span) by
about 30 to 40%. Mean life span can
be extended by reductions in tumorigenesis
or acute and sporadic diseases, not
generally regarded as a cause of aging.
The organisms with increased maximum
life span reported here showed longer
spans of normal vigorous activity (not
merely slowed metabolism, which can
also extend life span). The cellular roles
58 Aging and Sex, DNA Repair in
Tab. 2 Life span extension: increased life span from alterations in genes controlling DNA repair, apoptosis, or oxidant status.
Organism Genetic Pathway Aging Fertility Spontaneous Effect on
alteration phenotype phenotype cancer
phenotype Cellular Induced Spont. Induced
ROS DNA mutation apoptosis
damage
Mouse 100-fold excess
copies MGMT
gene
O6-meG DNA
repair
Life span
extension
n.t. Reduced n.t. Reduced
O6-meG
No effect at
H-ras
locus
n.t.
Fruit fly Excess SOD in
neurons or all
tissues or with
catalase in all
tissues
Removes ROS Life span
extension
No change n.t. Reduced
occurrence of
oxidized
proteins
n.t. n.t. n.t.
Fruit fly MsrA excess Methionine
sulfoxide
reductase
Life span
extension
Extended n.t. Repaired protein
oxidation
n.t. n.t. n.t.
Mouse p66shc defect Blocks oxidant
and apoptosis
parts of p53
pathways
Life span
extension
n.t. n.t. Reduced Reduced
oxidative
damage
n.t. Reduced
Human Higher specific
activity of PARP
BER Life span
extension
n.t. n.t. n.t. n.t. n.t. n.t.
Notes: ROS: reactive oxygen species; Spont.: spontaneous; MGMT: O6-methylguanine-DNA methyltransferase; n.t.: not tested; SOD: superoxide
dismutase.
Aging and Sex, DNA Repair in 59
of these genetic alterations are described
below.
1. MGMT. One frequent type of DNA
damage (see Table 1) isO6-methylguanine,
caused by low levels of alkylating agents
present in food, water, air, and tobacco
smoke, and formed by normal processes in
the body mediated by gastric bacteria and
macrophages.O6-methylguanine is specifically
repaired by a DNA repair enzyme
called O6-methylguanine-DNA methyltransferase
(MGMT). MGMT transfers the extra
methyl group from guanine in DNA to
a particular amino acid within itself and
becomes ‘‘used up’’ after the transfer occurs.
The MGMT gene codes for one of
the five DNA repair mechanisms listed
in Section 1.1. As indicated in Table 2,
when 100 copies of the MGMT gene were
inserted into the mouse genome, these
mice (under the usual conditions of mouse
maintenance) had their life span extended
and died at a considerably slower rate than
wild-type mice.
2. SOD. Another important type of
metabolically caused DNA damage is oxidative
damage, themost frequent damage
identified (Table 1). An apparently unavoidable
by-product of normal respiratory
metabolism is the production of reactive
oxygen species (ROS) from molecular
oxygen, and ROS cause oxidative damage.
ROS include free radicals (where
the symbol • indicates an unpaired electron):
the superoxide radical (O2
•−) and
the hydroxyl radical (OH•). Another oxygen
respiration by-product is hydrogen
peroxide (H2O2). H2O2, if not removed,
it diffuses fairly easily through the cell,
and when it encounters Fe2+ (the ferrous
ion), it can undergo the Fenton reaction
and produce OH• and other ROS.
ROS produce a number of lesions in
DNA, including base lesions, sugar lesions
(the deoxyribose sugar is in the
backbone of DNA), DNA–protein crosslinks,
single-strand breaks, double-strand
breaks, and abasic sites.
The major ROS produced by the cell
is O2
•−, formed in the mitochondria (the
energy-producing organelles of the cell).
Superoxide dismutase (SOD) occurs in
two forms, manganese SOD (MnSOD)
and copper/zinc SOD (Cu/ZnSOD). Both
forms of SODconvertO2
•− to the less damaging
H2O2, and then another enzyme,
catalase, converts H2O2 to molecular oxygen
and water. MnSOD occurs in the mitochondria
and Cu/ZnSOD occurs in the
cytoplasm.As shown in Table 2, a number
of investigators have found that inserting
genes producing higher than normal
levels of superoxide dismutase into the
fruit fly (Drosophila melanogaster) genome
results in life span extension. Insertion
of genes producing either MnSOD or
Cu/ZnSOD caused life span extension, although
the artificially inserted Cu/ZnSOD
only produced life span extension when
its expression was restricted to the motor
neurons, or solely to the adult phase of the
fruit fly life cycle.
Aging has been found to correlate with
increased levels of oxidative products, such
as protein carbonyls and 8-oxo-guanine in
DNA, and fruit flies lacking either catalase
or Cu/ZnSOD have a reduced life span.
Further, selection of a population of fruit
flies for increased life span correlates
with strongly increased expression of
both MnSOD and Cu/ZnSOD. Reverse
selection of these long life span flies to
a shorter life span resulted in reduced
expression of Cu/ZnSOD.
3. MsrA. In addition to DNA damage,
free radicals damage proteins, lipids,
and carbohydrates. Most proteins have
a short half-life (averaging about three
days in mouse liver). Oxidatively damaged
proteins and lipids are subject to both
60 Aging and Sex, DNA Repair in
degradation and some repair reactions.
If cellular genes that code for enzymes
involved in the replacement of damaged
proteins are themselves damaged, then
damaged proteins may not turn over
as rapidly, and protein damages may
become important as they accumulate with
age. Table 2 shows that insertion of an
extra gene encoding bovine methionine
sulfoxide reductase (MsrA) in the fruit fly
genome, which helps repair oxidatively
damaged proteins, leads to life span
extension. Consistent with this, MsrA,
when defective in the mouse, results in
early aging (Table 3).
4. p66Shc. The p53 gene has a central
role in response to DNA damage. The
p53 protein is directly active in three
forms of DNA repair (NER, BER, and
HRR).When there is no externally induced
DNA damage, p53 has a half-life of
only 5 to 40 minutes since specific
enzymes target p53 for degradation. Thus,
p53 is kept at a low level when there
is no DNA damage. However, upon
exposure of a cell to DNA-damaging
agents, p53 becomes metabolically stable
and, in addition, more copies of it are
produced in the cell. In the presence
of various types of DNA damage, p53
undergoes modifications at some of the
18 different sites within the protein. Some
of these modifications [phosphorylations,
acetylations, poly(ADP-ribosyl)ations, or
sumoylations (covalent attachments of
small ubiquitin-like proteins) allow the
p53 protein to act as a regulatory agent,
activating numerous other genes, carrying
out different responses to different kinds
or levels of DNA damage. The p53 protein
can regulate or act in at least four major
types of responses to DNA damage (acting
as a ‘‘master switch’’), and which action or
transactivation (regulating the induction
of other genes) it performs depends on
the level and type of DNA damage. p53
can (1) send the cell into cell cycle arrest
(to allow extra time for repair of DNA
damage); (2) act directly in DNA repair
(see Fig. 1 for where p53 acts in NER);
(3) cause the cell to switch into a cell
suicide mode (apoptosis); or (4) cause the
cell to produce higher levels of ROS
(apparently as a preliminary to entering
the cell suicide mode of apoptosis). When
acting to increase the internal level of ROS
and entry into apoptosis, p53 acts through
another gene it controls, p66Shc.
When a mouse embryo is produced
with both copies of its p66Shc gene
inactive (a p66Shc ‘‘knockout’’), mouse
embryo fibroblast cells derived from it
have intracellular levels of ROS reduced by
about 40%. Consistent with this reduction
in ROS, there is also greatly reduced
oxidative damage accumulation in both
nuclear and mitochondrial DNA of these
cells. A similar reduction in nuclear and
mitochondrial DNA damage is seen in
vivo in the tissues of lung, spleen, liver,
and skin in 3- and 24-month-old p66Shc
knockout mice, although there is no
reduction in the brain, where p66Shc is
not normally expressed. Cells of thesemice
are inhibited from undergoing apoptosis
after cellular oxidative damage (when
challenged with externally applied H2O2).
Knockout mice without p66Shc show
life span extension without any notable
increase in cancer or other pathological
defects (Table 2). Mice with a type of
overactive p53 (an increase in some
p53 functions) and intact p66Shc show
early aging (Table 3). On the other hand,
removal of all p53 functions (some of
which are protective in DNA repair) also
results in early aging (Table 3).
5. PARP. DNA damages caused by
alkylating agents (such as those that
methylate guanine, discussed above),
Aging and Sex, DNA Repair in 61
Tab. 3 Early aging: decreased life span from alterations in genes controlling DNA repair or protein oxidation.
Organism Genetic Pathway Aging Fertility Spontaneous Effect on
alteration phenotype cancer
Cellular Induced Spont. Induced
ROS DNA mutation apoptosis
damage
Human RECQ3 helicase and
exonuclease defect
(Werner syndrome)
HRR and NHEJ Early aging Reduced Increased n.t. Increased Increased n.t.
Human RECQ2 helicase defect
(Bloom syndrome)
HRR and NHEJ Early aging Reduced Increased n.t. Increased Increased n.t.
Human RECQ4 helicase defect
(Rothmund–Thomson
syndrome)
DNA repair
pathway,
unknown type
Early aging Reduced Increased n.t. n.t. Increased n.t.
Human and
Mouse
XPD helicase defective at
certain sites
(Trichothiodystrophy)
NER, also alters
transcription
initiation
Early aging Reduced No change n.t. n.t. n.t. Increased
Human CSB defect at 2 helicase
motifs or ATPase motif
(Cockayne syndrome)
BER if defective at
helicase motif V
or VI, TCR if
defective in
ATPase function
Early aging n.t. No change n.t. Increased n.t. Increased
Mouse Ku-80 (activator of Ku-70
helicase) defect
NHEJ Early aging n.t. Increased n.t. n.t. n.t.
Mouse Topoisomerase IIIβ
defect
Unknown, but
probably DNA
repair,
replication, or
recombination
Early aging n.t. n.t. n.t. n.t. n.t.
(continued overleaf )
62 Aging and Sex, DNA Repair in
Tab. 3 (continued)
Organism Genetic Pathway Aging Fertility Spontaneous Effect on
alteration phenotype cancer
Cellular Induced Spont. Induced
ROS DNA mutation apoptosis
damage
Mouse ERCC1 defect NER and interstrand
cross-link repair
(HRR)
Early aging Infertile No change n.t. Increased Increased
Mouse p53 overactivated Increases some p53
functions
Early aging n.t. Reduced n.t. n.t. n.t.
Mouse p53 defect Blocks all p53
functions,
including NER,
BER and HRR
Early aging Reduced Increased n.t. Increased Increased
Mouse MsrA Defect Methionine
sulfoxide
reductase
Early aging n.t. n.t. Increased
protein
oxidation
n.t. n.t.
Notes: ROS: reactive oxygen species; Spont.: spontaneous; n.t.: not tested.
Aging and Sex, DNA Repair in 63
ionizing radiation (which produces DNA
single- and double-strand breaks and
oxidative damages), and ROS result in
rapid activation of an enzyme called
poly(ADP-ribose) polymerase, or PARP.
PARP, similar to p53 discussed above,
has a role as a ‘‘master switch’’. PARP
can (1) act directly in one form of DNA
repair, BER, (2) control the function of
many other proteins by catalyzing the addition
of ADP-ribose branched polymers
onto them (either activating or repressing
their function), and (3) trigger apoptosis
(cell suicide). In addition, PARP controls
new transcription or activities of a number
of genes affecting survival or apoptosis,
including p53. It was found that centenarians
(humans who have lived for more
than 100 years) have a modified form
of PARP, which is more efficiently activated
than the PARP of noncentenarians
(Table 2), thereby apparently causing life
span extension. In addition, the maximal
poly(ADP-ribosyl)ation capacity (efficiency
of activation of PARP) in leukocytes of
13 mammalian species of different life
span was measured. There was a strong
correlation of PARP efficiency of activation
with species-specific life span.
четвъртък, 18 юни 2009 г.
Molecular medicine-Consequences of Unrepaired DNA Damage
If accumulated DNA damages are the
cause of aging, then repair processes
would be less than 100% efficient; some
types of unrepaired damages left each day
would gradually build up in nondividing or
slowly dividing cells.Most investigators examining
the presence of DNA damages in
tissues of young versus old mammals (usually
rodents) have found an accumulation
of damaged bases or single- or doublestrand
breaks with age. The tissues where
accumulation of DNA damage has been
shown include liver, kidney, heart,muscle,
and brain.
A number of different types of DNA
damage have been tested for their effects
on transcription and DNA replication. It
was found that transcription is blocked
by UV-induced damages (mainly pyrimidine
dimers) by adducts produced by
derivatives of benzo[a]pyrene, N-acetoxy-
2-fluorenylacetamide, or aflatoxin B1 and
also by the oxidized base, thymine glycol.
UV-induced DNA damages and thymine
glycol have also been shown to block DNA
replication. These findings suggest that
many types of DNA damage inhibit transcription
and replication.
A reduction in the ability to transcribe
mRNA should lead to a decline in the
function of the cells. In fact, in mammalian
Aging and Sex, DNA Repair in 57
Tab. 1 Endogenous DNA damages in mammalian cells.
Type of damage Approximate average incidence
(DNA damages/cell/day)
Oxidative 500,000 (young mouse brain)
2,000,000 (old mouse brain)
86,000 (rats, all tissues)
10,000 (humans, all tissues)
Depurinations 9,000 (humans and rats)
Single-strand break 7,200 (in vitro)
O6-methylguanine 2,000 (in vitro)
Double-strand break >40 (rats)a
>3 (humans)a
DNA cross-link >37 (rats)a
>3 (humans)a
Glucose 6-phosphate adduct 3 (humans)
aThese numbers were calculated from the values in the references
by methods indicated in the literature.
brain, it has been shown that as singlestrandDNAdamages
accumulate with age,
mRNA synthesis and protein synthesis decline,
neuron loss occurs, tissue function
is reduced, and functional impairments
directly related to the central processes
of aging (e.g. cognitive dysfunction and
decline in homeostatic regulation) occur.
Similarly, it has been shown in muscle
cells that as single-strand DNA damages
accumulate, mRNA and protein synthesis
decline, cellular structures deteriorate,
cells die, and this is accompanied by a reduction
in muscle strength and speed of
contraction. Thus, for brain and muscle,
accumulation ofDNAdamage is paralleled
by declines in function, suggesting a direct
cause-and-effect relationship between the
accumulation of DNA damage and major
features of aging. In other cells, including
those of liver and lymphocytes, evidence
for an increase in DNA damage paralleled
by a decline in gene expression and cellular
function has also been observed. In
general, it appears that tissues composed
of nondividing or slowly dividing cells accumulate
DNA damage and experience
functional declines with age.
cause of aging, then repair processes
would be less than 100% efficient; some
types of unrepaired damages left each day
would gradually build up in nondividing or
slowly dividing cells.Most investigators examining
the presence of DNA damages in
tissues of young versus old mammals (usually
rodents) have found an accumulation
of damaged bases or single- or doublestrand
breaks with age. The tissues where
accumulation of DNA damage has been
shown include liver, kidney, heart,muscle,
and brain.
A number of different types of DNA
damage have been tested for their effects
on transcription and DNA replication. It
was found that transcription is blocked
by UV-induced damages (mainly pyrimidine
dimers) by adducts produced by
derivatives of benzo[a]pyrene, N-acetoxy-
2-fluorenylacetamide, or aflatoxin B1 and
also by the oxidized base, thymine glycol.
UV-induced DNA damages and thymine
glycol have also been shown to block DNA
replication. These findings suggest that
many types of DNA damage inhibit transcription
and replication.
A reduction in the ability to transcribe
mRNA should lead to a decline in the
function of the cells. In fact, in mammalian
Aging and Sex, DNA Repair in 57
Tab. 1 Endogenous DNA damages in mammalian cells.
Type of damage Approximate average incidence
(DNA damages/cell/day)
Oxidative 500,000 (young mouse brain)
2,000,000 (old mouse brain)
86,000 (rats, all tissues)
10,000 (humans, all tissues)
Depurinations 9,000 (humans and rats)
Single-strand break 7,200 (in vitro)
O6-methylguanine 2,000 (in vitro)
Double-strand break >40 (rats)a
>3 (humans)a
DNA cross-link >37 (rats)a
>3 (humans)a
Glucose 6-phosphate adduct 3 (humans)
aThese numbers were calculated from the values in the references
by methods indicated in the literature.
brain, it has been shown that as singlestrandDNAdamages
accumulate with age,
mRNA synthesis and protein synthesis decline,
neuron loss occurs, tissue function
is reduced, and functional impairments
directly related to the central processes
of aging (e.g. cognitive dysfunction and
decline in homeostatic regulation) occur.
Similarly, it has been shown in muscle
cells that as single-strand DNA damages
accumulate, mRNA and protein synthesis
decline, cellular structures deteriorate,
cells die, and this is accompanied by a reduction
in muscle strength and speed of
contraction. Thus, for brain and muscle,
accumulation ofDNAdamage is paralleled
by declines in function, suggesting a direct
cause-and-effect relationship between the
accumulation of DNA damage and major
features of aging. In other cells, including
those of liver and lymphocytes, evidence
for an increase in DNA damage paralleled
by a decline in gene expression and cellular
function has also been observed. In
general, it appears that tissues composed
of nondividing or slowly dividing cells accumulate
DNA damage and experience
functional declines with age.
Molecular medicine-The DNA Damage Theory of Aging
Occurrence of DNA Damage and Pathways
of DNA Repair
Except for certain viruses with an RNA
genome, the genomes of most organisms
are composed of DNA. If DNA damage is
the cause of aging, thenDNAdamage is expected
to occur frequently in multicellular
organisms. Table 1 lists some important
types of DNA damage caused by normal
metabolic processes in mammals. These
data suggest, for instance, that in the rat at
least 95,000 DNA damages of various types
occur, averaged over all cell types, per cell
per day. The majority of these damages
alter the structure of only a single DNA
strand, so the redundant information in
the complementary strand can usually be
used to repair the damage. The damages
shown in Table 1 are the newly occurring
damages, most being rapidly repaired.
Five major DNA repair pathways known
to be utilized by cells to repair the damages
indicated in Table 1 are as follows:
• Nucleotide excision repair (NER) [with
two subpathways, largely using the
same enzymes: transcription coupled
repair (TCR) and global genomic repair
(GGR)]
• Base excision repair (BER)
• Nonhomologous end joining (NHEJ)
• Homologous recombinational repair
(HRR)
• O6-methylguanine-DNA methyltransferase
(MGMT)
of DNA Repair
Except for certain viruses with an RNA
genome, the genomes of most organisms
are composed of DNA. If DNA damage is
the cause of aging, thenDNAdamage is expected
to occur frequently in multicellular
organisms. Table 1 lists some important
types of DNA damage caused by normal
metabolic processes in mammals. These
data suggest, for instance, that in the rat at
least 95,000 DNA damages of various types
occur, averaged over all cell types, per cell
per day. The majority of these damages
alter the structure of only a single DNA
strand, so the redundant information in
the complementary strand can usually be
used to repair the damage. The damages
shown in Table 1 are the newly occurring
damages, most being rapidly repaired.
Five major DNA repair pathways known
to be utilized by cells to repair the damages
indicated in Table 1 are as follows:
• Nucleotide excision repair (NER) [with
two subpathways, largely using the
same enzymes: transcription coupled
repair (TCR) and global genomic repair
(GGR)]
• Base excision repair (BER)
• Nonhomologous end joining (NHEJ)
• Homologous recombinational repair
(HRR)
• O6-methylguanine-DNA methyltransferase
(MGMT)
Molecular medicine-Keywords
Aging
The progressive impairments of functions experienced by many organisms throughout
their life span.
Aging and Sex, DNA Repair in 55
DNA Damage
A DNA alteration that has an abnormal structure, which cannot itself be replicated
when the DNA is replicated, but which may be repaired.
DNA Repair
The process of removing damage from DNA and restoring the DNA structure.
Mutation
A change in the sequence of DNA base pairs, which may be replicated and
thus inherited.
Sex
The process by which genetic material (usually DNA) from two separate parents is
brought together in a common cytoplasm where recombination of the genetic material
ordinarily occurs, followed by the passage of the recombined genome(s) to progeny.
Complementation
The masking of the expression of mutant genes by corresponding wild-type genes
when two homologous chromosomes share a common cytoplasm.
A number of theories have been proposed to account for the biological phenomena
of aging and sexual reproduction (sex). An emerging unified theory that accounts
for a considerable amount of the data relating to both aging and sex is
presented here.
Aging appears to be a consequence of DNA damage, while sexual reproduction
(sex) appears to be an adaptation for coping with both DNA damage and mutation.
DNA, the genetic material of most organisms, is composed of molecular subunits
that are not endowed with any peculiar chemical stability. Thus, DNA is subject to
a wide variety of chemical reactions that might be expected of any such molecule
in a warm aqueous medium. DNA damages are known to occur very frequently,
and organisms have evolved enzyme-mediated repair processes to cope with them.
In any cell, however, some DNA damage may remain unrepaired despite repair
processes. Aging appears to be due to the accumulation of unrepaired DNA damage
in somatic cells, especially in nondividing cells such as those in mammalian brain
and muscle.
On the other hand, the primary function of sex appears to be the repair of damages
in germ cell DNA through efficient recombinational repair when chromosomes pair
during the sexual process. This allows a relatively undamaged genome to initiate the
next generation. In addition, in diploid organisms, sex allows chromosomes from
genetically unrelated individuals (parents) to come together in a common cytoplasm
(that of progeny). Since genetically unrelated parents ordinarily would not have
common mutations, the chromosomes present in the progeny should complement
each other, masking expression of any deleterious mutations that might be present.
56 Aging and Sex, DNA Repair in
Thus, aging and sex appear to be two sides of the same coin. Aging reflects the
accumulation of DNA damage and sex reflects the removal of DNA damage, and in
diploid organisms, the masking of mutations by complementation
The progressive impairments of functions experienced by many organisms throughout
their life span.
Aging and Sex, DNA Repair in 55
DNA Damage
A DNA alteration that has an abnormal structure, which cannot itself be replicated
when the DNA is replicated, but which may be repaired.
DNA Repair
The process of removing damage from DNA and restoring the DNA structure.
Mutation
A change in the sequence of DNA base pairs, which may be replicated and
thus inherited.
Sex
The process by which genetic material (usually DNA) from two separate parents is
brought together in a common cytoplasm where recombination of the genetic material
ordinarily occurs, followed by the passage of the recombined genome(s) to progeny.
Complementation
The masking of the expression of mutant genes by corresponding wild-type genes
when two homologous chromosomes share a common cytoplasm.
A number of theories have been proposed to account for the biological phenomena
of aging and sexual reproduction (sex). An emerging unified theory that accounts
for a considerable amount of the data relating to both aging and sex is
presented here.
Aging appears to be a consequence of DNA damage, while sexual reproduction
(sex) appears to be an adaptation for coping with both DNA damage and mutation.
DNA, the genetic material of most organisms, is composed of molecular subunits
that are not endowed with any peculiar chemical stability. Thus, DNA is subject to
a wide variety of chemical reactions that might be expected of any such molecule
in a warm aqueous medium. DNA damages are known to occur very frequently,
and organisms have evolved enzyme-mediated repair processes to cope with them.
In any cell, however, some DNA damage may remain unrepaired despite repair
processes. Aging appears to be due to the accumulation of unrepaired DNA damage
in somatic cells, especially in nondividing cells such as those in mammalian brain
and muscle.
On the other hand, the primary function of sex appears to be the repair of damages
in germ cell DNA through efficient recombinational repair when chromosomes pair
during the sexual process. This allows a relatively undamaged genome to initiate the
next generation. In addition, in diploid organisms, sex allows chromosomes from
genetically unrelated individuals (parents) to come together in a common cytoplasm
(that of progeny). Since genetically unrelated parents ordinarily would not have
common mutations, the chromosomes present in the progeny should complement
each other, masking expression of any deleterious mutations that might be present.
56 Aging and Sex, DNA Repair in
Thus, aging and sex appear to be two sides of the same coin. Aging reflects the
accumulation of DNA damage and sex reflects the removal of DNA damage, and in
diploid organisms, the masking of mutations by complementation
Molecular medicine-The Formation of Amyloid Fibrils and its Pathological Consequences
The formation of amyloid fibrils plays a key
role in the origin of several neurodegenerative
pathologies, such as spongiform
encephalopathies and Alzheimer’s disease.
Historically, the term amyloid was
introduced to describe fibrillar protein
deposits associated with diseases known
as amyloidoses that involve the extracellular
deposition of amyloid fibrils and
plaques with the aspect of starch. For
many of these diseases, the major fibrillar
46 Aggregation, Protein
protein component has been identified.
In the 1970s, it was demonstrated that
lysosomal proteins under acidic conditions
could form amyloid fibrils. It was
generally accepted at this time that proteolysis
was the amyloidogenic determinant.
Twenty years later, it was shown that
purified transthyretin is converted into
amyloid fibrils via an acid-induced conformational
change in vitro, demonstrating
that conformational changes alone were
responsible for producing an intermediate
generating amyloid structure. These
aberrant protein self-assemblies are at
the origin of more than hundred human
amyloid diseases, some of them being
lethal.
Twenty unrelated protein precursors
are known to form amyloid fibrils,
among them transthyretin, lysozyme, immunoglobulin
light chain, β2 microglobulin,
Alzheimer Aβ1–40 and Aβ1–42
peptides, the mammalian prion protein,
and the yeast prion-like proteins (Table 1).
Since they are subjects of another chapter,
prion proteins will not be discussed
here. Although they have no homology
in sequence and structure, all form amyloid
fibrils with a similar overall structure,
suggesting a common self-assembly
Tab. 1 Amyloidogenic proteins and the corresponding diseases.
Clinical syndrome Precursor protein Fibril component
Alzeimer’s disease APP β-peptide 1–40 to 1–43
Primary systemic amyloidosis Immunoglobulin light chain Intact light chain or fragments
Secondary systemic amyloidosis Serum amyloid A Amyloid A (76-residue
fragment)
Senile systemic amyloidosis Transthyretin Transthyretin or fragments
Familial amyloid polyneuropathy I Transthyretin Over 45 transthyretin variants
Hereditary cerebral amyloid
angiopathy
Cystatin C Cystatin C minus 10 residues
Hemodialysis-related β2-microglobulin β2-microglobulin
amyloidosis Apolipoprotein A1 Fragments of Apolipoprotein
A1
Familial amyloid polyneuropathy III Gelsosin 71-amino acid fragment of
gelsosin
Finnish hereditory systemic
amyloidosis
Islet amyloid polypeptide
(IAPP)
Fragment of IAPP
Type II diabetes Calcitonin Fragments of calcitonin
Medullary carcinoma of the thyroid
Spongiform encephalopathies Prion Prion or fragments thereof
Atrial amyloidosis Atrial natriuretic factor
(ANF)
ANF
Hereditary nonneuropathic systemic
amyloidosis
Lysozyme Lysozyme or fragments
thereof
Injection-localized amyloidosis Insulin Insulin
Hereditary renal amyloidosis Fibrinogen Fibrinogen fragments
Parkinson disease α-synuclein∗
Source: (According to Kelly, J.W. (1996) Alternative conformations of amyloidogenic proteins govern
their behavior, Curr. Opin. Struct. Biol. 6, 11–17); ∗From J.C. Rochet & P.T. Lansbury (2000) Curr.
Opin. Struct. Biol. 10, 60–68.
Aggregation, Protein 47
pathway. In all proteins known to form
amyloid fibrils, there is a conversion of
α- to β-structure. Amyloid fibrils are abnormal,
insoluble, and generally proteaseresistant
structures. They were first recognized
by their staining properties. The
most commonly used method to detect
amyloid is staining by Congo Red,
which exhibits a green birefringence. Amyloid
fibrils are generally 60 to 100 A˚ in
diameter and of variable length. X-ray
diffraction data on fibrils, solid-state NMR
studies, cryoelectron microscopy, and infrared
Fourier transform experiments have
shown that amyloid fibrils aremade of two
or more β-sheet filaments wound around
one another. They have a characteristic
cross-β repeat structure, the individual β-
strands being oriented perpendicular to
the long axis of the fibril.
Recently, progress has been made in
the knowledge of the mechanisms involved
in the formation of amyloid fibrils.
Oligomeric prefibrillar intermediates have
been extensively characterized with respect
to their structure and temporal
evolution. A well-documented example is
provided by the studies on transthyretin.
The biological role of this protein is the
transport of thyroxin by direct binding
and the transport of retinol via the retinol
binding protein. The wild-type protein is
very stable at neutral pH. In certain individuals,
however, it is converted into
amyloid fibrils, and this is associated
with the disease, senile systemic amyloidosis.
Several variants are associated
with familial polyneuropathies. In vitro
biophysical studies have identified conditions
leading to amyloid formation. The
three-dimensional structure of the protein
is known. The wild-type protein is
a tetramer at pH ranging between 5 and 7;
the tetramer dissociates into a monomer
when the pH decreases. The dissociation
is the rate-limiting step of the process.
The monomer exhibits an altered tertiary
structure, which aggregates in amyloid
Native protein Amylogenic intermediate
Protofibrils
Fibrils
n
Unfolded protein
Fig. 10 Schematic representation of the formation of amyloid fibrils from a
partially folded intermediate.
48 Aggregation, Protein
protofilaments, and then forms amyloid
fibrils. This formation is at its maximumat
pH 4.4. Using deuterium–proton
exchange monitored by two-dimensional
NMR spectroscopy on transthyretin at pH
5.75 and 4.5, Liu et al. have shown a selective
destabilization of one half of the
β-sandwich structure of the protein, increasing
the mobility of this region. These
studies have identified the residues that
undergo increased conformational fluctuations
under amyloidogenic conditions.
The mutations in the pathological variants
responsible for familial amyloid polyneuropathies
are localized in this region. A
strategy to delay the formation of amyloid
fibrils proposed by Saccheti & Kelly was
to developmolecules capable of stabilizing
the tetramer.
Fig. 11 Molecular model of an amyloid
fibril derived from cryoelectron
microscopy analysis of fibrils grown
from an SH3 domain by incubation of a
solution containing the protein at low
pH (reproduced from Dobson, C. (1999)
TIBS 24, 331, with permission).
Two variants of human lysozyme,
Ile56Thr andAsp67His have been reported
to be amyloidogenic; they are responsible
for fatal amyloidoses. Pepys and colleagues
have determined the precise structures and
properties of these mutants. The native
fold of the two amyloidogenic variants,
as resolved by X-ray crystallography, is
similar to that of the wild-type protein.
Both variants are enzymatically active, but
have been shown to be unstable. The replacement
of an aspartate by a histidine
suppresses a hydrogen bond formed in
the wild-type protein with a tyrosine in a
neighboring β-strand. This rupture opens
a large gap between two β-strands. In
the other variant, the replacement of an
isoleucine by a threonine suppresses a van
der Waals contact with a neighboring helix.
Consequently, changes in the interface
between the α- and β-domains occur in
both variants, destabilizing the molecule.
The mutations leading to amyloid fibril
formation are observed to result in a
decreased stability of the native state. In
all cases, the formation of fibrils occurs
from a partially structured molecule via
nucleation-dependent oligomerization. It
was observed for several proteins that
fibrillation takes place only after a lag
phase, which is abolished upon seeding.
Nucleation is followed by the formation
of protofibrils whose characteristics have
been determined (Fig. 10). Atomic force
microscopy and fluorescence correlation
spectroscopy have been used to monitor
transitions among the different types
of assemblies.
Aggregation, Protein 49
Recent observations from Dobson and
his group have shown that several proteins
unrelated to amyloid diseases are
able to aggregate in vitro into amyloid
fibrils when exposed to mild denaturing
conditions. These fibrils are indistinguishable
from those found in pathological
conditions. It was demonstrated for different
proteins such as normal lysozyme,
an SH3 domain of a phosphatidyl inositol
protein kinase (Fig. 11), an acyl
phosphatase, and an α-helical protein,
myoglobin suggesting a common mechanism
for the formation of amyloid.
These findings clearly indicate that amyloid
formation is a general property of
polypeptide chains rather than one restricted
to definite sequences as occurs
with chameleon sequences capable of
adopting either a β- or an α-helicoidal
structure depending on their environment.
Furthermore, these aggregates exhibited
an inherent toxicity when incubated with
mouse fibroblasts. Several groups suggest
that oligomeric intermediates rather
than fibrils themselves are responsible for
pathogenicity.
Significant progress has been made in
understanding the mechanisms involved
in the formation of amyloid fibrils. This
is an important step in guiding research
into the discovery of molecules with
therapeutic efficiency.
role in the origin of several neurodegenerative
pathologies, such as spongiform
encephalopathies and Alzheimer’s disease.
Historically, the term amyloid was
introduced to describe fibrillar protein
deposits associated with diseases known
as amyloidoses that involve the extracellular
deposition of amyloid fibrils and
plaques with the aspect of starch. For
many of these diseases, the major fibrillar
46 Aggregation, Protein
protein component has been identified.
In the 1970s, it was demonstrated that
lysosomal proteins under acidic conditions
could form amyloid fibrils. It was
generally accepted at this time that proteolysis
was the amyloidogenic determinant.
Twenty years later, it was shown that
purified transthyretin is converted into
amyloid fibrils via an acid-induced conformational
change in vitro, demonstrating
that conformational changes alone were
responsible for producing an intermediate
generating amyloid structure. These
aberrant protein self-assemblies are at
the origin of more than hundred human
amyloid diseases, some of them being
lethal.
Twenty unrelated protein precursors
are known to form amyloid fibrils,
among them transthyretin, lysozyme, immunoglobulin
light chain, β2 microglobulin,
Alzheimer Aβ1–40 and Aβ1–42
peptides, the mammalian prion protein,
and the yeast prion-like proteins (Table 1).
Since they are subjects of another chapter,
prion proteins will not be discussed
here. Although they have no homology
in sequence and structure, all form amyloid
fibrils with a similar overall structure,
suggesting a common self-assembly
Tab. 1 Amyloidogenic proteins and the corresponding diseases.
Clinical syndrome Precursor protein Fibril component
Alzeimer’s disease APP β-peptide 1–40 to 1–43
Primary systemic amyloidosis Immunoglobulin light chain Intact light chain or fragments
Secondary systemic amyloidosis Serum amyloid A Amyloid A (76-residue
fragment)
Senile systemic amyloidosis Transthyretin Transthyretin or fragments
Familial amyloid polyneuropathy I Transthyretin Over 45 transthyretin variants
Hereditary cerebral amyloid
angiopathy
Cystatin C Cystatin C minus 10 residues
Hemodialysis-related β2-microglobulin β2-microglobulin
amyloidosis Apolipoprotein A1 Fragments of Apolipoprotein
A1
Familial amyloid polyneuropathy III Gelsosin 71-amino acid fragment of
gelsosin
Finnish hereditory systemic
amyloidosis
Islet amyloid polypeptide
(IAPP)
Fragment of IAPP
Type II diabetes Calcitonin Fragments of calcitonin
Medullary carcinoma of the thyroid
Spongiform encephalopathies Prion Prion or fragments thereof
Atrial amyloidosis Atrial natriuretic factor
(ANF)
ANF
Hereditary nonneuropathic systemic
amyloidosis
Lysozyme Lysozyme or fragments
thereof
Injection-localized amyloidosis Insulin Insulin
Hereditary renal amyloidosis Fibrinogen Fibrinogen fragments
Parkinson disease α-synuclein∗
Source: (According to Kelly, J.W. (1996) Alternative conformations of amyloidogenic proteins govern
their behavior, Curr. Opin. Struct. Biol. 6, 11–17); ∗From J.C. Rochet & P.T. Lansbury (2000) Curr.
Opin. Struct. Biol. 10, 60–68.
Aggregation, Protein 47
pathway. In all proteins known to form
amyloid fibrils, there is a conversion of
α- to β-structure. Amyloid fibrils are abnormal,
insoluble, and generally proteaseresistant
structures. They were first recognized
by their staining properties. The
most commonly used method to detect
amyloid is staining by Congo Red,
which exhibits a green birefringence. Amyloid
fibrils are generally 60 to 100 A˚ in
diameter and of variable length. X-ray
diffraction data on fibrils, solid-state NMR
studies, cryoelectron microscopy, and infrared
Fourier transform experiments have
shown that amyloid fibrils aremade of two
or more β-sheet filaments wound around
one another. They have a characteristic
cross-β repeat structure, the individual β-
strands being oriented perpendicular to
the long axis of the fibril.
Recently, progress has been made in
the knowledge of the mechanisms involved
in the formation of amyloid fibrils.
Oligomeric prefibrillar intermediates have
been extensively characterized with respect
to their structure and temporal
evolution. A well-documented example is
provided by the studies on transthyretin.
The biological role of this protein is the
transport of thyroxin by direct binding
and the transport of retinol via the retinol
binding protein. The wild-type protein is
very stable at neutral pH. In certain individuals,
however, it is converted into
amyloid fibrils, and this is associated
with the disease, senile systemic amyloidosis.
Several variants are associated
with familial polyneuropathies. In vitro
biophysical studies have identified conditions
leading to amyloid formation. The
three-dimensional structure of the protein
is known. The wild-type protein is
a tetramer at pH ranging between 5 and 7;
the tetramer dissociates into a monomer
when the pH decreases. The dissociation
is the rate-limiting step of the process.
The monomer exhibits an altered tertiary
structure, which aggregates in amyloid
Native protein Amylogenic intermediate
Protofibrils
Fibrils
n
Unfolded protein
Fig. 10 Schematic representation of the formation of amyloid fibrils from a
partially folded intermediate.
48 Aggregation, Protein
protofilaments, and then forms amyloid
fibrils. This formation is at its maximumat
pH 4.4. Using deuterium–proton
exchange monitored by two-dimensional
NMR spectroscopy on transthyretin at pH
5.75 and 4.5, Liu et al. have shown a selective
destabilization of one half of the
β-sandwich structure of the protein, increasing
the mobility of this region. These
studies have identified the residues that
undergo increased conformational fluctuations
under amyloidogenic conditions.
The mutations in the pathological variants
responsible for familial amyloid polyneuropathies
are localized in this region. A
strategy to delay the formation of amyloid
fibrils proposed by Saccheti & Kelly was
to developmolecules capable of stabilizing
the tetramer.
Fig. 11 Molecular model of an amyloid
fibril derived from cryoelectron
microscopy analysis of fibrils grown
from an SH3 domain by incubation of a
solution containing the protein at low
pH (reproduced from Dobson, C. (1999)
TIBS 24, 331, with permission).
Two variants of human lysozyme,
Ile56Thr andAsp67His have been reported
to be amyloidogenic; they are responsible
for fatal amyloidoses. Pepys and colleagues
have determined the precise structures and
properties of these mutants. The native
fold of the two amyloidogenic variants,
as resolved by X-ray crystallography, is
similar to that of the wild-type protein.
Both variants are enzymatically active, but
have been shown to be unstable. The replacement
of an aspartate by a histidine
suppresses a hydrogen bond formed in
the wild-type protein with a tyrosine in a
neighboring β-strand. This rupture opens
a large gap between two β-strands. In
the other variant, the replacement of an
isoleucine by a threonine suppresses a van
der Waals contact with a neighboring helix.
Consequently, changes in the interface
between the α- and β-domains occur in
both variants, destabilizing the molecule.
The mutations leading to amyloid fibril
formation are observed to result in a
decreased stability of the native state. In
all cases, the formation of fibrils occurs
from a partially structured molecule via
nucleation-dependent oligomerization. It
was observed for several proteins that
fibrillation takes place only after a lag
phase, which is abolished upon seeding.
Nucleation is followed by the formation
of protofibrils whose characteristics have
been determined (Fig. 10). Atomic force
microscopy and fluorescence correlation
spectroscopy have been used to monitor
transitions among the different types
of assemblies.
Aggregation, Protein 49
Recent observations from Dobson and
his group have shown that several proteins
unrelated to amyloid diseases are
able to aggregate in vitro into amyloid
fibrils when exposed to mild denaturing
conditions. These fibrils are indistinguishable
from those found in pathological
conditions. It was demonstrated for different
proteins such as normal lysozyme,
an SH3 domain of a phosphatidyl inositol
protein kinase (Fig. 11), an acyl
phosphatase, and an α-helical protein,
myoglobin suggesting a common mechanism
for the formation of amyloid.
These findings clearly indicate that amyloid
formation is a general property of
polypeptide chains rather than one restricted
to definite sequences as occurs
with chameleon sequences capable of
adopting either a β- or an α-helicoidal
structure depending on their environment.
Furthermore, these aggregates exhibited
an inherent toxicity when incubated with
mouse fibroblasts. Several groups suggest
that oligomeric intermediates rather
than fibrils themselves are responsible for
pathogenicity.
Significant progress has been made in
understanding the mechanisms involved
in the formation of amyloid fibrils. This
is an important step in guiding research
into the discovery of molecules with
therapeutic efficiency.
Molecular medicine-Strategies for Refolding Inclusion Body Proteins
The recovery of the active protein from
inclusion bodies is crucial for industrial
purposes. In structural proteomics
today, efficient production of genetically
engineered proteins is a prerequisite for
exploiting the information contained in
the genome sequences. The strategy to recover
active proteins involves several steps
of purification. The first step, the separation
of the inclusion bodies from the
cell, consists of cell lysis monitored either
by high-pressure homogeneization, or by
a combination of mechanical, chemical,
and enzymatic techniques such as the use
of EDTA and lysozyme. The lysates are
then treated by low-speed centrifugation
or filtration to remove the soluble fraction
from the pellet containing inclusion
bodies and cell debris. The most difficult
task is to remove the contaminants; this
is achieved by the washing steps, which
commonly utilize EDTA and low concentrations
of denaturants or detergents such
as Triton X-100, deoxycholate, or octylglucoside.
Using centrifugation in a sucrose
gradient, it is generally possible to remove
cell debris and membrane proteins. When
the accumulation levels of aggregates are
very high, inclusion bodies may be directly
solubilized by treatment in a high concentration
of denaturant, eliminating the
need for gradient centrifugation. In this
44 Aggregation, Protein
case, the costs of production are considerably
reduced.
A variety of techniques are available
to solubilize purified inclusion bodies.
The most commonly used solubilizing
reagents are strong denaturants such as
guanidine hydrochloride and urea. Generally,
high denaturant concentrations are
employed, 4 to 6 M for guanidine hydrochloride,
and 5 to 10 M for urea to
allow the disruption of noncovalent intermolecular
interactions. Conditionsmay
differ somewhat according to the denaturant
and the protein. Lower denaturant
concentrations have been used to solubilize
cytokines from E. coli inclusion bodies.
The purity of the solubilized protein was
much higher at 1.5 to 2 M guanidinium
chloride than at 4 to 6 M guanidinium
chloride. At higher denaturant concentrations,
contaminating proteins were also
released from the particulate fractions.
Extremes of pH have also been used to
solubilize inclusion bodies and for growth
hormone, proinsulin, and some antifungal
recombinant peptides. However, exposure
to very low or very high pH may not be
applicable to many proteins andmay cause
irreversible chemical modifications.
Detergents such as sodium dodecylsulfate
(SDS) and n-cetyl trimethylammonium
bromide (CTAB), have also been
used to solubilize inclusion bodies. Extensive
washing may then be needed
to remove the solubilizing detergents.
They also may be extracted from the refolding
mixture by using cyclodextrins,
linear dextrins, or cycloamylose. Recent
developments include the use of high
hydrostatic pressure (1–2 kbar) for solubilization
and renaturation. For proteins
with disulfide bonds, the addition of a reducing
reagent such as dithiothreitol or
β-mercaptoethanol is necessary to disrupt
the incorrectly paired disulfide bonds. The
concentrations generally used are 0.1 M
for dithiothreitol and 0.1 to 0.3 M for β-
mercaptoethanol.
When expression levels are very high,
an in situ solubilization method can be
used. It consists of adding the solubilizing
reagent directly to the cells at the end
of the fermentation process. The main
disadvantage of this technique concerns
the release of contaminants.
The last step is the recovery of the active
protein. When inclusion bodies have
been solubilized, the refolding is achieved
by removal of the denaturant. This can
be done by different techniques including
dilution, dialysis, diafiltration, gel filtration,
chromatography, or immobilization
on a solid support. Dilution has been
extensively used. It considerably reduces
concentrations of both denaturant and
protein. This procedure, however, cannot
be applied to the commercial scale refolding
of recombinant proteins, because
large downstream processing volumes increase
the cost of products. Although
dialysis through semipermeable membranes
has been used successfully to
refold several proteins, it is not employed
in large-scale processes. This is because
it requires very long processing times,
and there is the risk that during dialysis,
the protein will remain too long
at a critical concentration of denaturant
and aggregate. The removal of the denaturant
may be accomplished through
gel filtration. However, here again, a
possible aggregation could lead to flow
restriction within the column. Dialfiltration
through a semipermeable membrane
allows the removal of denaturant and
other small molecules and retains the protein.
This procedure has been used for
large-scale processing and was particularly
efficient in the refolding of prorennin and
interferon-β.
Aggregation, Protein 45
During the refolding process, the formation
of incorrectly folded species and
aggregates usually decreases the refolding
yield. For disulfide-bridged proteins, the
renaturation buffer must contain redoxshuffling
mixture to allow the formation
of correctly paired disulfide bridges. Stabilizing
reagents may be added to improve
the refolding yield. An efficient strategy
is the addition of small molecules to suppress
intermolecular interactions leading
to aggregation. Sugar, alcohols, polyols
(including sucrose, glycerol, polyethylene
glycol, isopropanol), cyclodextrin, laurylmaltoside,
sulfobetains, L-arginine, and
low concentrations of denaturants and detergents,
have been used to increase the
refolding yield. L-arginine at a concentration
ranging from 0.4 M to 0.8 M is the
most widely used additive today.
Another important factor in the refolding
process is the rate of removal of the
denaturant. Since there is kinetic competition
between the correct folding and the
formation of aggregates from a folding intermediate,
conditions that favor folding
over the accumulation of aggregates must
be found. To optimize this selection, Vilick
and de Bernadez–Clark developed a
strategy for achieving high protein refolding
yields. They start from a model of
refolding, develop the equations of refolding
kinetics, characterize the rate-limiting
step of the process, determine the influence
of various environmental parameters,
and finally optimize the system of equations
in a scheme involving diafiltration to
remove the denaturant. The approach was
evaluated in the refolding of carbonic anhydrase
from 8 Murea. The yield obtained
after three diafiltration experiments was
69% whereas the model predicted a yield
of 73%.
The properties of molecular chaperones
have also been utilized to increase the
refolding yield. Altamiro and coworkers
have developed a systemfor refolding chromatography
that utilizes GroEL, DsbA, and
peptidyl–prolyl isomerase immobilized on
an agarose gel. Kohler and coworkers
have built a chaperone-assisted bioreactor;
however, it could only be used for
three cycles of refolding and needs to be
improved. Another strategy consists of the
co-overproduction of the DnaK–DnaJ or
GroEL–GroES chaperones with the desired
protein; this can greatly increase
the soluble yield of aggregation-prone proteins.
Fusion proteins have also been used
to minimize aggregation.
The recovery of active proteins from inclusion
bodies is a rather complex process.
Although some general strategies have
been developed, optimal conditions have to
be determined for each protein. Recently,
genetic strategies to improve recovery processes
for recombinant proteins have been
introduced. They consist of the introduction
of combinatorial protein engineering
to generate molecules highly specific to
a particular ligand. Such methods, which
allow efficient recovery of a recombinant
protein, will be increasingly used in industrial
scale bioprocesses as well
inclusion bodies is crucial for industrial
purposes. In structural proteomics
today, efficient production of genetically
engineered proteins is a prerequisite for
exploiting the information contained in
the genome sequences. The strategy to recover
active proteins involves several steps
of purification. The first step, the separation
of the inclusion bodies from the
cell, consists of cell lysis monitored either
by high-pressure homogeneization, or by
a combination of mechanical, chemical,
and enzymatic techniques such as the use
of EDTA and lysozyme. The lysates are
then treated by low-speed centrifugation
or filtration to remove the soluble fraction
from the pellet containing inclusion
bodies and cell debris. The most difficult
task is to remove the contaminants; this
is achieved by the washing steps, which
commonly utilize EDTA and low concentrations
of denaturants or detergents such
as Triton X-100, deoxycholate, or octylglucoside.
Using centrifugation in a sucrose
gradient, it is generally possible to remove
cell debris and membrane proteins. When
the accumulation levels of aggregates are
very high, inclusion bodies may be directly
solubilized by treatment in a high concentration
of denaturant, eliminating the
need for gradient centrifugation. In this
44 Aggregation, Protein
case, the costs of production are considerably
reduced.
A variety of techniques are available
to solubilize purified inclusion bodies.
The most commonly used solubilizing
reagents are strong denaturants such as
guanidine hydrochloride and urea. Generally,
high denaturant concentrations are
employed, 4 to 6 M for guanidine hydrochloride,
and 5 to 10 M for urea to
allow the disruption of noncovalent intermolecular
interactions. Conditionsmay
differ somewhat according to the denaturant
and the protein. Lower denaturant
concentrations have been used to solubilize
cytokines from E. coli inclusion bodies.
The purity of the solubilized protein was
much higher at 1.5 to 2 M guanidinium
chloride than at 4 to 6 M guanidinium
chloride. At higher denaturant concentrations,
contaminating proteins were also
released from the particulate fractions.
Extremes of pH have also been used to
solubilize inclusion bodies and for growth
hormone, proinsulin, and some antifungal
recombinant peptides. However, exposure
to very low or very high pH may not be
applicable to many proteins andmay cause
irreversible chemical modifications.
Detergents such as sodium dodecylsulfate
(SDS) and n-cetyl trimethylammonium
bromide (CTAB), have also been
used to solubilize inclusion bodies. Extensive
washing may then be needed
to remove the solubilizing detergents.
They also may be extracted from the refolding
mixture by using cyclodextrins,
linear dextrins, or cycloamylose. Recent
developments include the use of high
hydrostatic pressure (1–2 kbar) for solubilization
and renaturation. For proteins
with disulfide bonds, the addition of a reducing
reagent such as dithiothreitol or
β-mercaptoethanol is necessary to disrupt
the incorrectly paired disulfide bonds. The
concentrations generally used are 0.1 M
for dithiothreitol and 0.1 to 0.3 M for β-
mercaptoethanol.
When expression levels are very high,
an in situ solubilization method can be
used. It consists of adding the solubilizing
reagent directly to the cells at the end
of the fermentation process. The main
disadvantage of this technique concerns
the release of contaminants.
The last step is the recovery of the active
protein. When inclusion bodies have
been solubilized, the refolding is achieved
by removal of the denaturant. This can
be done by different techniques including
dilution, dialysis, diafiltration, gel filtration,
chromatography, or immobilization
on a solid support. Dilution has been
extensively used. It considerably reduces
concentrations of both denaturant and
protein. This procedure, however, cannot
be applied to the commercial scale refolding
of recombinant proteins, because
large downstream processing volumes increase
the cost of products. Although
dialysis through semipermeable membranes
has been used successfully to
refold several proteins, it is not employed
in large-scale processes. This is because
it requires very long processing times,
and there is the risk that during dialysis,
the protein will remain too long
at a critical concentration of denaturant
and aggregate. The removal of the denaturant
may be accomplished through
gel filtration. However, here again, a
possible aggregation could lead to flow
restriction within the column. Dialfiltration
through a semipermeable membrane
allows the removal of denaturant and
other small molecules and retains the protein.
This procedure has been used for
large-scale processing and was particularly
efficient in the refolding of prorennin and
interferon-β.
Aggregation, Protein 45
During the refolding process, the formation
of incorrectly folded species and
aggregates usually decreases the refolding
yield. For disulfide-bridged proteins, the
renaturation buffer must contain redoxshuffling
mixture to allow the formation
of correctly paired disulfide bridges. Stabilizing
reagents may be added to improve
the refolding yield. An efficient strategy
is the addition of small molecules to suppress
intermolecular interactions leading
to aggregation. Sugar, alcohols, polyols
(including sucrose, glycerol, polyethylene
glycol, isopropanol), cyclodextrin, laurylmaltoside,
sulfobetains, L-arginine, and
low concentrations of denaturants and detergents,
have been used to increase the
refolding yield. L-arginine at a concentration
ranging from 0.4 M to 0.8 M is the
most widely used additive today.
Another important factor in the refolding
process is the rate of removal of the
denaturant. Since there is kinetic competition
between the correct folding and the
formation of aggregates from a folding intermediate,
conditions that favor folding
over the accumulation of aggregates must
be found. To optimize this selection, Vilick
and de Bernadez–Clark developed a
strategy for achieving high protein refolding
yields. They start from a model of
refolding, develop the equations of refolding
kinetics, characterize the rate-limiting
step of the process, determine the influence
of various environmental parameters,
and finally optimize the system of equations
in a scheme involving diafiltration to
remove the denaturant. The approach was
evaluated in the refolding of carbonic anhydrase
from 8 Murea. The yield obtained
after three diafiltration experiments was
69% whereas the model predicted a yield
of 73%.
The properties of molecular chaperones
have also been utilized to increase the
refolding yield. Altamiro and coworkers
have developed a systemfor refolding chromatography
that utilizes GroEL, DsbA, and
peptidyl–prolyl isomerase immobilized on
an agarose gel. Kohler and coworkers
have built a chaperone-assisted bioreactor;
however, it could only be used for
three cycles of refolding and needs to be
improved. Another strategy consists of the
co-overproduction of the DnaK–DnaJ or
GroEL–GroES chaperones with the desired
protein; this can greatly increase
the soluble yield of aggregation-prone proteins.
Fusion proteins have also been used
to minimize aggregation.
The recovery of active proteins from inclusion
bodies is a rather complex process.
Although some general strategies have
been developed, optimal conditions have to
be determined for each protein. Recently,
genetic strategies to improve recovery processes
for recombinant proteins have been
introduced. They consist of the introduction
of combinatorial protein engineering
to generate molecules highly specific to
a particular ligand. Such methods, which
allow efficient recovery of a recombinant
protein, will be increasingly used in industrial
scale bioprocesses as well
сряда, 17 юни 2009 г.
Molecular medicine-Characteristics of Inclusion Bodies
Inclusion bodies can form in the cytoplasm
and in the periplasmic space of E. coli.
Wild-type β-lactamase expressed in E. coli
results in the formation of inclusion bodies
in the periplasm, whereas the protein
expressed without its signal sequence
aggregates in the cytoplasm.
The characteristics of the aggregates depend
on how the protein is expressed.
Different sizes and morphologies have
been observed.Generally, inclusion bodies
appear as dense isomorphous aggregates
of nonnative proteins separated from the
rest of the cytoplasm, but not surrounded
42 Aggregation, Protein
(a)
(b)
Fig. 9 Electron micrographs of (a) cytoplasmic β-lactamase inclusion
bodies in E. coli RB791(pGB1) and (b) purified inclusion bodies from the
same origin (courtesy of G.A. Bowden, A.M. Paredes & G. Georgiou).
Aggregation, Protein 43
by a membrane (Fig. 9). They look like
refractile inclusions, which can be easily
recognized by phase contrast microscopy
when large enough. For prochymosin expressed
in E. coli, the lack of birefringence
indicates that inclusion bodies are not crystalline.
The size distribution of inclusion
bodies has been studied for prochymosin
and interferon-γ , and Marston reported
the mean size of particles to be 0.81 and
1.28 μmrespectively, with a relatively high
void fraction. The void volume was about
70% of the total volume for interferon-
γ and 85% for prochymosin. Structural
characterization studies using ATR-FTIR
(attenuated total reflectance Fourier transformed
infrared spectroscopy) have shown
that the insoluble nature of inclusion bodies
may be due to their increased levels of
nonnative intramolecular β-sheet content.
Inclusion bodies consist mostly of
the overexpressed recombinant protein,
and can contain little contaminating
molecules. Thus, they can be used as a
source of relatively pure misfolded protein
when refolding yields the active protein.
However, some amorphous bodies incorporate
othermolecules, for example, inclusion
bodies from E. coli cells overexpressing
β-lactamase contain only between 35
and 95% intact β-lactamase. The rest consists
of a variety of intracellular proteins,
some lipids, and a small amount of nucleic
acids. Homogeneous inclusion bodies
were obtained by expressing β-lactamase
without its leader peptide. Under these
conditions, aggregation occurs within the
cytoplasm. The extent of incorporation of
other macromolecules in inclusion bodies
depends upon the overexpressed protein.
The formation of inclusion bodies generally
appears to be a disadvantage, since
it requires the dissolving of the aggregates
in denaturant and subsequent refolding of
the protein. However, when the recovery
of the active product can be obtained with
a sufficient yield, certain advantages may
accrue. Indeed, aggregation generally prevents
proteolytic attack, except when the
protein coaggregates with a protease. The
formation of inclusion bodies is also an advantage
for the production of proteins that
are toxic for the host cells. Furthermore,
these aggregates contain a great quantity
of the overexpressed protein.
and in the periplasmic space of E. coli.
Wild-type β-lactamase expressed in E. coli
results in the formation of inclusion bodies
in the periplasm, whereas the protein
expressed without its signal sequence
aggregates in the cytoplasm.
The characteristics of the aggregates depend
on how the protein is expressed.
Different sizes and morphologies have
been observed.Generally, inclusion bodies
appear as dense isomorphous aggregates
of nonnative proteins separated from the
rest of the cytoplasm, but not surrounded
42 Aggregation, Protein
(a)
(b)
Fig. 9 Electron micrographs of (a) cytoplasmic β-lactamase inclusion
bodies in E. coli RB791(pGB1) and (b) purified inclusion bodies from the
same origin (courtesy of G.A. Bowden, A.M. Paredes & G. Georgiou).
Aggregation, Protein 43
by a membrane (Fig. 9). They look like
refractile inclusions, which can be easily
recognized by phase contrast microscopy
when large enough. For prochymosin expressed
in E. coli, the lack of birefringence
indicates that inclusion bodies are not crystalline.
The size distribution of inclusion
bodies has been studied for prochymosin
and interferon-γ , and Marston reported
the mean size of particles to be 0.81 and
1.28 μmrespectively, with a relatively high
void fraction. The void volume was about
70% of the total volume for interferon-
γ and 85% for prochymosin. Structural
characterization studies using ATR-FTIR
(attenuated total reflectance Fourier transformed
infrared spectroscopy) have shown
that the insoluble nature of inclusion bodies
may be due to their increased levels of
nonnative intramolecular β-sheet content.
Inclusion bodies consist mostly of
the overexpressed recombinant protein,
and can contain little contaminating
molecules. Thus, they can be used as a
source of relatively pure misfolded protein
when refolding yields the active protein.
However, some amorphous bodies incorporate
othermolecules, for example, inclusion
bodies from E. coli cells overexpressing
β-lactamase contain only between 35
and 95% intact β-lactamase. The rest consists
of a variety of intracellular proteins,
some lipids, and a small amount of nucleic
acids. Homogeneous inclusion bodies
were obtained by expressing β-lactamase
without its leader peptide. Under these
conditions, aggregation occurs within the
cytoplasm. The extent of incorporation of
other macromolecules in inclusion bodies
depends upon the overexpressed protein.
The formation of inclusion bodies generally
appears to be a disadvantage, since
it requires the dissolving of the aggregates
in denaturant and subsequent refolding of
the protein. However, when the recovery
of the active product can be obtained with
a sufficient yield, certain advantages may
accrue. Indeed, aggregation generally prevents
proteolytic attack, except when the
protein coaggregates with a protease. The
formation of inclusion bodies is also an advantage
for the production of proteins that
are toxic for the host cells. Furthermore,
these aggregates contain a great quantity
of the overexpressed protein.
Molecular medicine-Occurrence of Inclusion Bodies
Inclusion bodies were first identified in
the blood cells of patients with abnormal
hemoglobins, the resulting pathology being
anemia. Pathological point mutants of
hemoglobin aggregate into inclusion bodies;
this is the case for hemoglobin K¨oln
(Val98Met on the β chain) and hemoglobin
Sabine (Leu91Pro on the β chain). Similar
deposits have been described in studies on
the metabolism of abnormal proteins subjected
to covalent modification in E. coli.
The formation of aggregates also occurs
when cells are subjected to heat shock.
The in vivo folding pathway of tailspike
endorhamnosidase of Salmonella phage
22 is a well-documented system studied
by J.King’s group. Furthermore, it is one
of the few systems in which the in vivo
folding pathway has been compared with
the in vitro refolding pathway. The protein
is a trimer of 666 amino acids. The secondary
structure is predominantly β-sheet.
Newly synthesized polypeptide chains released
from the ribosome generate an
early partially folded intermediate. This intermediate
further evolves into a species
sufficiently structured for chain–chain
recognition. In the following step, an incompletely
folded trimer is formed upon
close association with the latter species.
The protrimer is then transformed into
the native tailspike. A clear difference between
the physicochemical properties of
the intermediates and the native state has
Aggregation, Protein 41
allowed their identification. Figure 3 illustrates
the folding pathway of the protein.
The native protein is highly thermostable
with a Tm of 88 ◦C; it is also resistant
to detergents and proteases. During the
in vivo folding process, the intermediates
are sensitive to these factors, allowing
their identification. At low temperature,
almost 100% of the newly synthesized
chains reach the native trimer conformation.
When the temperature increases in
the cells, the number of polypeptide chains
achieving the native state decreases. At
39 ◦C, the maturation proceeds with 30%
efficiency, while the remainder aggregates
into inclusion bodies. It has been shown
that the aggregation does not result from
an intracellular denaturation of the native
protein, but is generated from an early
thermolabile intermediate. The aggregated
chains cannot recover their proper folding
by lowering the temperature. But when
polypeptide chains that have been synthesized
at high temperatures are shifted to
low temperature early enough, they can
refold correctly.
A set of mutations that alter protein
folding without modifying the properties
and stability of native P22 tailspike
has been identified; they are referred
to as temperature-sensitive folding (tsf)
mutants. These mutations have been supposed
to destabilize the already thermolabile
intermediate and are located at more
than 30 sites in the central region of
the polypeptide chain. Starting from mutants
kinetically blocked in their folding, a
second set of mutants capable of correcting
the folding defects was selected, and
the sequences surrounding the suppressor
mutations were identified. Only two substitution
positions on the 666 amino acids
of the polypeptide chain were sufficient to
prevent inclusion body formation. Thus,
single temperature mutations that affect
the folding pathway but not the native
conformation of a protein are efficient in
preventing off-pathway and subsequent aggregation.
A similar result has been found
for heterodimeric luciferase. For recombinant
proteins such as interferon-γ and
interleukin 1β, as well as for P22 tailspike,
amino acid substitutions that can
decrease or increase the formation of inclusion
bodies without alteration of the
functional structure were found by Wetzel
and coworkers.
The formation of inclusion bodies is
frequently observed in the production of
recombinant proteins. High levels of expression
of these proteins result in the
formation of inactive amorphous aggregates,
and has been reported for proteins
expressed in E. coli and also in several
host cells, gram-negative as well as grampositive
bacteria, and eukaryotic cells such
as Saccharomyces cerevisiae, insect cells,
and even animal cells. The production
of recombinant proteins, among them
human insulin, interferon-γ, interleukin
1β, β-lactamase, prochymosin, tissue plasminogen
activator, basic fibroblast growth
hormone, and somatotropin, gives rise to
inclusion bodies.
the blood cells of patients with abnormal
hemoglobins, the resulting pathology being
anemia. Pathological point mutants of
hemoglobin aggregate into inclusion bodies;
this is the case for hemoglobin K¨oln
(Val98Met on the β chain) and hemoglobin
Sabine (Leu91Pro on the β chain). Similar
deposits have been described in studies on
the metabolism of abnormal proteins subjected
to covalent modification in E. coli.
The formation of aggregates also occurs
when cells are subjected to heat shock.
The in vivo folding pathway of tailspike
endorhamnosidase of Salmonella phage
22 is a well-documented system studied
by J.King’s group. Furthermore, it is one
of the few systems in which the in vivo
folding pathway has been compared with
the in vitro refolding pathway. The protein
is a trimer of 666 amino acids. The secondary
structure is predominantly β-sheet.
Newly synthesized polypeptide chains released
from the ribosome generate an
early partially folded intermediate. This intermediate
further evolves into a species
sufficiently structured for chain–chain
recognition. In the following step, an incompletely
folded trimer is formed upon
close association with the latter species.
The protrimer is then transformed into
the native tailspike. A clear difference between
the physicochemical properties of
the intermediates and the native state has
Aggregation, Protein 41
allowed their identification. Figure 3 illustrates
the folding pathway of the protein.
The native protein is highly thermostable
with a Tm of 88 ◦C; it is also resistant
to detergents and proteases. During the
in vivo folding process, the intermediates
are sensitive to these factors, allowing
their identification. At low temperature,
almost 100% of the newly synthesized
chains reach the native trimer conformation.
When the temperature increases in
the cells, the number of polypeptide chains
achieving the native state decreases. At
39 ◦C, the maturation proceeds with 30%
efficiency, while the remainder aggregates
into inclusion bodies. It has been shown
that the aggregation does not result from
an intracellular denaturation of the native
protein, but is generated from an early
thermolabile intermediate. The aggregated
chains cannot recover their proper folding
by lowering the temperature. But when
polypeptide chains that have been synthesized
at high temperatures are shifted to
low temperature early enough, they can
refold correctly.
A set of mutations that alter protein
folding without modifying the properties
and stability of native P22 tailspike
has been identified; they are referred
to as temperature-sensitive folding (tsf)
mutants. These mutations have been supposed
to destabilize the already thermolabile
intermediate and are located at more
than 30 sites in the central region of
the polypeptide chain. Starting from mutants
kinetically blocked in their folding, a
second set of mutants capable of correcting
the folding defects was selected, and
the sequences surrounding the suppressor
mutations were identified. Only two substitution
positions on the 666 amino acids
of the polypeptide chain were sufficient to
prevent inclusion body formation. Thus,
single temperature mutations that affect
the folding pathway but not the native
conformation of a protein are efficient in
preventing off-pathway and subsequent aggregation.
A similar result has been found
for heterodimeric luciferase. For recombinant
proteins such as interferon-γ and
interleukin 1β, as well as for P22 tailspike,
amino acid substitutions that can
decrease or increase the formation of inclusion
bodies without alteration of the
functional structure were found by Wetzel
and coworkers.
The formation of inclusion bodies is
frequently observed in the production of
recombinant proteins. High levels of expression
of these proteins result in the
formation of inactive amorphous aggregates,
and has been reported for proteins
expressed in E. coli and also in several
host cells, gram-negative as well as grampositive
bacteria, and eukaryotic cells such
as Saccharomyces cerevisiae, insect cells,
and even animal cells. The production
of recombinant proteins, among them
human insulin, interferon-γ, interleukin
1β, β-lactamase, prochymosin, tissue plasminogen
activator, basic fibroblast growth
hormone, and somatotropin, gives rise to
inclusion bodies.
Molecular medicine-Protein Aggregation in the Cellular Environment
The Formation of Inclusion Bodies
The overexpression of genes introduced in
foreign hosts frequently results in aggregated
nonnative proteins called inclusion
bodies. In cells, inclusion bodies appear as
unordered amorphous aggregates clearly
separated from the rest of the cytoplasm;
they form a highly refractive area when
observed microscopically. A great variety
of experimental studies indicates that the
formation of inclusion bodies results from
partially folded intermediates in the intracellular
folding pathway and not from
either totally unfolded or native proteins.
The overexpression of genes introduced in
foreign hosts frequently results in aggregated
nonnative proteins called inclusion
bodies. In cells, inclusion bodies appear as
unordered amorphous aggregates clearly
separated from the rest of the cytoplasm;
they form a highly refractive area when
observed microscopically. A great variety
of experimental studies indicates that the
formation of inclusion bodies results from
partially folded intermediates in the intracellular
folding pathway and not from
either totally unfolded or native proteins.
Molecular medicine-The Role of Molecular Chaperones
The discovery of a ubiquitous class of
proteins mediating the correct folding in
cellular environment has led to a reconsideration
of the mechanism of protein
folding in vivo. Historically, the term
molecular chaperone was introduced by
Laskyard and coworkers in 1987 to describe
the function of nucleoplasmin,
which mediates the in vitro assembly
of nucleosomes from separated histones
and DNA. The concept was further extended
by Ellis to define a class of
proteins whose function is to ensure
the correct folding and assembly of
proteins through a transient association
with the nascent polypeptide chain. Studies
on heat-shock proteins have widely
contributed to the development of this
concept.
Today, more than 20 protein families
have been identified as molecular chaperones.
Molecular chaperones comprise
Aggregation, Protein 35
several highly conserved families of related
proteins. They can be divided into two
classes according to their size. Small chaperones
are less than 200 kDa, whereas
large chaperones are more than 800 kDa.
During the past few years, a large amount
of biochemical, biophysical, and low- and
high-resolution structural data have provided
mechanistic insights into the machinery
of protein folding as assisted by
molecular chaperones.
Molecular chaperones are involved in
diverse cellular functions. The constitutive
members of the heat-shock protein family
(Hsp70) can stabilize nascent polypeptide
chains during their elongation in
ribosomes. The large cylindrical chaperonins
GroEL in bacteria, mitochondria,
and chloroplasts and the corresponding
TriC in eukaryotes and archaebacteria
provide a sequestered environment for
productive folding. Several chaperones are
stress-dependent; their expression is induced
under conditions such as high
temperatures, which provoke protein unfolding
and aggregation. The members of
the Hsp90 and Hsp100 families, as well
as small Hsp, play a role in preventing
protein aggregation under stress. Chaperone
interactions are also important for
the translocation of polypeptide chains
into membranes.
Within cells, the nascent polypeptide
chain is synthesized sequentially on the
ribosome by a vectorial process. For
many proteins, the rate of this process
is slower than the rate of folding. Synthesis
times range from 20 s for a 400
residue–polypeptide chain in E. coli at
37 ◦C to 10 times as long for such a
chain in an eukaryotic cell.Many unfolded
proteins refold completely in 20 s under
the same conditions. Thus, there is the
possibility for the elongating polypeptide
either to misfold before completion or to
be degraded by proteolytic enzymes. Chaperones
prevent such unfavorable events by
protecting the nascent chain. Hsp70 and
its prokaryotic homolog DnaK recognize
extended hydrophobic regions of the elongating
polypeptides. These interactions are
not specific.Hsp70 andDnaK interactwith
most unfolded polypeptide chains that expose
hydrophobic residues. They do not
recognize folded proteins. Binding and release
of unfolded proteins from Hsp70
are ATP-dependent and require the presence
of various cochaperones such asDnaJ
and GrpE. The basic mechanism ofHsp70
(DnaK in E. coli) is represented in Fig. 6.
In E. coli, DnaJ binds the nascent unfolded
polypeptide, U; then the complex
binds to the ATP-bound state of DnaK.
ATP is hydrolyzed in the ternary complex
DnaJ
ATP
DnaK
ATP ADP
ATP
ATP
Or towards GroEL
ADP
GrpE
Pi; DnaJ
U
U N
Fig. 6 Schematic representation of the basic mechanism of DnaK (see text).
36 Aggregation, Protein
allowing the release of DnaJ and Pi. In
the following step, GrpE acts as an exchange
factor to regenerate the ATP-bound
state of DnaK. The unfolded polypeptide
chain is released into the bulk solution.
Thus, Hsp70 systems bind and release the
polypeptide in an unfolded conformation.
The unfolded protein has the possibility
either to fold or to be transferred to
the GroEL system, as illustrated in Fig. 6.
Significant insights into this mechanism
were obtained from structural data. The
three-dimensional structures of Hsp70
and DnaJ as well as those of a complex
(a)
(b)
Fig. 7 Crystal structure of
GroEL–GroES–(ADP)7 complex
determined by Sigler et al. (a) view
along the axis and (b) view from the top
of the complex. (Reproduced from the
PDB web site.)
DnaK–polypeptide and a complex of GrpE
with the ATP binding domain of DnaK
are known. DnaK and its homologs are
composed of two domains, a C-terminal
domain that binds ATP and an N-terminal
domain that binds peptides. GrpE is a
tight homodimer associated along twolong
helices. It binds DnaK–ATPase domain
through its proximal monomer. DnaJ activates
the ATP hydrolysis by DnaK. It
was shown that a conformational change
may occur upon ATP binding, opening the
polypeptide binding cleft in the polypeptide
binding domain of DnaK. The closed
state may correspond to the ADP-bound
conformation. The ADP-bound state of
DnaK binds the peptide tightly. Peptide
release requires the dissociation of ADP,
which is mediated by GrpE. DnaK then
rebinds ATP.
The GroEL–GroES system acts by a different
mechanism in which the unfolded
protein is sequestered. The chaperonins
are large cylindrical protein complexes.
The crystal structure of E. coli chaperonin
GroEL was determined in 1994 and that
of the asymmetric GroEL–GroES–(ADP)7
complex in 1997 by Sigler and his group.
GroEL consists of two heptameric rings
of 58-kDa subunits stacked back to back
with a dyad symmetry and forming a
porous cylinder (Fig. 7). Each subunit is
organized in three structural domains. A
large equatorial domain forms the foundation
of the assembly and holds the rings
together. It contains the nucleotide binding
site. A large apical domain forms
the end of the cylinder. The apical domain
contains a number of hydrophobic
Aggregation, Protein 37
residues exposed to the solvent. A small
intermediate domain connects the two
large domains. The intermediate segments
have some flexibility allowing a hingelike
opening of the apical domains, which
occurs upon nucleotide binding. These
movements are large and have been visualized
by three-dimensional reconstruction
from cryoelectron microscopy by Sebil and
her group.
GroES is a heptamer of 10 kDa subunits
forming a flexible dome-shaped structure
with an internal cavity large enough
to accommodate proteins up to 70 kDa.
Each subunit is folded into a single
domain containing β-sheets and flexible
loop regions. The loop regions are critical
for the interactions between GroEL and
GroES. It was deduced from electron
microscopy studies that GroES binding
to GroEL induces large movements in
the apical GroEL domains. This provokes
a significant increase in the volume
of the central cavity in which protein
folding proceeds. NMR coupled with the
study of hydrogen-exchange techniques
has indicated that small proteins are
essentially unfolded in their GroEL-bound
states. Mass spectroscopy has revealed
the presence of fluctuating elements of
secondary structure for several proteins.
In a way, the GroEL–GroES system
recognizes nonnative proteins.
The reaction cycle of the GroEL–GroES
system is represented in Fig. 8. The
nonnative protein binds to the apical
domain of the upper ring of GroEL
through hydrophobic interactions. Then,
the equatorial domain of the same ring
binds ATP, and GroES caps the upper
ring, sequestering the protein inside the
internal chamber in which the protein
folding proceeds. The binding of GroES
induces a conformational change in GroEL
and ATP hydrolysis, which is a cooperative
process that produces a conformational
change in the lower ring, allowing it
to bind a nonnative protein molecule.
This promotes subsequent binding of
ATP and GroES in the lower ring, and
the dissociation of the upper complex,
releasing the protein and ejecting GroES.
If the protein has not reached the native
state, it is subjected to a new cycle.
ADP ADP
ATP
ATP ATP ADP ADP
ATP ATP
ADP
ATP
t ∼ 6 s
Inf N
ADP
A
E
I
GroEL
GroES
(I) (II) (III) (IV)
Fig. 8 The reaction cycle of GroEL–GroES. Inf
is the unfolded protein, N the folded one, A is
the apical domain, (in blue), I the intermediate
domain (in red) and E the equatorial domain (in
magenta). (Reproduced from Wang & Weissman
(1999) Nat. Struct. Biol. 6, 597, with permission.)
(See color plate p. xxii).
38 Aggregation, Protein
The hydrolysis of ATP by GroEL is used
only to induce conformational changes
of the chaperone, which permits the release
of the folded protein. The molecular
chaperones, by their transient association
through hydrophobic interactions with
nascent, stress-destabilized, or translocated
proteins, have a role in preventing
improper folding and subsequent aggregation.
They do not interact with folded
proteins. They do not carry information
capable of directing a protein to assume a
structure different from that dictated by its
amino acid sequence. Therefore, molecular
chaperones assist the folding in the
cells without violation of the Anfinsen postulate.
They increase the yield but not the
rate of folding reactions; in this respect
they do not act as catalysts. Furthermore,
themajority of newly synthesized polypeptide
chains in both bacterial and eukaryotic
cells fold spontaneously without the assistance
of molecular chaperones.
Many proteins from prokaryotic and
eukaryotic organisms are produced with
an amino-terminal propeptide, which is
removed by limited proteolysis during
the activation process. Several of these
propeptides consist of a long polypeptide
chain; for example, there are 174 amino
acids in the propeptide of pro-α-lytic protease,
91 in that of procarboxypeptidase
Y, and 77 in that of prosubtilisin. Several
studies have shown that the propeptide
is required for proper folding of these
proteins. The mature enzymes are not
able to refold correctly. They seem to
have kinetic stability only, whereas the
proenzymes have thermodynamic stability.
Since propeptides perform the function
of mediating protein folding, they have
been classified as intramolecular chaperones.
However, this terminology is not
appropriate since the nascent protein is
the proenzyme, not the enzyme that has
undergone proteolytic cleavage. Thus, it
is not surprising that the proenzyme refolds
spontaneously, whereas the mature
protein does not. Indeed, the information
is contained in the totality of the proenzyme
sequence.
Two other classes of proteins play
the role of helpers during protein folding
in vivo: protein disulfide isomerases
(PDIs) and peptidyl–prolyl cis – trans isomerases.
Protein disulfide isomerase is an
abundant component of the lumen of the
endoplasmic reticulum in secretory cells.
The enzymewas discovered independently
in 1963 by two research groups: in rat
and ox by Anfinsen and coworkers, and
in chicken and pigeon pancreas by Straub
and coworkers. Proteins destined to be secreted
enter the endoplasmic reticulum in
an unfolded state. In this environment,
the folding process is associated with the
formation of disulfide bonds, which is
catalyzed by PDI through thiol–disulfide
interchange. The first PDI cDNA was sequenced
in 1985 by Edman et al. It displays
sequence homologies implying a multidomain
architecture. PDI consists of four
structural domains arranged in the order
a, b, b, a, with the b and a domains
being connected by a linker region. Furthermore,
it possesses an acidic C-terminal
extension. The a and a domains contain
the active site motif – W-C-G-H-C-. They
display significant sequence identity to
thioredoxin, a small cytoplasmic protein
involved in several redox functions, and
they have a similar active site sequence.
Recombinants of the a and b domains
have been obtained and studied by highresolution
NMR. The a domain has the
same overall fold as thioredoxin, an α/β
fold with a central core made up of a
five-stranded β-sheet surrounded by four
helices. As in thioredoxin, the active site
is located at the N-terminus of helix
Aggregation, Protein 39
α2. Preliminary NMR data of the a
domain confirm its structural similarity
to the a domain. The b and b domains
have significant sequence similarity to
each other, but no similarity with the a
domain. Nevertheless, NMR studies of
the b domain have indicated a similar
overall fold. From its sequence, it could
be inferred that b also has the same fold.
Neither b nor b contain the active site.
The folding pathway of disulfide-bound
proteins involves isomerizations between
a number of species containing disulfide
bonds. In vitro experimental studies were
performed using the isolated a and a domains,
and the resultswere comparedwith
those obtained with the holoenzyme. It
was concluded that the activity of long
length PDI is not simply the sum of
the activities of the isolated a and a
domains. Using a series of constructs
including nearly every linear combination
of domains, the contribution of each
domain was investigated. It was determined
that the thiol-disulfide chemistry
requires only the a and a domains, and
that simple isomerization requires one of
these in a linear combination including b,
whereas complex isomerization involving
large conformational changes requires all
the PDI domains except the C-terminal
extension. Thus, it appears that the b domain
is the principal peptide binding site,
but all domains contribute to the binding
of larger polypeptide chains holding
them in a partially unfolded conformation
while the catalytic sites acts synergistically
to perform the thiol-disulfide exchange.
Since PDI has binding properties, it has
been proposed that it acts as a molecular
chaperone. However, as underlined by
Freedman and coworkers, this property
does not represent a chaperone activity
and instead reflects its role as a catalyst to
accelerate the formation of native disulfide
bridges during protein folding.
Several gene products with similarity
to PDI have been identified in higher
eukaryotes. All are probably localized in
the endoplasmic reticulum and have thioldisulfide
exchange activity.
In prokaryotes, the disulfide formation
occurs in the periplasm and is catalyzed by
a protein called DsbA, which exchanges
its Cys30–Cys33 to a pair of thiols in
the target protein, leaving DsbA in its
reduced state. The crystal structure of
oxidized DsbA displays a domain with a
thioredoxin-like fold and another domain,
which caps the thioredoxin-like active site
C30-P31-H32-C33, located at the domain
interface. Reoxidation of DsbA is catalyzed
by a cytoplasmic membrane protein
called DsbB, which contains four cysteine
residues essential for catalysis. DsbB
transfers the electrons from the reduced
DsbA to membrane embedded quinones.
The reduced quinones are then oxidized
enzymatically either aerobically or anaerobically.
Thus, DsbA is found in normal
cells in its oxidized state. E. coli also
has a complex reductive system including
another periplasmic protein DsbC,
which is a homodimer. The molecule
consists of two thioredoxin-like domains
with a CxxC motif, joined via hinged
linker helices to an N-terminal dimerization
domain. The hinge regions allow
movement of the active site, and a broad
hydrophobic cleft between the two domains
may bind the polypeptide chain.
Its function consists of reducing proteins
with incorrect disulfide bonds. DsbC is
maintained in its reduced form by a membrane
protein called DsbD, which contains
six essential cysteine residues. Then, the
electrons are transferred to thioredoxin
and ultimately to NADPH by thioredoxin
reductase.
40 Aggregation, Protein
All these enzymes, which catalyze the
pairing of cysteine residues in disulfidebridged
proteins, have functional domains
pertaining to the thioredoxin superstructure.
Another type of enzyme, peptidyl–prolyl
cis–trans isomerases, facilitates the folding
of some proteins by catalyzing the
cis–trans isomerization of X-Pro peptide
bonds. Two classes of unrelated proteins
demonstrate this activity, those that
bind cyclosporin, which are known as cyclophilins,
and those that bind FK506. The
cellular function of these enzymes is important,
since cyclosporin and FK506 are
potent immunosuppressors that regulate
T-cells activation. Both classes of peptidyl–
prolyl isomerases are ubiquitous,
and abundant in prokaryotes and eukaryotes.
The sequences of several members
of each family are known, and the threedimensional
structures of at least one
member of each family have been elucidated
by X-ray crystallography andmultidimensional
NMR. Their role is to accelerate
the cis-trans isomerization of X-pro peptide
bonds when this process is the ratelimiting
step in protein folding. Although
they do not present structural similarity,
both exhibit a hydrophobic binding cleft
favoring the rotamase activity by excluding
water molecules.
proteins mediating the correct folding in
cellular environment has led to a reconsideration
of the mechanism of protein
folding in vivo. Historically, the term
molecular chaperone was introduced by
Laskyard and coworkers in 1987 to describe
the function of nucleoplasmin,
which mediates the in vitro assembly
of nucleosomes from separated histones
and DNA. The concept was further extended
by Ellis to define a class of
proteins whose function is to ensure
the correct folding and assembly of
proteins through a transient association
with the nascent polypeptide chain. Studies
on heat-shock proteins have widely
contributed to the development of this
concept.
Today, more than 20 protein families
have been identified as molecular chaperones.
Molecular chaperones comprise
Aggregation, Protein 35
several highly conserved families of related
proteins. They can be divided into two
classes according to their size. Small chaperones
are less than 200 kDa, whereas
large chaperones are more than 800 kDa.
During the past few years, a large amount
of biochemical, biophysical, and low- and
high-resolution structural data have provided
mechanistic insights into the machinery
of protein folding as assisted by
molecular chaperones.
Molecular chaperones are involved in
diverse cellular functions. The constitutive
members of the heat-shock protein family
(Hsp70) can stabilize nascent polypeptide
chains during their elongation in
ribosomes. The large cylindrical chaperonins
GroEL in bacteria, mitochondria,
and chloroplasts and the corresponding
TriC in eukaryotes and archaebacteria
provide a sequestered environment for
productive folding. Several chaperones are
stress-dependent; their expression is induced
under conditions such as high
temperatures, which provoke protein unfolding
and aggregation. The members of
the Hsp90 and Hsp100 families, as well
as small Hsp, play a role in preventing
protein aggregation under stress. Chaperone
interactions are also important for
the translocation of polypeptide chains
into membranes.
Within cells, the nascent polypeptide
chain is synthesized sequentially on the
ribosome by a vectorial process. For
many proteins, the rate of this process
is slower than the rate of folding. Synthesis
times range from 20 s for a 400
residue–polypeptide chain in E. coli at
37 ◦C to 10 times as long for such a
chain in an eukaryotic cell.Many unfolded
proteins refold completely in 20 s under
the same conditions. Thus, there is the
possibility for the elongating polypeptide
either to misfold before completion or to
be degraded by proteolytic enzymes. Chaperones
prevent such unfavorable events by
protecting the nascent chain. Hsp70 and
its prokaryotic homolog DnaK recognize
extended hydrophobic regions of the elongating
polypeptides. These interactions are
not specific.Hsp70 andDnaK interactwith
most unfolded polypeptide chains that expose
hydrophobic residues. They do not
recognize folded proteins. Binding and release
of unfolded proteins from Hsp70
are ATP-dependent and require the presence
of various cochaperones such asDnaJ
and GrpE. The basic mechanism ofHsp70
(DnaK in E. coli) is represented in Fig. 6.
In E. coli, DnaJ binds the nascent unfolded
polypeptide, U; then the complex
binds to the ATP-bound state of DnaK.
ATP is hydrolyzed in the ternary complex
DnaJ
ATP
DnaK
ATP ADP
ATP
ATP
Or towards GroEL
ADP
GrpE
Pi; DnaJ
U
U N
Fig. 6 Schematic representation of the basic mechanism of DnaK (see text).
36 Aggregation, Protein
allowing the release of DnaJ and Pi. In
the following step, GrpE acts as an exchange
factor to regenerate the ATP-bound
state of DnaK. The unfolded polypeptide
chain is released into the bulk solution.
Thus, Hsp70 systems bind and release the
polypeptide in an unfolded conformation.
The unfolded protein has the possibility
either to fold or to be transferred to
the GroEL system, as illustrated in Fig. 6.
Significant insights into this mechanism
were obtained from structural data. The
three-dimensional structures of Hsp70
and DnaJ as well as those of a complex
(a)
(b)
Fig. 7 Crystal structure of
GroEL–GroES–(ADP)7 complex
determined by Sigler et al. (a) view
along the axis and (b) view from the top
of the complex. (Reproduced from the
PDB web site.)
DnaK–polypeptide and a complex of GrpE
with the ATP binding domain of DnaK
are known. DnaK and its homologs are
composed of two domains, a C-terminal
domain that binds ATP and an N-terminal
domain that binds peptides. GrpE is a
tight homodimer associated along twolong
helices. It binds DnaK–ATPase domain
through its proximal monomer. DnaJ activates
the ATP hydrolysis by DnaK. It
was shown that a conformational change
may occur upon ATP binding, opening the
polypeptide binding cleft in the polypeptide
binding domain of DnaK. The closed
state may correspond to the ADP-bound
conformation. The ADP-bound state of
DnaK binds the peptide tightly. Peptide
release requires the dissociation of ADP,
which is mediated by GrpE. DnaK then
rebinds ATP.
The GroEL–GroES system acts by a different
mechanism in which the unfolded
protein is sequestered. The chaperonins
are large cylindrical protein complexes.
The crystal structure of E. coli chaperonin
GroEL was determined in 1994 and that
of the asymmetric GroEL–GroES–(ADP)7
complex in 1997 by Sigler and his group.
GroEL consists of two heptameric rings
of 58-kDa subunits stacked back to back
with a dyad symmetry and forming a
porous cylinder (Fig. 7). Each subunit is
organized in three structural domains. A
large equatorial domain forms the foundation
of the assembly and holds the rings
together. It contains the nucleotide binding
site. A large apical domain forms
the end of the cylinder. The apical domain
contains a number of hydrophobic
Aggregation, Protein 37
residues exposed to the solvent. A small
intermediate domain connects the two
large domains. The intermediate segments
have some flexibility allowing a hingelike
opening of the apical domains, which
occurs upon nucleotide binding. These
movements are large and have been visualized
by three-dimensional reconstruction
from cryoelectron microscopy by Sebil and
her group.
GroES is a heptamer of 10 kDa subunits
forming a flexible dome-shaped structure
with an internal cavity large enough
to accommodate proteins up to 70 kDa.
Each subunit is folded into a single
domain containing β-sheets and flexible
loop regions. The loop regions are critical
for the interactions between GroEL and
GroES. It was deduced from electron
microscopy studies that GroES binding
to GroEL induces large movements in
the apical GroEL domains. This provokes
a significant increase in the volume
of the central cavity in which protein
folding proceeds. NMR coupled with the
study of hydrogen-exchange techniques
has indicated that small proteins are
essentially unfolded in their GroEL-bound
states. Mass spectroscopy has revealed
the presence of fluctuating elements of
secondary structure for several proteins.
In a way, the GroEL–GroES system
recognizes nonnative proteins.
The reaction cycle of the GroEL–GroES
system is represented in Fig. 8. The
nonnative protein binds to the apical
domain of the upper ring of GroEL
through hydrophobic interactions. Then,
the equatorial domain of the same ring
binds ATP, and GroES caps the upper
ring, sequestering the protein inside the
internal chamber in which the protein
folding proceeds. The binding of GroES
induces a conformational change in GroEL
and ATP hydrolysis, which is a cooperative
process that produces a conformational
change in the lower ring, allowing it
to bind a nonnative protein molecule.
This promotes subsequent binding of
ATP and GroES in the lower ring, and
the dissociation of the upper complex,
releasing the protein and ejecting GroES.
If the protein has not reached the native
state, it is subjected to a new cycle.
ADP ADP
ATP
ATP ATP ADP ADP
ATP ATP
ADP
ATP
t ∼ 6 s
Inf N
ADP
A
E
I
GroEL
GroES
(I) (II) (III) (IV)
Fig. 8 The reaction cycle of GroEL–GroES. Inf
is the unfolded protein, N the folded one, A is
the apical domain, (in blue), I the intermediate
domain (in red) and E the equatorial domain (in
magenta). (Reproduced from Wang & Weissman
(1999) Nat. Struct. Biol. 6, 597, with permission.)
(See color plate p. xxii).
38 Aggregation, Protein
The hydrolysis of ATP by GroEL is used
only to induce conformational changes
of the chaperone, which permits the release
of the folded protein. The molecular
chaperones, by their transient association
through hydrophobic interactions with
nascent, stress-destabilized, or translocated
proteins, have a role in preventing
improper folding and subsequent aggregation.
They do not interact with folded
proteins. They do not carry information
capable of directing a protein to assume a
structure different from that dictated by its
amino acid sequence. Therefore, molecular
chaperones assist the folding in the
cells without violation of the Anfinsen postulate.
They increase the yield but not the
rate of folding reactions; in this respect
they do not act as catalysts. Furthermore,
themajority of newly synthesized polypeptide
chains in both bacterial and eukaryotic
cells fold spontaneously without the assistance
of molecular chaperones.
Many proteins from prokaryotic and
eukaryotic organisms are produced with
an amino-terminal propeptide, which is
removed by limited proteolysis during
the activation process. Several of these
propeptides consist of a long polypeptide
chain; for example, there are 174 amino
acids in the propeptide of pro-α-lytic protease,
91 in that of procarboxypeptidase
Y, and 77 in that of prosubtilisin. Several
studies have shown that the propeptide
is required for proper folding of these
proteins. The mature enzymes are not
able to refold correctly. They seem to
have kinetic stability only, whereas the
proenzymes have thermodynamic stability.
Since propeptides perform the function
of mediating protein folding, they have
been classified as intramolecular chaperones.
However, this terminology is not
appropriate since the nascent protein is
the proenzyme, not the enzyme that has
undergone proteolytic cleavage. Thus, it
is not surprising that the proenzyme refolds
spontaneously, whereas the mature
protein does not. Indeed, the information
is contained in the totality of the proenzyme
sequence.
Two other classes of proteins play
the role of helpers during protein folding
in vivo: protein disulfide isomerases
(PDIs) and peptidyl–prolyl cis – trans isomerases.
Protein disulfide isomerase is an
abundant component of the lumen of the
endoplasmic reticulum in secretory cells.
The enzymewas discovered independently
in 1963 by two research groups: in rat
and ox by Anfinsen and coworkers, and
in chicken and pigeon pancreas by Straub
and coworkers. Proteins destined to be secreted
enter the endoplasmic reticulum in
an unfolded state. In this environment,
the folding process is associated with the
formation of disulfide bonds, which is
catalyzed by PDI through thiol–disulfide
interchange. The first PDI cDNA was sequenced
in 1985 by Edman et al. It displays
sequence homologies implying a multidomain
architecture. PDI consists of four
structural domains arranged in the order
a, b, b, a, with the b and a domains
being connected by a linker region. Furthermore,
it possesses an acidic C-terminal
extension. The a and a domains contain
the active site motif – W-C-G-H-C-. They
display significant sequence identity to
thioredoxin, a small cytoplasmic protein
involved in several redox functions, and
they have a similar active site sequence.
Recombinants of the a and b domains
have been obtained and studied by highresolution
NMR. The a domain has the
same overall fold as thioredoxin, an α/β
fold with a central core made up of a
five-stranded β-sheet surrounded by four
helices. As in thioredoxin, the active site
is located at the N-terminus of helix
Aggregation, Protein 39
α2. Preliminary NMR data of the a
domain confirm its structural similarity
to the a domain. The b and b domains
have significant sequence similarity to
each other, but no similarity with the a
domain. Nevertheless, NMR studies of
the b domain have indicated a similar
overall fold. From its sequence, it could
be inferred that b also has the same fold.
Neither b nor b contain the active site.
The folding pathway of disulfide-bound
proteins involves isomerizations between
a number of species containing disulfide
bonds. In vitro experimental studies were
performed using the isolated a and a domains,
and the resultswere comparedwith
those obtained with the holoenzyme. It
was concluded that the activity of long
length PDI is not simply the sum of
the activities of the isolated a and a
domains. Using a series of constructs
including nearly every linear combination
of domains, the contribution of each
domain was investigated. It was determined
that the thiol-disulfide chemistry
requires only the a and a domains, and
that simple isomerization requires one of
these in a linear combination including b,
whereas complex isomerization involving
large conformational changes requires all
the PDI domains except the C-terminal
extension. Thus, it appears that the b domain
is the principal peptide binding site,
but all domains contribute to the binding
of larger polypeptide chains holding
them in a partially unfolded conformation
while the catalytic sites acts synergistically
to perform the thiol-disulfide exchange.
Since PDI has binding properties, it has
been proposed that it acts as a molecular
chaperone. However, as underlined by
Freedman and coworkers, this property
does not represent a chaperone activity
and instead reflects its role as a catalyst to
accelerate the formation of native disulfide
bridges during protein folding.
Several gene products with similarity
to PDI have been identified in higher
eukaryotes. All are probably localized in
the endoplasmic reticulum and have thioldisulfide
exchange activity.
In prokaryotes, the disulfide formation
occurs in the periplasm and is catalyzed by
a protein called DsbA, which exchanges
its Cys30–Cys33 to a pair of thiols in
the target protein, leaving DsbA in its
reduced state. The crystal structure of
oxidized DsbA displays a domain with a
thioredoxin-like fold and another domain,
which caps the thioredoxin-like active site
C30-P31-H32-C33, located at the domain
interface. Reoxidation of DsbA is catalyzed
by a cytoplasmic membrane protein
called DsbB, which contains four cysteine
residues essential for catalysis. DsbB
transfers the electrons from the reduced
DsbA to membrane embedded quinones.
The reduced quinones are then oxidized
enzymatically either aerobically or anaerobically.
Thus, DsbA is found in normal
cells in its oxidized state. E. coli also
has a complex reductive system including
another periplasmic protein DsbC,
which is a homodimer. The molecule
consists of two thioredoxin-like domains
with a CxxC motif, joined via hinged
linker helices to an N-terminal dimerization
domain. The hinge regions allow
movement of the active site, and a broad
hydrophobic cleft between the two domains
may bind the polypeptide chain.
Its function consists of reducing proteins
with incorrect disulfide bonds. DsbC is
maintained in its reduced form by a membrane
protein called DsbD, which contains
six essential cysteine residues. Then, the
electrons are transferred to thioredoxin
and ultimately to NADPH by thioredoxin
reductase.
40 Aggregation, Protein
All these enzymes, which catalyze the
pairing of cysteine residues in disulfidebridged
proteins, have functional domains
pertaining to the thioredoxin superstructure.
Another type of enzyme, peptidyl–prolyl
cis–trans isomerases, facilitates the folding
of some proteins by catalyzing the
cis–trans isomerization of X-Pro peptide
bonds. Two classes of unrelated proteins
demonstrate this activity, those that
bind cyclosporin, which are known as cyclophilins,
and those that bind FK506. The
cellular function of these enzymes is important,
since cyclosporin and FK506 are
potent immunosuppressors that regulate
T-cells activation. Both classes of peptidyl–
prolyl isomerases are ubiquitous,
and abundant in prokaryotes and eukaryotes.
The sequences of several members
of each family are known, and the threedimensional
structures of at least one
member of each family have been elucidated
by X-ray crystallography andmultidimensional
NMR. Their role is to accelerate
the cis-trans isomerization of X-pro peptide
bonds when this process is the ratelimiting
step in protein folding. Although
they do not present structural similarity,
both exhibit a hydrophobic binding cleft
favoring the rotamase activity by excluding
water molecules.
Molecular medicine-Protein Folding in the Cellular Environment
Molecular Crowding in the Cells
The main rules of protein folding have
been deduced from a considerable body of
in vitro and in silico studies. It has been
accepted that the same mechanisms are
involved in in vitro refolding and in the
folding of a nascent polypeptide chain in
the cell. However, the intracellular environment
differs markedly from that of
the test tube where low protein concentrations
are used. The interior of a cell is
highly crowded withmacromolecules. The
concentration is so high that a significant
proportion of the volume is occupied. As
mentioned by Ellis, in general, 20 to 30%
volume of the interior of the cells are occupied
by macromolecules; for example,
the concentration of total protein inside
cells ranges from 200 to 300 g L−1. The
total concentration of proteins and RNA
inside Escherichia coli ranges from 300 to
400 g L−1 depending on the growth phase.
Polysaccharides also contribute to the
crowding. It can be predicted practically
that diffusion coefficients will be reduced
by factors up to 10-fold due to crowding.
Since the average time for a molecule to
move a certain distance varies by D−2,
D being the diffusion coefficient, it will
take 100 times longer to move this distance
in the cell as would be necessary
under low concentration conditions. Another
prediction indicates that equilibrium
constants for macromolecular associations
may be increased by two to three orders
of magnitude.
Molecular crowding inside cells also has
consequences for protein folding, favoring
the association of partly folded polypeptide
chains into aggregates. This could explain
why cells contain molecular chaperones,
even though most denatured proteins
refold spontaneously in the test tube.
The main rules of protein folding have
been deduced from a considerable body of
in vitro and in silico studies. It has been
accepted that the same mechanisms are
involved in in vitro refolding and in the
folding of a nascent polypeptide chain in
the cell. However, the intracellular environment
differs markedly from that of
the test tube where low protein concentrations
are used. The interior of a cell is
highly crowded withmacromolecules. The
concentration is so high that a significant
proportion of the volume is occupied. As
mentioned by Ellis, in general, 20 to 30%
volume of the interior of the cells are occupied
by macromolecules; for example,
the concentration of total protein inside
cells ranges from 200 to 300 g L−1. The
total concentration of proteins and RNA
inside Escherichia coli ranges from 300 to
400 g L−1 depending on the growth phase.
Polysaccharides also contribute to the
crowding. It can be predicted practically
that diffusion coefficients will be reduced
by factors up to 10-fold due to crowding.
Since the average time for a molecule to
move a certain distance varies by D−2,
D being the diffusion coefficient, it will
take 100 times longer to move this distance
in the cell as would be necessary
under low concentration conditions. Another
prediction indicates that equilibrium
constants for macromolecular associations
may be increased by two to three orders
of magnitude.
Molecular crowding inside cells also has
consequences for protein folding, favoring
the association of partly folded polypeptide
chains into aggregates. This could explain
why cells contain molecular chaperones,
even though most denatured proteins
refold spontaneously in the test tube.
вторник, 16 юни 2009 г.
Molecular medicine-Mechanisms of Protein Aggregation
A substantial body of information supports
the idea that protein aggregation
arises from partially folded intermediates
through hydrophobic interactions. The
formation of aggregates has often been
considered as a trivial phenomenon, a
nonspecific association of partially folded
polypeptide chains to form a disordered
precipitate. However, several analyses indicate
that aggregation occurs by specific
intramolecular associations involving the
recognition of a sequence partner in another
molecule rather than in the same
molecule during the folding process. Analyses
of the aggregation mechanisms of
various proteins, such as bovine growth
hormone and phosphoglycerate kinase,
has permitted the identification of specific
sites that are critical in the association.
An elegant demonstration of the specificity
of aggregation was provided by King
Aggregation, Protein 31
and coworkers. The authors showed that
during the in vitro refolding of a mixture
of two proteins, tailspike endorhamnidase
and coat protein from phage P22, no heterogeneous
aggregates were formed. Tailspike
endorhamnidase is a thermostable
trimer whose folding intermediates are
thermolabile and either undergo productive
folding or formmultimeric aggregates
(Fig. 3). The P22 coat protein, which comprises
the capsid shell of phage P22, yields
either a correct fold or ‘‘off-pathway’’ aggregates
upon refolding. Both proteins were
intensively studied by King and coworkers
who first denatured the two proteins
in urea and then chose refolding conditions
such that aggregation competes
with correct folding. Folding and soluble
aggregates of the two proteins were characterized
either separately ormixed together.
No heterogeneous aggregates were found,
clearly indicating that only self-association
of transient refolding molecules occurs in
the formation of soluble multimers.
One mechanism that accounts for the
formation of aggregates during refolding
of multidomain proteins is domain swapping.
This was first suggested by Monod
and later proposed by Goldberg and colleagues
to account for the formation of
aggregates during the refolding of tryptophanase.
The concept was foreshadowed
by the results of Crestfield and coworkers
in 1962. From their experiments based
on chemical modification of bovine pancreatic
ribonuclease, the authors proposed
that the dimer is formed by exchanging
the N-terminal fragments. The term 3D
domain swapping was introduced in 1994
by Bennett and coworkers to describe the
structure of a diphtheria toxin dimer. The
mechanism involves the replacement of
one domain of a monomeric protein by the
same domain of an identical neighboring
Aggregate = inclusion body
Suppressor mutation
action
(I*)
(I)
40 °C
tsf 30 °C
Substitution
Early-folding
intermediate
Native
tailspike
Protrimer
Fig. 3 The folding pathway of the P22 tailspike protein. (From Mitraki, A., King, J. (1992) FEBS Lett.
307, 20–25; reproduced with permission.)
32 Aggregation, Protein
(a)
(b)
(c)
(d)
Fig. 4 Schematic
representation of
domain swapping. (a)
monomeric protein, (b) and (c)
partially unfolded monomers,
(d) domain-swapped dimer.
molecule, thus resulting in an intertwined
dimer or oligomer, as defined by Eisenberg
and colleagues (Fig. 4). When the
exchange is reciprocated, domain-swapped
dimers are formed. However, if the exchange
is not reciprocated but propagated
along multiple polypeptide chains, higher
order assemblies or aggregates may form.
Domain-swapped oligomers are divided
into two types, open and closed. The open
oligomers are linear and have one closed
interface (closed in themonomer) exposed
to the solvent, whereas closed oligomers
are cyclic and do not expose a closed
interface. Eisenberg and coworkers have
defined the structure of the monomer as
the ‘‘closed monomer’’ and the conformation
of the polypeptide chain in the
domain-swapped oligomer as the ‘‘open
monomer.’’
The ability of monomeric proteins to
swap structural elements requires the presence
of a hinge or linker region that
permits the protein to attain the native
fold with parts of two polypeptide chains.
In fact, domain-swapped structures reveal
regions of protein structure that are
flexible. Bergdoll and coworkers have suggested
that a proline in the linker region,
by rigidifying the hinge region in intermediate
states, might facilitate domain
swapping. Baker and colleagues proposed
that strain in a hairpin loop might predispose
a protein to domain swapping.
The possible role of 3D domain swapping
in the evolution of oligomeric proteins
has been discussed in several reviews.
In the past years, the number of known
domain-swapped proteins has increased
and today about 40 such structures are
solved. One common feature of these proteins
is that all the swapped domains
are from either the N-terminus or the
C-terminus of the polypeptide chain. In
this regard, an interesting example arises
from the work of Eisenberg and his group
on the dimerization of ribonuclease A.
This protein forms two types of dimers
upon concentration in mild acid. The minor
dimer is formed by swapping of its
N-terminal α-helix with that of an identical
molecule. The major dimer results
from the swapping of its C-terminal β-
strand. RNase A was also reported to form
trimers. On the basis of the structure of
the N- and C-terminal swapped dimers,
a model was proposed (Fig. 5). This indicates
that two types of swapping can occur
simultaneously in the same oligomer. Further
biochemical studies have supported
this model. A less abundant trimer in
which only the C-terminal β-strand is
swapped and exhibits a cyclic structure
was also found. RNase represents the
Aggregation, Protein 33
first protein found to form both linear
and cyclic domain-swapped oligomers.
This protein also was described to form
tetramers. Models based on the structures
of dimers and trimers were proposed for
these tetramers. Two linear models exhibit
both types of swapping that occur in
one molecule, and a cyclic tetramer shows
the swapping of the C-terminal β-strand
only. A trimeric domain-swapped barnase
was obtained at low pH and high protein
concentration. Crystallographic studies revealed
a structure suggesting a probable
folding intermediate. Domain swapping
was described for the cell cycle regulatory
protein p13suc1, a small protein of 113
amino acids.
Folding studies as well as molecular
dynamics simulations have shown
that domain swapping occurs in the unfolded
state. Eisenberg and his colleagues
have proposed a free energy diagram for
the pathway of domain swapping. The
free energy difference between the closed
monomer and domain-swapped oligomer
is small since they share the same structures
except at the hinge loop, but the
energy barrier can be reduced under certain
conditions making domain swapping
more favorable. Several molecular or environmental
events may favor the formation
of extended domain-swapped polymers.
Genetic mutations introducing a deletion
in the hinge loop can destabilize the
monomeric form of a protein. The replacement
of only one amino acid can also favor
Fig. 5 Domain swapping in
ribonuclease. Ribbon diagram of the
structures of (a) the ribonuclease A
monomer (2.0 A˚ ), (b) the N-terminal
swapped dimer (2.1 A˚ ), (c) the
C-terminal swapped dimer (1.75 A˚ ), (d)
the N- and C-terminal trimer model, and
(e) the cyclic C-terminal swapped trimer
(2.2 A˚ ) (reproduced from Liu et al. Prot.
Sci. 11, 371, 2002 with permission).
(a)
(d) (e)
(b)
(c)
the polymerization of the mutated protein.
Three-dimensional domain-swapped
oligomers are expected to be increasingly
favored as the protein concentration increases.
Thus, a metabolic change that
increases the concentration of a protein
will favor aggregation. Charge effects,
caused either by mutations or by pH
change or salt concentration, can induce
domain swapping; for example, in RNase
A, a decrease in pH, by protonating the
residues involved in hydrogen bonds and
in salt bridges, lowers the energy barrier
of the formation of the open monomer,
hence inducing domain swapping.
There is great diversity of swapped domains,
with different sizes and sequences.
They can consist of entire tertiary domains
or smaller structural elementsmade
of several residues. No specific sequence
motif seems to be involved among the
swapped domains. Three-dimensional domain
swapping has also been proposed as
a mechanism for amyloid formation. This
aspect will be discussed in Sect. 4.2.
34 Aggregation, Protein
As can be seen here, several mechanisms
exist, which lead to the formation
of aggregates. It is recognized that aggregation
results from the association of
incompletely or incorrectly folded intermediates
through hydrophobic interactions.
In the energy landscape of protein folding,
the presence of local minima separated
by an energy barrier allows the accumulation
of intermediates. If the barrier is
high enough, these intermediates cannot
easily reach the native state, and kinetic
competition thus favors the formation
of aggregates.
the idea that protein aggregation
arises from partially folded intermediates
through hydrophobic interactions. The
formation of aggregates has often been
considered as a trivial phenomenon, a
nonspecific association of partially folded
polypeptide chains to form a disordered
precipitate. However, several analyses indicate
that aggregation occurs by specific
intramolecular associations involving the
recognition of a sequence partner in another
molecule rather than in the same
molecule during the folding process. Analyses
of the aggregation mechanisms of
various proteins, such as bovine growth
hormone and phosphoglycerate kinase,
has permitted the identification of specific
sites that are critical in the association.
An elegant demonstration of the specificity
of aggregation was provided by King
Aggregation, Protein 31
and coworkers. The authors showed that
during the in vitro refolding of a mixture
of two proteins, tailspike endorhamnidase
and coat protein from phage P22, no heterogeneous
aggregates were formed. Tailspike
endorhamnidase is a thermostable
trimer whose folding intermediates are
thermolabile and either undergo productive
folding or formmultimeric aggregates
(Fig. 3). The P22 coat protein, which comprises
the capsid shell of phage P22, yields
either a correct fold or ‘‘off-pathway’’ aggregates
upon refolding. Both proteins were
intensively studied by King and coworkers
who first denatured the two proteins
in urea and then chose refolding conditions
such that aggregation competes
with correct folding. Folding and soluble
aggregates of the two proteins were characterized
either separately ormixed together.
No heterogeneous aggregates were found,
clearly indicating that only self-association
of transient refolding molecules occurs in
the formation of soluble multimers.
One mechanism that accounts for the
formation of aggregates during refolding
of multidomain proteins is domain swapping.
This was first suggested by Monod
and later proposed by Goldberg and colleagues
to account for the formation of
aggregates during the refolding of tryptophanase.
The concept was foreshadowed
by the results of Crestfield and coworkers
in 1962. From their experiments based
on chemical modification of bovine pancreatic
ribonuclease, the authors proposed
that the dimer is formed by exchanging
the N-terminal fragments. The term 3D
domain swapping was introduced in 1994
by Bennett and coworkers to describe the
structure of a diphtheria toxin dimer. The
mechanism involves the replacement of
one domain of a monomeric protein by the
same domain of an identical neighboring
Aggregate = inclusion body
Suppressor mutation
action
(I*)
(I)
40 °C
tsf 30 °C
Substitution
Early-folding
intermediate
Native
tailspike
Protrimer
Fig. 3 The folding pathway of the P22 tailspike protein. (From Mitraki, A., King, J. (1992) FEBS Lett.
307, 20–25; reproduced with permission.)
32 Aggregation, Protein
(a)
(b)
(c)
(d)
Fig. 4 Schematic
representation of
domain swapping. (a)
monomeric protein, (b) and (c)
partially unfolded monomers,
(d) domain-swapped dimer.
molecule, thus resulting in an intertwined
dimer or oligomer, as defined by Eisenberg
and colleagues (Fig. 4). When the
exchange is reciprocated, domain-swapped
dimers are formed. However, if the exchange
is not reciprocated but propagated
along multiple polypeptide chains, higher
order assemblies or aggregates may form.
Domain-swapped oligomers are divided
into two types, open and closed. The open
oligomers are linear and have one closed
interface (closed in themonomer) exposed
to the solvent, whereas closed oligomers
are cyclic and do not expose a closed
interface. Eisenberg and coworkers have
defined the structure of the monomer as
the ‘‘closed monomer’’ and the conformation
of the polypeptide chain in the
domain-swapped oligomer as the ‘‘open
monomer.’’
The ability of monomeric proteins to
swap structural elements requires the presence
of a hinge or linker region that
permits the protein to attain the native
fold with parts of two polypeptide chains.
In fact, domain-swapped structures reveal
regions of protein structure that are
flexible. Bergdoll and coworkers have suggested
that a proline in the linker region,
by rigidifying the hinge region in intermediate
states, might facilitate domain
swapping. Baker and colleagues proposed
that strain in a hairpin loop might predispose
a protein to domain swapping.
The possible role of 3D domain swapping
in the evolution of oligomeric proteins
has been discussed in several reviews.
In the past years, the number of known
domain-swapped proteins has increased
and today about 40 such structures are
solved. One common feature of these proteins
is that all the swapped domains
are from either the N-terminus or the
C-terminus of the polypeptide chain. In
this regard, an interesting example arises
from the work of Eisenberg and his group
on the dimerization of ribonuclease A.
This protein forms two types of dimers
upon concentration in mild acid. The minor
dimer is formed by swapping of its
N-terminal α-helix with that of an identical
molecule. The major dimer results
from the swapping of its C-terminal β-
strand. RNase A was also reported to form
trimers. On the basis of the structure of
the N- and C-terminal swapped dimers,
a model was proposed (Fig. 5). This indicates
that two types of swapping can occur
simultaneously in the same oligomer. Further
biochemical studies have supported
this model. A less abundant trimer in
which only the C-terminal β-strand is
swapped and exhibits a cyclic structure
was also found. RNase represents the
Aggregation, Protein 33
first protein found to form both linear
and cyclic domain-swapped oligomers.
This protein also was described to form
tetramers. Models based on the structures
of dimers and trimers were proposed for
these tetramers. Two linear models exhibit
both types of swapping that occur in
one molecule, and a cyclic tetramer shows
the swapping of the C-terminal β-strand
only. A trimeric domain-swapped barnase
was obtained at low pH and high protein
concentration. Crystallographic studies revealed
a structure suggesting a probable
folding intermediate. Domain swapping
was described for the cell cycle regulatory
protein p13suc1, a small protein of 113
amino acids.
Folding studies as well as molecular
dynamics simulations have shown
that domain swapping occurs in the unfolded
state. Eisenberg and his colleagues
have proposed a free energy diagram for
the pathway of domain swapping. The
free energy difference between the closed
monomer and domain-swapped oligomer
is small since they share the same structures
except at the hinge loop, but the
energy barrier can be reduced under certain
conditions making domain swapping
more favorable. Several molecular or environmental
events may favor the formation
of extended domain-swapped polymers.
Genetic mutations introducing a deletion
in the hinge loop can destabilize the
monomeric form of a protein. The replacement
of only one amino acid can also favor
Fig. 5 Domain swapping in
ribonuclease. Ribbon diagram of the
structures of (a) the ribonuclease A
monomer (2.0 A˚ ), (b) the N-terminal
swapped dimer (2.1 A˚ ), (c) the
C-terminal swapped dimer (1.75 A˚ ), (d)
the N- and C-terminal trimer model, and
(e) the cyclic C-terminal swapped trimer
(2.2 A˚ ) (reproduced from Liu et al. Prot.
Sci. 11, 371, 2002 with permission).
(a)
(d) (e)
(b)
(c)
the polymerization of the mutated protein.
Three-dimensional domain-swapped
oligomers are expected to be increasingly
favored as the protein concentration increases.
Thus, a metabolic change that
increases the concentration of a protein
will favor aggregation. Charge effects,
caused either by mutations or by pH
change or salt concentration, can induce
domain swapping; for example, in RNase
A, a decrease in pH, by protonating the
residues involved in hydrogen bonds and
in salt bridges, lowers the energy barrier
of the formation of the open monomer,
hence inducing domain swapping.
There is great diversity of swapped domains,
with different sizes and sequences.
They can consist of entire tertiary domains
or smaller structural elementsmade
of several residues. No specific sequence
motif seems to be involved among the
swapped domains. Three-dimensional domain
swapping has also been proposed as
a mechanism for amyloid formation. This
aspect will be discussed in Sect. 4.2.
34 Aggregation, Protein
As can be seen here, several mechanisms
exist, which lead to the formation
of aggregates. It is recognized that aggregation
results from the association of
incompletely or incorrectly folded intermediates
through hydrophobic interactions.
In the energy landscape of protein folding,
the presence of local minima separated
by an energy barrier allows the accumulation
of intermediates. If the barrier is
high enough, these intermediates cannot
easily reach the native state, and kinetic
competition thus favors the formation
of aggregates.
Molecular medicine-Irreversible Aggregation
Thermal unfolding of proteins is frequently
accompanied by the formation of
aggregates and therefore behaves as an
irreversible process. It occurs at temperatures
that vary widely according to the
protein, since the temperature of optimum
stability depends on the balance between
hydrogen bonds and hydrophobic interactions.
Generally, the products of thermal
denaturation are not completely unfolded
and retain some structured regions. At the
end of the thermal transition, the addition
of a denaturant such as urea or GdnHCl
frequently induces further unfolding.
An apparent irreversibility at a critical
concentration of denaturant has been observed
during the refolding of monomeric
as well as oligomeric proteins. It was reported
for the first time by M.Goldberg
and coworkers for the refolding of β-
galactosidase, and for tryptophanase. It
was also observed for a two-domain protein,
horse muscle phosphoglycerate kinase
by Yon and coworkers. In the latter
study, when the enzyme activity was used
as a conformational probe of the native
structure, an irreversibility was observed
for a critical concentration of denaturant
equal to 0.7 M± 0.1 MGdnHCl, a concentration
very close to the end of the transition
curve. Such irreversibility was found
to be concentration dependent. For protein
concentrations higher than 30 μM, restoration
of enzyme activity was practically null.
The formation of irreversible nonnative
species was found to be temperature dependent;
it was practically abolished at
4 ◦C, suggesting that aggregation occurs
through hydrophobic interactions. The aggregation
also depends on the time of
exposure of the protein to the denaturant.
When the unfolding–refolding process
was observed using structural signals such
as fluorescence or circular dichroism, it appeared
completely reversible whatever the
final denaturant concentration.
Another example is provided by rhodanese,
a two-domain monomeric protein.
During refolding at low denaturant concentration,
an intermediate accumulates
with partially structured domains and apolar
surfaces exposed to the solvent, leading
to the formation of aggregates. The aggregation
can be prevented by refolding the
protein in the presence of lauryl maltoside.
Most of the examples discussed above
are related to multidomain proteins. Another
degree of complexity appears in the
folding of oligomeric proteins. It is generally
accepted that the early steps of the
process are practically identical to the folding
ofmonomeric proteins. In the last step,
subunit association and subsequent conformational
readjustments yield the native
and functional oligomeric protein. The correct
recognition of subunit interfaces is
required to achieve the process. The overall
process of the folding of oligomeric proteins
was extensively studied by Jaenicke
and his coworkers for several enzymes and
described in reviews. As with monomeric
proteins, the formation of aggregates is
concentration dependent. The kinetics of
aggregation are complex and multiphasic,
indicating that several rate-limiting reactions
are involved in the process. In an
attempt to characterize these aggregates, it
was shown that noncovalent interactions
occur between monomeric species with
30 Aggregation, Protein
partially restored secondary structures.
The aggregates formed by either heat or
pH denaturation can be disrupted in 6 M
GdnHCl into monomeric unfolded species
and then renatured under optimal conditions
to yield an active enzyme.Only strong
denaturants such as high concentrations
of guanidine hydrochloride are efficient in
this disruption process.
The presence of covalent cross-links
such as disulfide bridges in a protein
molecule can complicate the refolding of
the denatured and reduced protein resulting
in the formation of incorrect and
intramolecular disulfide bridges leading
to further aggregation. The first welldocumented
studies were performed by
Anfinsen and his group on the refolding
of reduced ribonuclease. The authors
showed that the reoxidation of the enzyme
produces a great number of species with
incorrectly paired disulfide bonds. This
scrambled ribonuclease is capable of regaining
its native structure in a slow step,
a process that is accelerated by the addition
of a small quantity of reducing reagent
such as β-mercaptoethanol yielding about
100% of active enzyme. The reshuffling
of a protein’s disulfide bonds takes place
through a series of redox equilibria according
to either an intramolecular or
an intermolecular exchange. To prevent
a wrong pairing of half-cystine and further
aggregation, the addition of small
amounts of reducing reagents or redox
mixture is frequently used as investigated
by Wetlaufer.
The detection and characterization of
aggregates represent an important aspect
of folding studies. The aggregation phenomenon
can occur without precipitation.
Indeed, the degree of association of protein
intermediates during folding might
be small, depending on the intermolecular
interactions, and does not necessarily lead
to a visible insolubility. The association
state may be determined in several ways.
The most common methods, available
in any biochemistry laboratory, are gel
permeation and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDSPAGE),
used both with and without crosslinking.
The detection of aggregates can
also be monitored by other hydrodynamic
methods suchas analytical ultracentrifugation
or classical light scattering. The latter
method also gives information on the size
of the aggregates. Quasi-elastic light scattering
is a dynamic technique that can be
used to determine macromolecule diffusion
coefficients as a function of time, that
is, to follow the kinetics of aggregation.
Neutron scattering can also be used to
detect protein aggregates, and mass spectrometry
has become a useful tool as well.
accompanied by the formation of
aggregates and therefore behaves as an
irreversible process. It occurs at temperatures
that vary widely according to the
protein, since the temperature of optimum
stability depends on the balance between
hydrogen bonds and hydrophobic interactions.
Generally, the products of thermal
denaturation are not completely unfolded
and retain some structured regions. At the
end of the thermal transition, the addition
of a denaturant such as urea or GdnHCl
frequently induces further unfolding.
An apparent irreversibility at a critical
concentration of denaturant has been observed
during the refolding of monomeric
as well as oligomeric proteins. It was reported
for the first time by M.Goldberg
and coworkers for the refolding of β-
galactosidase, and for tryptophanase. It
was also observed for a two-domain protein,
horse muscle phosphoglycerate kinase
by Yon and coworkers. In the latter
study, when the enzyme activity was used
as a conformational probe of the native
structure, an irreversibility was observed
for a critical concentration of denaturant
equal to 0.7 M± 0.1 MGdnHCl, a concentration
very close to the end of the transition
curve. Such irreversibility was found
to be concentration dependent. For protein
concentrations higher than 30 μM, restoration
of enzyme activity was practically null.
The formation of irreversible nonnative
species was found to be temperature dependent;
it was practically abolished at
4 ◦C, suggesting that aggregation occurs
through hydrophobic interactions. The aggregation
also depends on the time of
exposure of the protein to the denaturant.
When the unfolding–refolding process
was observed using structural signals such
as fluorescence or circular dichroism, it appeared
completely reversible whatever the
final denaturant concentration.
Another example is provided by rhodanese,
a two-domain monomeric protein.
During refolding at low denaturant concentration,
an intermediate accumulates
with partially structured domains and apolar
surfaces exposed to the solvent, leading
to the formation of aggregates. The aggregation
can be prevented by refolding the
protein in the presence of lauryl maltoside.
Most of the examples discussed above
are related to multidomain proteins. Another
degree of complexity appears in the
folding of oligomeric proteins. It is generally
accepted that the early steps of the
process are practically identical to the folding
ofmonomeric proteins. In the last step,
subunit association and subsequent conformational
readjustments yield the native
and functional oligomeric protein. The correct
recognition of subunit interfaces is
required to achieve the process. The overall
process of the folding of oligomeric proteins
was extensively studied by Jaenicke
and his coworkers for several enzymes and
described in reviews. As with monomeric
proteins, the formation of aggregates is
concentration dependent. The kinetics of
aggregation are complex and multiphasic,
indicating that several rate-limiting reactions
are involved in the process. In an
attempt to characterize these aggregates, it
was shown that noncovalent interactions
occur between monomeric species with
30 Aggregation, Protein
partially restored secondary structures.
The aggregates formed by either heat or
pH denaturation can be disrupted in 6 M
GdnHCl into monomeric unfolded species
and then renatured under optimal conditions
to yield an active enzyme.Only strong
denaturants such as high concentrations
of guanidine hydrochloride are efficient in
this disruption process.
The presence of covalent cross-links
such as disulfide bridges in a protein
molecule can complicate the refolding of
the denatured and reduced protein resulting
in the formation of incorrect and
intramolecular disulfide bridges leading
to further aggregation. The first welldocumented
studies were performed by
Anfinsen and his group on the refolding
of reduced ribonuclease. The authors
showed that the reoxidation of the enzyme
produces a great number of species with
incorrectly paired disulfide bonds. This
scrambled ribonuclease is capable of regaining
its native structure in a slow step,
a process that is accelerated by the addition
of a small quantity of reducing reagent
such as β-mercaptoethanol yielding about
100% of active enzyme. The reshuffling
of a protein’s disulfide bonds takes place
through a series of redox equilibria according
to either an intramolecular or
an intermolecular exchange. To prevent
a wrong pairing of half-cystine and further
aggregation, the addition of small
amounts of reducing reagents or redox
mixture is frequently used as investigated
by Wetlaufer.
The detection and characterization of
aggregates represent an important aspect
of folding studies. The aggregation phenomenon
can occur without precipitation.
Indeed, the degree of association of protein
intermediates during folding might
be small, depending on the intermolecular
interactions, and does not necessarily lead
to a visible insolubility. The association
state may be determined in several ways.
The most common methods, available
in any biochemistry laboratory, are gel
permeation and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDSPAGE),
used both with and without crosslinking.
The detection of aggregates can
also be monitored by other hydrodynamic
methods suchas analytical ultracentrifugation
or classical light scattering. The latter
method also gives information on the size
of the aggregates. Quasi-elastic light scattering
is a dynamic technique that can be
used to determine macromolecule diffusion
coefficients as a function of time, that
is, to follow the kinetics of aggregation.
Neutron scattering can also be used to
detect protein aggregates, and mass spectrometry
has become a useful tool as well.
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