REVIEW ARTICLE
The small heat shock proteins and their role in human
disease
Yu Sun and Thomas H. MacRae
Department of Biology, Dalhousie University, Halifax, Canada
Within the molecular chaperone family, sHSPs consti-
tute a structurally divergent group characterized by
a conserved sequence of 80–100 amino acid residues
termed the a-crystallin domain [1–8]. The a-crystallin
domain, duplicated in the unusual example of parasi-
tic flatworms (Platyhelminthes) [9], is located toward
a highly flexible, variable, C-terminal extension, and
is usually preceded by a poorly conserved N-terminal
region. The molecular mass of sHSP subunits ranges
from 12 to 43 kDa, and they assemble into large,
dynamic complexes up to 1 MDa. sHSP secondary
structure is dominated by b-strands with limited
a-helical content, and b-sheets within the a-crystallin
domain mediate dimer formation. Crystallization of
two sHSPs has contributed significantly to the des-
cription of oligomerization, quaternary structure,
subunit exchange, and chaperone activity. Characteri-
zation of a highly conserved arginine is also an
important outcome of crystallization and related stud-
ies because mutation of this residue has profound
effects on sHSP function and contributes to certain
diseases [10–16].
The sHSPs are molecular chaperones, storing aggre-
gation prone proteins as folding competent intermedi-
ates and conferring enhanced stress resistance on cells
by suppressing aggregation of denaturing proteins,

fusion injury due to heart attack and stroke. On the other hand, mutated
sHSPs are implicated in diseases such as desmin-related myopathy and they
have an uncertain relationship to neurological disorders including Parkin-
son’s and Alzheimer’s disease. This review explores the involvement of
sHSPs in disease and their potential for therapeutic intervention.
Abbreviations
17-AAG, 17-allylamino-17-demethoxygeldanamycin; Ab, amyloid-b; AGE, advanced glycation end-product; ALS, amyotrophic lateral sclerosis;
CAT, cancer ⁄ testis antigen; GFAP, glial fibrillary acidic protein; HMM, high molecular weight; IFN-c, interferon-c; MS, multiple sclerosis;
sHSP, small heat shock protein; SOD, superoxide dismutase.
FEBS Journal 272 (2005) 2613–2627 ª 2005 FEBS 2613
more limited than for other chaperones, but this is
changing as the application of genomics and proteo-
mics reveals sHSP characteristics and their medical
importance emerges. In this context, 10 sHSPs, termed
HspB1–10, many of which are constitutively present at
high levels in muscle and implicated in disease, are
found in humans [2,21–23]. Intracellular quantities and
cellular localizations of sHSPs change in response to
development, physiological stressors such as anoxia ⁄
hypoxia, heat and oxidation, and in relation to patho-
logical status. sHSPs interact with many essential cell
structures and it follows from such promiscuity that
functional disruption and inappropriate association of
these molecular chaperones with substrates will foster
disease. Therefore, this review considers the role of
sHSPs in several human medical conditions and it ends
with a discussion of their therapeutic potential.
sHSPs and cataract
sHSP mutation and post-translational change con-
tribute to cataract development in the mammalian

Cataract and a-crystallin post-
translational changes
Posttranslational modifications of aA- and aB-crystal-
lin, including truncation [33–37], deamidation [36,
38–42], oxidation [40,43–46], glycation [46–53], phos-
phorylation [33] and racemization ⁄ isomerization [54,
55], promote cataract formation in aging organisms
through modification of chaperone activity and solubil-
ity [24,35,40,41,47,56]. a-crystallin post-translational
changes, with a corresponding effect on lens transpar-
ency, occur during diabetes where chaperone activity
decreases in reverse correlation to glucose levels [52].
Glycation, the nonenzymatic addition of sugars to pro-
teins, is enhanced in rat and human lenses during dia-
betes, causing protein cross-linking and advanced
glycation end-products (AGE), a change engendered
by methylglyoxal interaction with lysine and arginine
residues [51]. Glycation in vitro limits the chaperone
activity of human, calf and rabbit lens a-crystallins
[46,51], as does methylglyoxal treatment of calf lens in
organ culture, with corresponding reduction in protein
stability [48,49]. However, in other studies, glycation
of C-terminal lysines does not disrupt a-crystallin
chaperoning [53] and activity increases when the pro-
tein is modified in vitro [48,50], suggesting in contrast
to prevailing theories that post-translational modifica-
tions are an aging related protective mechanism for
long-lived lens proteins.
Demonstrating definitive causal relationships
between sHSP post-translational modifications and

contributing to functional changes and to cataract.
Site-directed mutagenesis was employed to examine
oxidation of aA-crystallin, a protein with two cysteine
residues [44] and where intrapolypeptide disulfides [45]
and mixed glutathione disulfides [59] curtail chaperone
activity. Exposing wild-type a-crystallin and mutants
C113I, C142I and C131I ⁄ C142I to hydrogen peroxide
demonstrates disulfide-dependent dimerizations are less
important in production of high molecular mass
(HMM) protein aggregates accompanying cataract
than are secondary structural changes generated upon
tryptophan and tyrosine oxidation. Additionally,
a-crystallin dimerization promoted by calcium-activa-
ted transglutaminase eliminates chaperone activity,
suggesting a role in reduced lens transparency and
cataract [56]. Oxidation and transglutaminase induced
cross-linking may coordinately transform lens a-crys-
tallin chaperone activity and packing, magnifying the
consequences of these changes and promoting cataract
formation more than anticipated.
Evidence linking cataract and a-crystallin post-trans-
lational changes is compelling, but there are examples
of extensive a-crystallin modification before disease
appears, and cataract associated protein changes may
occur subsequent to lens a-crystallin denaturation rather
than before [24,42]. In spite of these observations,
the prevalence of post-translational changes in lens
a-crystallins argues forcefully for a major role in cata-
ract and their study remains important if the disease is
to be fully understood. Potential exists for development

by modified a-crystallins, results obtained by mamma-
lian two-hybrid analyses demonstrate that interaction
of aA- and aB-crystallin with one another is about
three times stronger than the engagement of either
chaperone with the prominent lens proteins, bB2-crys-
tallin or cC-crystallin [65,66]. Moreover, aB-crystallin
self-interaction occurs essentially independent of the
polypeptide’s N-terminus, but self-association of aA-
crystallin requires this domain [66]. Attachment of
R116C aA-crystallin to Hsp27 and aB-crystallin
increases in comparison to wild type, while binding to
cC-crystallin and bB2-crystallin decreases. Reaction of
R120G aB-crystallin with bB2-crystallin is moderately
enhanced, but there is no change in recognition of
cC-crystallin and Hsp27, and association with aA- and
aB-crystallin declines. The altered interplay with other
crystallins illustrates that R116C aA-crystallin and
R120G aB-crystallin, both observed in congenital cata-
ract, maintain lens protein solubility less effectively
and promote cataract development.
Lens size drops off in mice homozygous for aA-crys-
tallin gene loss [aA(–⁄ –)], a characteristic correlated
with 50% reduction in lens epithelial cell growth and
enhanced sensitivity to apoptotic death [67,68]. The
lenses of aA(–⁄ –) mice become opaque with age and
contain many inclusion bodies reactive with antibody
to aB-crystallin, but not to b- and c-crystallin, suggest-
ing an important role for aA-crystallin in maintaining
lens transparency [69]. Over-expression of aA-crystallin
protects stably transfected cells against UVA radiation,

aggregates with aB-crystallin [63]. The mutation dis-
rupts aB-crystallin structure, chaperone activity and
intermediate filament interaction, demonstrating the
functional importance of residue R120 [14–16,62,74].
This was the first sHSP mutation shown to cause
inherited human muscle disease, but two additional
dominant negative aB-crystallin mutations have since
been linked to myofibrillar myopathy, but not cardio-
myopathy [75]. The aB-crystallin C-terminus is trun-
cated by 13 residues in one case and 25 in another, a
region important for sHSP solubilization, chaperone
activity and oligomer formation.
R120G aB-crystallin synthesis in hearts of trans-
genic mice induces desmin-related cardiomyopathy
[74,76], potentiating desmin and aB-crystallin aggre-
gation, myofibril derangement, compromised muscle
action, and heart failure. Study of transgenic mice
containing mutations in both desmin and aB-crystal-
lin signifies that the sHSP prevents aggregation of
misfolded desmin [77]. A nuclear role for aB-crystal-
lin during cardiomyopathy is also possible because
the R120G mutant inhibits speckle formation by the
wild-type chaperone in several transfected cell lines
[78]. Speckles are thought to participate in RNA
transcription and splicing. Cardiomyocyte transfection
with adenovirus encoding R120G aB-crystallin pro-
motes microtubule-dependent production of intracellu-
lar aggresomes [79]. These structures, appearing in
cardiomyocytes of dilated and hypertrophic cardio-
myopathies, are characteristic of amyloid-related

myocytes, the latter by transfection with adenovirus
vectors, shields heart cells against apoptosis and necro-
sis upon ischemia ⁄ reperfusion injury [74,81–84]. Over
expressed wild-type and nonphosphorylatable Hsp27
were equally effective in safeguarding contractile activ-
ity and cell integrity, as determined by retention of cre-
atine kinase activity in transgenic mice hearts during
ischemia ⁄ reperfusion [81]. sHSP phosphorylation sta-
tus may have little influence on the ability of Hsp27 to
protect myocardial cells of these transgenic mice dur-
ing ischemia ⁄ reperfusion, although nonphosphorylata-
ble Hsp27 variants produce larger oligomers on
average than wild type, a trend accentuated by the
stress of ischemia ⁄ reperfusion, and there is a potential
effect on how well cells cope with oxidative stress.
Gene deletion experiments indicate sHSPs defend
cells against ischemia ⁄ reperfusion injury. That is, the
hearts of double knock-out mice lacking the abundant
sHSPs, aB-crystallin and HspB2, develop as expected
under nonstress conditions and show normal contrac-
tility [85]. However, when exposed to ischemia and
Small heat shock proteins and disease Y. Sun and T. H. MacRae
2616 FEBS Journal 272 (2005) 2613–2627 ª 2005 FEBS
reperfusion, hearts from these animals display reduced
contractility and less glutathione, accompanied by
greater necrosis and apoptosis due to free radical pro-
duction. The need for either or both aB-crystallin and
HspB2 for optimal recovery from heart attack is
apparent. Phosphorylated Hsp20, known to associate
with and stabilize actin [86], and aB-crystallin [87],

Alzheimer’s is characterized by amyloid-b peptide
(Ab) in extracellular senile plaques and tau in neuro-
fibrillary tangles, aggregates that are major morpho-
logical indicators of the disease [103]. Alzheimer’s
disease is the most common tauopathy, a group of
familial neurodegenerative conditions distinguished by
intracellular filamentous bodies composed of tau, a
low molecular weight microtubule-associated protein
[104]. Neurons are the predominant location of tau
pathology in Alzheimer’s, but glial pathology manifests
in corticobasal degeneration and progressive supra-
nuclear palsy. Increased aB-crystallin, and to a lesser
extent Hsp27, appear in the latter, conceivably in
response to aberrant tau. aB-Crystallin and Hsp27,
up-regulated in Alzheimer’s brains and localizing to
astrocytes and degenerating neurons [104–109], interact
with Ab and occur in amyloid plaque, thereby affect-
ing amyloid production [107,110,111].
Mass spectrometry reveals that three Hsp16 family
members, in addition to other molecular chaperones,
coimmunoprecipitate with human Ab in transgenic
Caenorhabditis elegans [112]. sHSP expression is
induced by the presence of Ab, which is associated
with progressive worm paralysis, and the proteins colo-
calize intracellularly, suggesting a role for molecular
chaperones in Ab toxicity and metabolism. Human
recombinant aB-crystallin also interacts with Ab
in vitro, and as shown by thioflavin T fluorescence and
far-CD measurements, aB-crystallin promotes b-sheet
formation by Ab [110]. Samples were not examined by

of sHSPs in motor neurons and up-regulation in astro-
cytes. Mouse Hsp25 colocalizes with mutant SOD-1
[117], similar to results obtained with a cultured neur-
onal cell line [118]. Interaction with mutant, but not
wild-type SOD-1 may limit antiapoptotic potential and
decrease cell protection by Hsp25. In another example,
Hsp27 and aB-crystallin appear in Parkinson’s disease
Y. Sun and T. H. MacRae Small heat shock proteins and disease
FEBS Journal 272 (2005) 2613–2627 ª 2005 FEBS 2617
with severe dementia [119]. sHSPs and neurological
diseases are evidently linked, but consequences are
uncertain. Chaperoning can prevent or promote aggre-
gate creation, and either outcome may be favorable or
unfavorable, depending on the disease. As a case in
point, formation of huntingtin-containing inclusion
bodies in Huntington’s disease encourages cell survival,
whereas monomers and small inclusion bodies of hunt-
ingtin, a protein possessing abnormal polyQ repeats,
are toxic, an effect potentially mediated by transcrip-
tion factor destabilization [96,99,120]. Prevention of
abnormal protein aggregation obviously does not
always benefit cells, an observation with important
implications when choosing therapeutic approaches to
neurological diseases.
Nerve demyelination presents in multiple sclerosis
(MS), a chronic autoimmune neurological condition
involving brain and spinal cord inflammation. T cells
from MS patients express a dominant response to
aB-crystallin, a major autoantigen affiliated with cen-
tral nervous system myelin, the disease target

neuropathies, nor is HspB8 function understood, how-
ever, mutations to K141 are linked to motor neuro-
pathies. Mutations S135F, R127W, T151I and P182L
in HspB1 (Hsp27) were subsequently discovered in
families with distal hereditary motor neuropathy [128].
Individuals with the genetically and clinically hetero-
geneous syndrome, Charot–Marie–Tooth disease, the
most common inherited motor and sensory neuro-
pathy, contain HspB8 K141N, as in distal hereditary
motor neuropathy [126], as well as S135F and
R136W in HspB1 [128]. All HspB1 mutations, with
exception of P182L in the C-terminal extension, are
quartered in the a-crystallin domain near residue
R140. Neuronal N2a cells transfected with S135F
HspB1 are less viable than cells expressing wild-type
HspB1, symptomatic of distal motor neuropathies and
Charot–Marie–Tooth disease being caused by muta-
tion induced, premature axonal degeneration. Multi-
nucleated cells almost double upon expression of the
S135F HspB1 mutant and intermediate filament
arrangement is affected adversely in an adrenal carci-
noma cell line, implicating cytoskeleton disruption in
these diseases.
sHSPs and cancer
Based on the consequences of molecular chaperone
induction in diseased (stressed) cells, the relationship
between cancer and sHSPs is worthy of examination.
One area receiving attention is sHSP value in clinical
prognosis of individual cancers and of cancers at dif-
ferent developmental stages. By example, a strong cor-

cells prevents E-cadherin loss, and synthesis of the
adhesion molecule MUC18 ⁄ MCAM, which correlates
with metastatic potential, is disrupted [142]. The cumu-
lative data indicate Hsp27 slows A375 melanoma cell
growth in vitro, lowers tumor appearance rate in mice
[143] and inhibits tumor progression. In another exam-
ple, Hsp27 increases MDA-MB-231 breast cancer cell
metastasis [135]. Concurrently, MMP-9, a zinc depend-
ent endoprotease capable of degrading several extra-
cellular matrix proteins and enhancing tumor cell
invasion, is amplified, while Yes, a Src tyrosine kinase
related to cell adhesion and invasion, declines. Recon-
stitution of Yes in Hsp27 over-expressing cells by
transfection reduces MMP-9, signifying mediation of
Hsp27 effects by the Yes signaling cascade. Intrigu-
ingly, enhancing chondrocyte Hsp25 lowers growth
rate, modifies morphology, lessens adhesion and dis-
rupts differentiation, but leaves actin distribution unaf-
fected. These observations have implications for
metastatic potential as reduced adhesion leads to cell
release from tumors and spreading throughout the
organism [144].
sHSP induced drug resistance is of concern for
patients undergoing cancer chemotherapy [145,146].
Rat sarcoma cells exhibit less cell death than either
rat lymphoma or mouse breast carcinoma cells upon
treatment with the anticancer drugs doxorubicin and
lovastatin [132]. Among the three cancers, sarcoma
cells possess the most Hsp25, the rodent equivalent of
human Hsp27, and the protein builds up upon drug

These observations suggest sHSP utility as early diag-
nostic markers and therapeutic targets. Novel approa-
ches include the use of reagents that modify
chaperones structurally and functionally, the modula-
tion of signaling pathways regulating sHSP properties
such as phosphorylation, and changing the level of
sHSP synthesis [26].
Suppression of sHSPs indicating poor cancer prog-
nosis could be important for treatment. For example,
the down regulation of Hsp27 by interferon-c (IFN-c)
in oral squamous cell carcinoma lines enhances drug
effectiveness [134]. Hsp27 is thought to protect
against drug induced apoptosis and once either
removed or reduced by IFN-c exposure, cells gain
sensitivity to anticancer drugs such as cisplatin. The
importance of combination therapy consisting of
sHSP reduction and drug exposure is demonstrated,
however, INF-c induced lowering of Hsp27 may be
specific to oral squamous cell carcinomas, conse-
quently limiting this potential therapeutic approach.
The metabolite, pantethine, increases a-crystallin
chaperone activity and aids prevention of rat lens
opacification [26,151]. Other therapeutic possibilities
include alteration of cellular Ca
2+
balance through
membrane transport protein effectors and changing
sHSP function by nucleotide and anti-inflammatory
drug application [26]. SAPK2 ⁄ p38 kinase stimulation
leads to sHSP phosphorylation and oligomer size

heat shock transcription factor-1 (HSF-1) binds gene
promoters, presumably increasing HSPs and protecting
cells from protein misfolding. The macrocyclic antifun-
gal antibiotic, radicicol, induces HSP expression in
neonatal rat cardiomyocytes and shelters cells from the
effects of simulated ischemia [156]. Radicicol frees
HSF-1 from Hsp90. In contrast to many Hsp90 cli-
ents, liberated HSF-1 evades degradation, undergoes
activation and enhances HSP gene expression, thereby
inducing heat shock response. The HSPs increased
upon radicicol exposure of rat neonatal cardiomyo-
cytes are unknown, but protection from simulated isc-
hemia is independent of Hsp90 over-expression [156].
Stimulation of HSP synthesis by drug-induced disrup-
tion of Hsp90 may promote sHSP synthesis leading to
beneficial therapeutic effects.
sHSP delivery by gene therapy is being tested in
animal models and a catheter-based clinical approach
for infusion of adenoviral vectors has promise for
treatment of congestive heart failure [157]. In a proce-
dural variation, recombinant adeno-associated virus
vectors containing an extracellular superoxide dis-
mutase (SOD) are administered by intramyocardial
injection, yielding long lasting protection against isc-
hemia ⁄ reperfusion injury in rats [158]. Pre-emptive
gene therapy strategies, where SOD or other thera-
peutic proteins are produced in patients at high risk
for ischemic ⁄ reperfusion injury associated with coron-
ary artery disease and related chronic ailments, hold
medical potential.

Charot–Marie–Tooth disease Hsp25 S135F [128]
Hsp25 R136W [128]
Hsp22 K141N [126]
Small heat shock proteins and disease Y. Sun and T. H. MacRae
2620 FEBS Journal 272 (2005) 2613–2627 ª 2005 FEBS
recognition and initiating internal signaling cascades.
Many peptides generated by degradation of self and
nonself bind HSPs noncovalently, indicating cells of
origin and cause of destruction, while effectively sti-
mulating the immune system [159–164]. Tumor cell
HSPs and client proteins ⁄ peptides have been used to
synthesize oncophage vaccines, and when injected
into patients immune responses against cells contain-
ing HSP-associated proteins are promoted, an
approach that may facilitate cancer treatment. The
delivery of constitutively active HSF-1 enhances
tumor cell HSP expression and augments tumor
immunoantigenicity, perhaps by limiting phagocytosis
of apoptotic cells [161]. If HSF-1 is employed thera-
peutically only one gene must be introduced to effect
expression of several HSP genes, all with the capa-
city to enhance HSP synthesis and immunogenecity.
sHSPs have also been considered for delivery of
antigens and the design of vaccines directed against
protein targets in HIV infection [163]. The therapeu-
tic implications associated with HSPs, are provocat-
ive, and efforts to exploit molecular chaperones,
including the sHSPs, in disease amelioration are
underway.
Conclusions

Engineering Research Council of Canada Discovery
Grant, a Nova Scotia Health Research Founda-
tion ⁄ Canadian Institutes of Health Research Regional
Partnership Plan Grant, and a Heart and Stroke Foun-
dation of Nova Scotia Grant to THM and a NSHRF
Student Fellowship to YS.
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