Sick Chaperones, Cellular Stress, and Disease
http://www.100md.com
《新英格兰医药杂志》
Cells maintain a complete set of functionally competent proteins normally and in the face of injury or stress with the use of various mechanisms, including systems of proteins called molecular chaperones.1 The typical function of a chaperone is to assist a nascent polypeptide chain to attain a functional conformation as a new protein and then to assist the protein's arrival at the site in the cell where the protein carries out its functions. It has become increasingly clear that disruption of chaperoning mechanisms contributes to aging and disease. This review outlines the involvement of defective chaperones in senescence and in several diseases. Since chaperones are ubiquitous, their deficiencies and defects are bound to affect diverse tissues and, hence, to be of interest to those in internal medicine, ophthalmology, neurology, immunology, endocrinology, pediatrics, and gerontology.
Protein Quality Control and Antistress Mechanisms
Cellular Stress and Protein Damage
The stressors listed in Table 1 cause modifications of the intracellular milieu that are conducive to damage of DNA and proteins. They also induce the stress response; this response inhibits many housekeeping genes and activates stress genes, which are not transcribed or are transcribed at low levels in the absence of stress.2,3 As a result, the intracellular concentration of stress proteins, including chaperones, increases. Stress proteins can also increase in response to stress by mechanisms other than a boost in gene transcription; among these alternative mechanisms are increases in the stability of chaperone messenger RNA (mRNA) and chaperone proteins and the efficiency of translation of the nucleotide sequences of mRNA into the amino acids of polypeptides.
Table 1. Cell Stressors.
Stress to the cell also causes protein denaturation: the protein molecule loses its native functional conformation when it unfolds. Chaperones assist the damaged molecule to regain its functional conformation. If cellular stress proceeds unchecked by such antistress mechanisms as the protein-refolding action of chaperones, intracellular proteins become denatured and insoluble. These denatured proteins tend to stick to one another, precipitate, and form inclusion bodies. The development of inclusion bodies is a common pathologic process in Parkinson's, Alzheimer's, and Huntington's diseases, even in the absence of cellular stress.4,5,6,7,8,9 Denatured and aggregated proteins cannot function and must either be rescued or eliminated with the help of chaperones10,11,12,13,14,15,16,17,18 (Figure 1).
Figure 1. Diagram of the Life of a Protein.
Translation of messenger RNA (mRNA) occurs on the ribosome (1). Folding of the nascent polypeptide begins, assisted by chaperones (2). The polypeptide proceeds to post-translational folding (3). The new protein is correctly folded into its tertiary structure (4). Some proteins are translocated into organelles, such as mitochondria (5). Unfolded and misfolded polypeptides aggrgate (6) and precipitate in an inclusion body (7). Unfolded or partially folded polypeptides are freed from the aggregates by chaperones (8) and refolded (4) or degraded in the proteasome (9). Red arrows denote the pathway toward folding. The black dashed arrow denotes the pathway toward unfolding and aggregation initiated by cell stressors. The green arrow denotes the pathway toward degradation. The pathway from step 8 to step 4 is very active in protein-misfolding disorders and offers a convenient target for devising therapeutic strategies based on the administration of chaperones or chaperone genes.
Heat-Shock Proteins and Chaperones
Only a fraction of chaperones are encoded in genes that are inducible by stressors and thus belong to the large class of stress proteins.3 If the stressor is heat shock, the induced chaperones are named heat-shock proteins (HSPs).19 For historical reasons, the term "HSP" is used even if the parent gene is not induced by heat shock. Conversely, many HSPs are not chaperones. Therefore, these terms have to be used carefully to avoid misunderstandings.
Chaperones and HSPs are classified into groups according to phylogeny and structure20 or molecular mass in kilodaltons (a classifier useful for clinical laboratory analyses). For HSPs, the groups are as follows: high-molecular-mass HSPs (100 kD), HSP90 (81 to 99 kD), HSP70 (65 to 80 kD), HSP60 (55 to 64 kD), HSP40 (35 to 54 kD), and small HSPs (34 kD).9,21,22
Chaperoning Machines
A chaperone does not act alone but, rather, acts as a member of a team of various molecules, including chaperones and cochaperones. The cochaperones interact with chaperones, such as HSP70 and HSP90, and help them in their various roles; for example, the cochaperone nucleotide-exchange factor B-cell lymphoma 2–associated athanogene 1 (BAG-1) facilitates the ATP–ADP cycle that HSP70 undergoes as it assists protein folding. Chaperone–cochaperone complexes help nascent polypeptide chains along the folding pathway, refold damaged molecules, or direct them to proteases for degradation if they cannot be rescued.11,19,23,24,25,26,27,28,29,30,31,32,33,34 Distinctive functional domains in chaperone molecules recognize a polypeptide in need of assistance, interact with a teammate to build a chaperoning complex, and network with other chaperoning complexes or with a protein-degrading machine like the ubiquitin–proteasome system. Some chaperones bind ATP and possess ATPase activity.29,30,31,32,33,34
The main chaperoning teams of the cell are the molecular chaperone machine (consisting of HSP70, HSP40, and nucleotide-exchange factor); the chaperonin-containing tailless complex polypeptide 1 (TCP-1) complex of eight subunits (also called CCT; a chaperonin is a subtype of chaperones); the prefoldin chaperone with five subunits; the small HSP chaperones that form multimers of various sizes; and the Cpn60–Cpn10 complex of molecular chaperones, in which Cpn represents chaperonin (also known as HSP60–HSP10). The Cpn60–Cpn10 complex resides in the mitochondrion, whereas the others are in the cytosol, the nucleus, or other cellular compartments (Figure 2).9,21,22,23,24,32
Figure 2. Chaperoning Teams.
The chaperone machine is a team of three proteins: heat-shock protein (HSP) 70, HSP40, and nucleotide-exchange factor. HSP70 binds a client (unfolded or misfolded) polypeptide and, in collaboration with the other members of the team, assists the polypeptide to fold correctly and thus achieve its native conformation. Prefoldin consists of five distinct subunits arranged like a medusa. The mitochondrial chaperonin consists of two multimeric assemblages. The larger assemblage has 14 HSP60 subunits; the smaller one has 7 HSP10 subunits. The chaperonin-containing TCP-1 (tailless complex polypeptide 1) complex is similar in overall structure to the mitochondrial chaperonin, but eight distinct subunits (A through H) form each of its two rings. The small HSPs are chaperones, such as the -crystallins, that are normally monomers. In response to cellular stress, they form multimers that participate in the protection of unfolded polypeptides.
Chaperonopathies
Figure 1 shows what would happen if a mutation rendered a chaperone structurally defective and unable to function (genetic defect) or if post-transcriptional modification of the chaperone were aberrant (acquired defect). A genetic or acquired defect could affect one or more of the specialized chaperone domains. The manifestations of a chaperonopathy depend on the domain or function that is impaired or abolished.35,36,37,38
The cost of a defective chaperone to the cell depends on the functional range of the affected chaperone. Some chaperones assist only a narrow range of clients (i.e., polypeptides in need of assistance for folding or refolding), whereas other chaperones serve a wide array of polypeptides. The proteins known as cofactors A through E, for example, are dedicated chaperones involved specifically in tubulin folding and assembly. In contrast, HSP70 and HSP90 participate in numerous activities involving various molecules. A functional impairment of such nonspecific chaperones can affect a multiplicity of cellular and organismic processes.
Acquired Chaperonopathies
Acquired chaperonopathies are associated with post-translational modifications of the chaperone and usually become clinically evident late in life. They may, however, start subclinically in early childhood or young adulthood and then progress with aging.
Mechanisms of post-translational modifications of proteins are diverse. They include: oxidation of amino acids, deamidation, glycation, phosphorylation, acetylation, nitrosylation, and truncation of the protein (cleavage or deletion of N-terminal or C-terminal residues).39 During senescence, these modifications damage many proteins, including chaperones. As a result, functionally incompetent chaperones are unable to deal with an excessive demand for the repair of proteins.37,40,41 In the chaperonopathies that are associated with aging or disease, chaperones can be structurally altered, increased or decreased, or distributed abnormally in tissues and cells (Table 2).
Table 2. Examples of Chaperonopathies Associated with Aging and Disease.
It is not certain how these changes occur. Quantitative changes could be due to up-regulation or down-regulation of chaperone genes, mutation of these genes, or post-translational modifications of the chaperone itself. Heat-shock factors (HSF), which are the proteins that regulate chaperone-gene transcription,57,58 could also be altered by mutation, post-translational modifications, or both, resulting in quantitative changes in the production of chaperones. Sequestration in inclusion bodies can decrease the amount of chaperone molecules in the fluid phase of a cell compartment. The presence of chaperones in body fluids is sometimes associated with disease or senescence and with antichaperone autoantibodies (Table 3).
Table 3. Occurrence of HSPs and Chaperones and Autoantibodies against HSPs and Chaperones in Biologic Fluids.
Acquired Chaperonopathies Associated with Aging and Disease
Hsp70 and Hsp90 in Aging
The amount and distribution of HSP70 in various cells and tissues in relation to disease and aging (Table 2) have been widely studied.2,22,44,49,50,62,65,66,67,68 Heat shock of rat hepatocytes increased the levels of the chaperones heat-shock cognate protein 70 (HSC70) and HSP70, an effect that was attributed to increased transcription of the respective genes relative to the levels in cells maintained at 37°C.42 The increase in the levels of HSP70 was less in hepatocytes from old rats than in those from young-adult rats.
Similar studies were performed with cultures of early-passage and late-passage human lung fibroblasts and epidermal melanocytes and of skin fibroblasts taken from younger donors (27 to 44 years of age) and older donors (78 to 92 years of age).43 The levels of HSP70 protein in lung fibroblasts were lower in late-passage than in early-passage cells, and the heat-induced increase of HSP70 and its mRNA was lower in late-passage cells (Table 2). These results were confirmed in cultured melanocytes.
The basal levels of HSP70 were lower in cultures of fibroblasts from the older donors than in similar cultures from the younger donors.43 Likewise, the increase in HSP70 after heat shock was lower in fibroblasts from older donors than in those from their younger counterparts. Heat-shock factor 1 (HSF-1) was present in fibroblasts from the younger and older donors, but the levels in the older donors were lower43 (Table 2). The levels of the transcription factor HSF-1, a regulator of chaperone-gene expression, decreased as the number of passages increased for both fibroblast populations, suggesting that the amount of the transcription factor decreases as a cell ages, even in a cell from a younger donor. Similar observations were made in retinal cells44 (Table 2). HSC70 and its parent mRNA were present in smaller amounts in retinal cells from an 80-year-old donor than in cells from a 4-year-old child.
HSP90 levels and the chaperoning capacity of hepatocyte cytosol from older rats were found to be lower than those in hepatocytes from younger rats.48 This finding is interesting because a functional assay was used to evaluate chaperoning capacity directly in cells from young and old animals, and it showed that the levels of HSC70 and HSP70 in cells from young and old rats did not differ. Thus, this report contradicts others that have found a decrease in these two chaperones occurring with age.
A decrease in serum HSP70 levels was observed in a population of Chinese persons 30 to 50 years of age, as compared with persons 25 to 30 years of age62 (Table 3). The highest levels were found in persons 25 to 30 years of age. Likewise, a decrease in the HSP70 levels was found in the lymphocytes of subjects older than 40 years, relative to younger controls. These findings indicate that serum chaperones are potential biomarkers of health status, particularly in the elderly. We can conjecture, considering the multiple roles of chaperones, that people with low levels of chaperones in serum will have a more pronounced decline in vital functions with age than people with higher levels of chaperones. However, it is not yet certain why, or by what mechanism, chaperones — currently considered typical intracellular proteins — appear in serum and other biologic fluids (Table 3).
Perhaps the levels of chaperones in serum reflect the degree of cell-membrane damage accompanied by leakage of intracellular proteins, such as that in vascular endothelial cells in old people. In this circumstance, high chaperone levels in serum indicate poor health rather than good health. Clinical studies are necessary to clarify this critical point. To understand chaperones in disease and aging, it will be useful to determine whether their presence in biologic fluids is always the result of leakage from sick cells or is due to an active mechanism that fulfills a need for circulating chaperones.
Crystallins in Neurodegenerative Diseases, Myositis, Cataracts, and Retinopathy
Crystallins comprise proteins from several groups (, , and ) and subgroups (e.g., A, B, A1, A2, A3, A4, B1, B2, and B3) and occur in various tissues, not just in the lens of the eye.69,70,71 Only the -crystallins have chaperoning abilities.71,72,73,74,75,76,77,78,79,80,81,82,83 The B-crystallin gene is expressed constitutively in muscle, heart, and the lens of the eye but not in lymphoid tissue. However, expression occurs in lymphocytes under stress (e.g., viral infection).
Neurodegenerative Disease
Abnormal tissue distribution of B-crystallin, possibly due to aberrant gene expression in different areas of the brain, has been detected in the brains of patients with Alzheimer's disease.51 Also, pathologic deposition of B-crystallin has been found in the glial inclusions of tauopathies (neurodegenerative diseases with tangled tau, a microtubule-associated protein)55 (Table 2). These abnormalities in the distribution and quantity of chaperones reflect attempts by the chaperoning systems to correct the folding defects inherent in the abnormal proteins in Alzheimer's disease and other neurodegenerative disorders.4,5,6,7,8 In these disorders, also called protein-misfolding diseases, the hallmark is the occurrence of molecules with a tendency to misfold and to precipitate — for example, -amyloid in Alzheimer's disease, -synuclein in Parkinson's disease, and huntingtin in Huntington's disease.4,5,6,7,8,22,84 These abnormal proteins are targeted by chaperones for refolding (Figure 1). If the tendency of the abnormal proteins to misfold and precipitate is overwhelming because of critical structural defects in them and because of the large quantity of these abnormal molecules, the chaperones, though capable of binding the defective proteins, are ineffective in assisting them to refold, and the chaperones precipitate together with the abnormal proteins.6,22,49,50,66
Inclusion-Body Myositis
In inclusion-body myositis, the commonest myopathy in persons older than 50 years of age,84 B-crystallin has been found to be increased in muscle fibers, whether inclusion bodies are present or absent52 (Table 2). These observations suggest that an increase in B-crystallin precedes the formation of the inclusion bodies and could be a result of overexpression of the B-crystallin gene in response to a stressor that is still to be identified.
Cataracts
The transparency and refractive capacity of the lens depend on the short-range interactions, concentration, and configuration of water-soluble proteins, the crystallins, within the fiber cells of the lens.69 Aging entails post-translational modifications of crystallins, including progressive loss of solubility and a tendency to aggregate that results in lens opacity.69 Although there is no evidence that the - and -crystallins function as chaperones, they are discussed together with the chaperones -crystallins because of their roles in maintaining transparency of the lens and because they all contribute to lens opacity if damaged.38,69,85,86,87
-Crystallins have two domains and variable N-terminal and C-terminal extensions.69,87 These extensions probably stabilize the -crystallin oligomers normally found in the lens, serve as spacers for preventing excessive polymerization, and regulate short-range, nonspecific protein interactions. During aging, the extensions undergo proteolysis, a likely cause of lens opacification.87 The chaperoning capacity of the -crystallins diminishes with aging (Table 2), just when they are most needed to stabilize the other aging crystallins.69,73 The extent of this acquired, age-related chaperoning deficiency is comparable to the deficiency caused by -crystallin mutations in hereditary cataracts.38,69,88
Retinopathy
An age-related decrease in the expression of A-crystallin and post-translational modifications of the protein (such as phosphorylation, acetylation or glycation of lysine, deamidation of asparagine or glutamine, and glutathionylation of cysteine) occur in the retina45 (Table 2). These modifications alter the charge distribution across the molecule, impair the chaperone function of A-crystallin, and promote aggregation of the chaperone; all these phenomena contribute to the development of age-related retinopathy.
Truncation is an additional modification of A-crystallin in the lens and retina associated with aging.45 In the lens, a truncation occurs at the C-terminal; 1 or more residues (up to 22) are deleted. In the retina, C-terminal truncations have been detected in young-adult and middle-aged rats, but not in old rats.45 In contrast, N-terminal truncations were present in the retinal A-crystallin from all three age groups and were most frequent in the oldest animals.
Other Pathologic Conditions Involving Chaperones
Von Hippel–Lindau Disease
One function of chaperones is the interaction with other chaperones to form multimolecular chaperoning machines; these, in turn, may assist in the assembly of other multimeric complexes with diverse functions. If the client molecule that the chaperone machine recognizes lacks a chaperone-recognition site (e.g., because of a mutation), the assembly process will not start, and the multimeric complex will not be built. An example is the chaperone complex TCP-1 that mediates the assembly of the von Hippel–Lindau (VHL) tumor suppressor protein with elongin B and elongin C.89 The assemblage VHL-elongin BC interacts with the suppressor of kinetochore protein 1 (Skp1)–Cullin 1 (Cul1)–F box (SCF) ubiquitin ligase complex to promote the destruction of a set of proteins necessary for vascularization and growth of tumors.89,90 If the entire multimolecular structure is not assembled correctly, the tumor-suppressor activity of VHL is lost, allowing the development and growth of tumors.
The VHL protein has a 55-amino-acid segment for CCT binding. A mutation in or deletion of this VHL segment is associated with von Hippel–Lindau disease, in which pheochromocytoma, renal carcinoma, and densely vascularized retinal and cerebellar tumors develop.90 Two mutations in the segment of the VHL protein that is the target of CCT recognition interfere with the binding of CCT and, consequently, the folding of the VHL protein.89
Autoantibodies against Chaperones
HSPs and molecular chaperones are immunogenic. Structural alterations in a chaperone caused by mutation or post-translational modification can transform it into an autoantigen, which can elicit an autoimmune response if the altered chaperone gains access to the immune system. It is possible that antichaperone antibodies cross-react with normal antigens. During senescence, a mechanism involving abnormal chaperones that leak into the extracellular space and circulation and evoke antichaperone autoantibodies that cause tissue damage is thought to develop and to contribute to the general functional decline accompanying aging59,60 (Table 3). Anti-HSP47 antibodies were found to be elevated in serum from patients with systemic lupus erythematosus and other autoimmune disorders (Table 3), but not in serum from patients with a nonautoimmune disease.61
These and other findings point to the need to determine the prevalence and pathogenetic roles of antichaperone autoantibodies. Antichaperone autoantibodies may be involved in the pathogenesis of autoimmune diseases by cross-reactions with other molecules61,63 and could contribute to the development of diseases that are not predominantly autoimmune but that have an autoimmune component, such as inflammatory bowel diseases.91 Such antibodies could also disrupt chaperoning mechanisms if they form a complex with a chaperone molecule and interfere with its functions.59,60,61,62,63,64,91 Interference with chaperone functions could also be caused by antibodies against a foreign (microbial) chaperone that cross-reacts with the human counterpart.92 Even though the likelihood of chaperone–antibody complex formation is low because chaperones function mainly intracellularly, exploring the potential of antichaperone autoantibodies as markers of disease progression and senescence would be worthwhile. After the pathogenetic role of antichaperone antibodies is established, it should become possible to develop strategies for treatment (e.g., immunosuppression).
Chaperonopathies and Malfunction of the Immune System
Chaperones are involved in various stages of the immune response: initiation of proinflammatory cytokine production, antigen recognition and processing, activation of antigen-presenting cells, and chaperoning of peptides during antigen presentation.91,92,93,94 The molecular mechanisms by which HSP70 participates in the immune response have not been elucidated. It is clear, however, that it binds viral, bacterial, and tumor peptides through its peptide-binding domain near the middle of the molecule and makes the peptides immunogenic; HSP70 has adjuvant properties.91,92,93,94 Other effects of HSP70 on the immune system, such as stimulation of interleukin 12, tumor necrosis factor , and nitric oxide production, are mediated by its C-terminal domain (consisting of 10 amino acids).94 It follows that if the peptide-binding domain or the C-terminal domain of the chaperone is impaired because of mutation or age-related post-translational modifications, immune responses will be defective.
In a rat model, exposure to stressors was shown to increase the extracellular levels of HSP72, presumably signaling the immune system to mount a response against a bacterial pathogen.95 How this signaling occurs was not ascertained in that study, but it was shown that stimulation of splenocytes and macrophages with HSP72 increased the levels of nitric oxide, tumor necrosis factor , interleukin-1, and interleukin-6. These observations suggested that extracellular HSP72 triggers the production of nitric oxide along with the immune response to bacterial antigens. Thus, if HSP72 is defective, the immune response to microbial pathogens will be adversely affected.
Chaperone-Gene Polymorphisms
Chaperone-gene polymorphisms are promising predictors of longevity because of the key roles of chaperones in cell physiology and antistress mechanisms. The frequency of allele (A ) –110 (the A/A genotype) in the HSP70-1 promoter region was decreased among long-lived women in southern Italy96 (Table 4). In that study, the A/A genotype was thought to contribute to a functional state unfavorable to longevity in women, perhaps in relation to female hormones. However, a similar study in another group, from Denmark, showed that the A/A genotype was more prevalent among persons with higher self-rated health scores, who are predicted to live longer, and no sex-specific association was observed97 (Table 4).
Table 4. Polymorphisms of Human HSP (Molecular Chaperone) Genes with Possible Effects on Longevity, the Risk of Disease, or Both.
There are three HSP70 genes within the HLA class III region.98 One of these, HSP70-HOM , is not heat-inducible, protects from oxidative stress, and exhibits a functional polymorphism (a methionine-to-threonine substitution at position 493 of the HSP70-Hom protein) (Table 4). A comparative study revealed that the T/T genotype (encoding methionine) was more frequent among older than among younger subjects, whereas the reverse held true for the T/C genotype (encoding threonine).98 These results show that methionine at HSP70-Hom position 493 is accompanied by an increase in the probability of living a long life. Position 493 is in the peptide-binding domain of the chaperone. Peptide binding is optimal when this position is occupied by methionine (a nonpolar and hydrophobic amino acid), but it is impaired when methionine is replaced by threonine (a polar and neutral amino acid), which results in chaperone malfunction. The adverse effect of this malfunction can be predicted to be widespread, affecting many cellular processes, such as the response to stressors, and allowing the accumulation of defective proteins, particularly those with post-translational modifications that increase with aging. These effects would be cumulative over a period of time and finally cause a shortening of the life span. This type of chaperonopathy is a good candidate for replacement therapy with, for example, recombinant HSP70-Hom with the optimal amino acid at position 493 or its gene (i.e., gene therapy).
The association of polymorphisms with Parkinson's disease was investigated by genotyping DNA samples obtained from peripheral-blood leukocytes99 (Table 4). No association with the disease was detected among the polymorphisms in the coding regions, with one possible exception. The frequencies of the HSP70-1 –110CC and +190CC genotypes were higher among the patients with Parkinson's disease than among the controls; in in vitro assays, –110A was a better HSP70-1 transcriptional inducer than –110C. The same patterns were observed in heat-shocked and nonstressed cells.
Genetic Chaperonopathies
At least 15 conditions have been reported that are associated with mutations in genes encoding chaperones or chaperone-like molecules (Table 5). These conditions have been reviewed elsewhere and can be considered "inborn errors of development,"38 a term coined to designate collectively human malformations associated with gene mutations.100
Table 5. Genetic Chaperonopathies.
Prospects for the Future
The way is open for basic, applied, and clinical investigations directed at discovering new chaperonopathies, expanding knowledge about those already identified, and elucidating the mechanisms by which defective chaperones contribute to disease. Chaperones are so universally distributed and perform so many different functions that the probability is high that they are involved in the initiation and progression of disease. Exciting challenges for the next few years will be to determine the prevalence and full extent of medical consequences of chaperonopathies, to standardize diagnostic tests, and to develop strategies for prevention and treatment.
We are indebted to Tara Garcia-Collins and Brian W. Meneghan for their help with the comparative and phylogenetic analyses of human genes, to Phil Grimley and Alex Macario for their critical reading of the manuscript.
Source Information
From the Wadsworth Center, Division of Molecular Medicine, New York State Department of Health (A.J.L.M., E.C.M.); and the Department of Biomedical Sciences, School of Public Health, State University of New York at Albany (A.J.L.M.) — both in Albany.
Address reprint requests to Dr. A.J.L. Macario at the Wadsworth Center, Room B-749, Division of Molecular Medicine, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, or at macario@wadsworth.org.
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Kapphahn RJ, Ethen CM, Peters EA, Higgins LA, Ferrington DA. Modified alpha A crystallin in the retina: altered expression and truncation with aging. Biochemistry 2003;42:15310-15325.
Ma ZX, Hanson SRA, Lampi KJ, David LL, Smith DL, Smith JB. Age-related changes in human lens crystallins identified by HPLC and mass spectrometry. Exp Eye Res 1998;67:21-30.
Fonager J, Beedholm R, Clark BF, Rattan SI. Mild stress-induced stimulation of heat-shock protein synthesis and improved functional ability of human fibroblasts undergoing aging in vitro. Exp Gerontol 2002;37:1223-1228.
Nardai G, Csermely P, S?ti C. Chaperone function and chaperone overload in the aged: a preliminary analysis. Exp Gerontol 2002;37:1257-1262.
Hamos JE, Oblas B, Pulaski-Salo D, Welch WJ, Bole DG, Drachman DA. Expression of heat shock proteins in Alzheimer's disease. Neurology 1991;41:345-350.
Perez N, Sugar J, Charya S, et al. Increased synthesis and accumulation of heat shock 70 proteins in Alzheimer's disease. Brain Res Mol Brain Res 1991;11:249-254.
Yoo BC, Kim SH, Cairns N, Fountoulakis M, Lubec G. Deranged expression of molecular chaperones in brains of patients with Alzheimer's disease. Biochem Biophys Res Commun 2001;280:249-258.
Banwell BL, Engel AG. Alpha-B-crystallin immunolocalization yields new insights into inclusion body myositis. Neurology 2000;54:1033-1041.
Zabel C, Chamrad DC, Priller J, et al. Alterations in the mouse and human proteome caused by Huntington's disease. Mol Cell Proteomics 2002;1:366-375.
Hay DG, Sathasivam K, Tobaben S, et al. Progressive decrease in chaperone protein levels in a mouse model of Huntington's disease and induction of stress proteins as a therapeutic approach. Hum Mol Genet 2004;13:1389-1405.
Dabir DV, Trojanowski JQ, Richter-Landsberg C, Lee VML, Forman MS. Expression of the small heat-shock protein alpha-B-crystallin in tauopathies with glial pathology. Am J Pathol 2004;164:155-166.
Maatkamp A, Vlug A, Haasdijk E, Troost D, French PJ, Jaarsma D. Decrease of Hsp25 protein expression precedes degeneration of motoneurons in ALS-SOD1 mice. Eur J Neurosci 2004;20:14-28.
Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 1998;12:3788-3796.
Pirkkala L, Nykanen P, Sistonen L. Roles of the heat shock transcription factors in the regulation of the heat shock response and beyond. FASEB J 2001;15:1118-1131.
Pockley AG, Bulmer J, Hanks BM, Wright BH. Identification of human heat shock protein 60 (Hsp60) and anti-Hsp60 antibodies in the peripheral circulation of normal individuals. Cell Stress Chaperones 1999;4:29-35.
Rea IM, McNerlan S, Pockley AG. Serum heat shock protein and anti-heat shock protein antibody levels in aging. Exp Gerontol 2001;36:341-352.
Yokota S, Kubota H, Matsuoka Y, et al. Prevalence of HSP47 antigen and autoantibodies to HSP47 in the sera of patients with mixed connective tissue disease. Biochem Biophys Res Commun 2003;303:413-418.
Jin X, Wang R, Xiao C, et al. Serum and lymphocyte levels of heat shock protein 70 in aging: a study in the normal Chinese population. Cell Stress Chaperones 2004;9:69-75.
Yang M, Zheng J, Yang Q, et al. Frequency-specific association of antibodies against heat shock proteins 60 and 70 with noise-induced hearing loss in Chinese workers. Cell Stress Chaperones 2004;9:207-213.
Zhang J, Goodlett DR, Peskind ER, et al. Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid. Neurobiol Aging 2005;26:207-227.
Jin X, Xiao C, Tanguay RM, et al. Correlation of lymphocyte heat shock protein 70 levels with neurologic deficits in elderly patients with cerebral infarction. Am J Med 2004;117:406-411.
Macario AJL. Heat-shock proteins and molecular chaperones: implications for pathogenesis, diagnostics, and therapeutics. Int J Clin Lab Res 1995;25:59-70.
Snoeckx LH, Cornelussen RN, van Nieuwenhoven FA, Reneman RS, Van Der Vusse GJ. Heat shock proteins and cardiovascular pathophysiology. Physiol Rev 2001;81:1461-1497.
Macario AJL, Conway de Macario E. Stress and molecular chaperones in disease. Int J Clin Lab Res 2000;30:49-66.
Bloemendal H, de Jong W, Jaenicke R, Lubsen NH, Slingsby C, Tardieu A. Ageing and vision: structure, stability and function of lens crystallins. Prog Biophys Mol Biol 2004;86:407-485.
Iwaki T, Kime-Iwaki A, Liem RKH, Goldman JE. Alpha-B-crystallin is expressed in non-lenticular tissues and accumulates in Alexander's disease brain. Cell 1989;57:71-78.
Iwaki T, Iwaki A, Tateishi J, Sakaki Y, Goldman JE. Alpha-B-crystallin and 27-kd heat-shock protein are regulated by stress conditions in the central nervous system and accumulate in Rosenthal fibers. Am J Pathol 1993;143:487-495.
Horwitz J. Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 1992;89:10449-10453.
Cherian M, Abraham EC. Decreased molecular chaperone property of alpha-crystallins due to posttranslational modifications. Biochem Biophys Res Commun 1995;208:675-679.
Andley UP, Mathur S, Griest TA, Petrash JM. Cloning, expression, and chaperone-like activity of human alphaA-crystallin. J Biol Chem 1996;271:31973-31980.
Djabali K, de Nechaud B, Landon F, Portier MM. AlphaB-crystallin interacts with intermediate filaments in response to stress. J Cell Sci 1997;110:2759-2769.
Lindner RA, Kapur A, Mariani M, Titmuss SJ, Carver JA. Structural alterations of alpha-crystallin during its chaperone action. Eur J Biochem 1998;258:170-183.
Koyama Y, Goldman JE. Formation of GFAP cytoplasmic inclusions in astrocytes and their disaggregation by alphaB-crystallin. Am J Pathol 1999;154:1563-1572.
Muchowski PJ, Valdez MM, Clark JL. AlphaB-crystallin selectively targets intermediate filament proteins during thermal stress. Invest Ophthalmol Vis Sci 1999;40:951-958.
Cobb BA, Petrash JM. Alpha-crystallin chaperone-like activity and membrane binding in age-related cataracts. Biochemistry 2002;41:483-490.
Narberhaus F. Alpha-crystallin-type heat-shock proteins: socializing minichaperones in the context of a multichaperone network. Microbiol Mol Biol Rev 2002;66:64-93.
Derham BK, Ellory JC, Bron AJ, Harding JJ. The molecular chaperone alpha-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress. Eur J Biochem 2003;270:2605-2611.
Liang JJ-N. Interactions and chaperone function of alphaA-crystallin with T5P gammaC-crystallin mutant. Protein Sci 2004;13:2476-2482.
Rekas A, Adda CG, Aquilina JA, et al. Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity. J Mol Biol 2004;340:1167-1183.
Askanas V, Engel WK. Proposed pathogenetic cascade of inclusion-body myositis: importance of amyloid-beta, misfolded proteins, predisposing genes, and aging. Curr Opin Rheumatol 2003;15:737-744.
Brady JP, Garland D, Duglas-Tabor Y, Robison WG Jr, Groome A, Wawrousek EF. Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin. Proc Natl Acad Sci U S A 1997;94:884-889.
Heon E, Priston M, Schorderet DF, et al. The gamma-crystallins and human cataracts: a puzzle made clearer. Am J Hum Genet 1999;65:1261-1267.
Werten PJL, Vos E, de Jong WW. Truncation of beta3/A1-crystallin during aging of the bovine lens: possible implications for lens optical quality. Exp Eye Res 1999;68:99-103.
Cobb BA, Petrash JM. Structural and functional changes in the alpha A-crystallin R116C mutant in hereditary cataracts. Biochemistry 2000;39:15791-15798.
Feldman DE, Thulasiraman V, Ferreyra RG, Frydman J. Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol Cell 1999;4:1051-1061.
Maher ER. VHL and von Hippel-Lindau disease. In: Epstein CJ, Erickson RP, Wynshaw-Boris A, eds. Inborn errors of development. Oxford, England: Oxford University Press, 2004:823-7.
Kumaraguru U, Gouffon CA Jr, Ivey RA III, Rouse BT, Bruce BD. Antigenic peptides complexed to phylogenically diverse HSP70s induce differential immune responses. Cell Stress Chaperones 2003;8:134-143.
Kaufmann SHE, Schoel B. Heat shock proteins as antigens in immunity against infection and self. In: Morimoto RI, Tissieres A, Georgopoulos C, eds. The biology of heat shock proteins and molecular chaperones. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, 1994:495-531.
Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu Rev Immunol 2002;20:395-425.
Lehner T, Wang Y, Whittall T, McGowan E, Kelly CG, Singh M. Functional domains of HSP70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity. Biochem Soc Trans 2004;32:629-632.
Campisi J, Leem TH, Fleshner M. Stress-induced extracellular Hsp72 is a functionally significant danger signal to the immune system. Cell Stress Chaperones 2003;8:272-286.
Altomare K, Greco V, Bellizzi D, et al. The allele (A)(-110) in the promoter region of the HSP70-1 gene is unfavorable to longevity in women. Biogerontology 2003;4:215-220.
Singh R, Kolvraa S, Bross P, et al. Association between low self-rated health and heterozygosity for -110A > C polymorphism in the promoter region of HSP70-1 in aged Danish twins. Biogerontology 2004;5:169-176.
Ross OA, Curran MD, Crum KA, Rea IM, Barnett YA, Middleton D. Increased frequency of the 2437T allele of the heat shock protein 70-Hom gene in an aged Irish population. Exp Gerontol 2003;38:561-565.
Wu Y-R, Wang C-K, Chen C-M, et al. Analysis of heat-shock protein 70 gene polymorphisms and the risk of Parkinson's disease. Hum Genet 2004;114:236-241.
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Protein Quality Control and Antistress Mechanisms
Cellular Stress and Protein Damage
The stressors listed in Table 1 cause modifications of the intracellular milieu that are conducive to damage of DNA and proteins. They also induce the stress response; this response inhibits many housekeeping genes and activates stress genes, which are not transcribed or are transcribed at low levels in the absence of stress.2,3 As a result, the intracellular concentration of stress proteins, including chaperones, increases. Stress proteins can also increase in response to stress by mechanisms other than a boost in gene transcription; among these alternative mechanisms are increases in the stability of chaperone messenger RNA (mRNA) and chaperone proteins and the efficiency of translation of the nucleotide sequences of mRNA into the amino acids of polypeptides.
Table 1. Cell Stressors.
Stress to the cell also causes protein denaturation: the protein molecule loses its native functional conformation when it unfolds. Chaperones assist the damaged molecule to regain its functional conformation. If cellular stress proceeds unchecked by such antistress mechanisms as the protein-refolding action of chaperones, intracellular proteins become denatured and insoluble. These denatured proteins tend to stick to one another, precipitate, and form inclusion bodies. The development of inclusion bodies is a common pathologic process in Parkinson's, Alzheimer's, and Huntington's diseases, even in the absence of cellular stress.4,5,6,7,8,9 Denatured and aggregated proteins cannot function and must either be rescued or eliminated with the help of chaperones10,11,12,13,14,15,16,17,18 (Figure 1).
Figure 1. Diagram of the Life of a Protein.
Translation of messenger RNA (mRNA) occurs on the ribosome (1). Folding of the nascent polypeptide begins, assisted by chaperones (2). The polypeptide proceeds to post-translational folding (3). The new protein is correctly folded into its tertiary structure (4). Some proteins are translocated into organelles, such as mitochondria (5). Unfolded and misfolded polypeptides aggrgate (6) and precipitate in an inclusion body (7). Unfolded or partially folded polypeptides are freed from the aggregates by chaperones (8) and refolded (4) or degraded in the proteasome (9). Red arrows denote the pathway toward folding. The black dashed arrow denotes the pathway toward unfolding and aggregation initiated by cell stressors. The green arrow denotes the pathway toward degradation. The pathway from step 8 to step 4 is very active in protein-misfolding disorders and offers a convenient target for devising therapeutic strategies based on the administration of chaperones or chaperone genes.
Heat-Shock Proteins and Chaperones
Only a fraction of chaperones are encoded in genes that are inducible by stressors and thus belong to the large class of stress proteins.3 If the stressor is heat shock, the induced chaperones are named heat-shock proteins (HSPs).19 For historical reasons, the term "HSP" is used even if the parent gene is not induced by heat shock. Conversely, many HSPs are not chaperones. Therefore, these terms have to be used carefully to avoid misunderstandings.
Chaperones and HSPs are classified into groups according to phylogeny and structure20 or molecular mass in kilodaltons (a classifier useful for clinical laboratory analyses). For HSPs, the groups are as follows: high-molecular-mass HSPs (100 kD), HSP90 (81 to 99 kD), HSP70 (65 to 80 kD), HSP60 (55 to 64 kD), HSP40 (35 to 54 kD), and small HSPs (34 kD).9,21,22
Chaperoning Machines
A chaperone does not act alone but, rather, acts as a member of a team of various molecules, including chaperones and cochaperones. The cochaperones interact with chaperones, such as HSP70 and HSP90, and help them in their various roles; for example, the cochaperone nucleotide-exchange factor B-cell lymphoma 2–associated athanogene 1 (BAG-1) facilitates the ATP–ADP cycle that HSP70 undergoes as it assists protein folding. Chaperone–cochaperone complexes help nascent polypeptide chains along the folding pathway, refold damaged molecules, or direct them to proteases for degradation if they cannot be rescued.11,19,23,24,25,26,27,28,29,30,31,32,33,34 Distinctive functional domains in chaperone molecules recognize a polypeptide in need of assistance, interact with a teammate to build a chaperoning complex, and network with other chaperoning complexes or with a protein-degrading machine like the ubiquitin–proteasome system. Some chaperones bind ATP and possess ATPase activity.29,30,31,32,33,34
The main chaperoning teams of the cell are the molecular chaperone machine (consisting of HSP70, HSP40, and nucleotide-exchange factor); the chaperonin-containing tailless complex polypeptide 1 (TCP-1) complex of eight subunits (also called CCT; a chaperonin is a subtype of chaperones); the prefoldin chaperone with five subunits; the small HSP chaperones that form multimers of various sizes; and the Cpn60–Cpn10 complex of molecular chaperones, in which Cpn represents chaperonin (also known as HSP60–HSP10). The Cpn60–Cpn10 complex resides in the mitochondrion, whereas the others are in the cytosol, the nucleus, or other cellular compartments (Figure 2).9,21,22,23,24,32
Figure 2. Chaperoning Teams.
The chaperone machine is a team of three proteins: heat-shock protein (HSP) 70, HSP40, and nucleotide-exchange factor. HSP70 binds a client (unfolded or misfolded) polypeptide and, in collaboration with the other members of the team, assists the polypeptide to fold correctly and thus achieve its native conformation. Prefoldin consists of five distinct subunits arranged like a medusa. The mitochondrial chaperonin consists of two multimeric assemblages. The larger assemblage has 14 HSP60 subunits; the smaller one has 7 HSP10 subunits. The chaperonin-containing TCP-1 (tailless complex polypeptide 1) complex is similar in overall structure to the mitochondrial chaperonin, but eight distinct subunits (A through H) form each of its two rings. The small HSPs are chaperones, such as the -crystallins, that are normally monomers. In response to cellular stress, they form multimers that participate in the protection of unfolded polypeptides.
Chaperonopathies
Figure 1 shows what would happen if a mutation rendered a chaperone structurally defective and unable to function (genetic defect) or if post-transcriptional modification of the chaperone were aberrant (acquired defect). A genetic or acquired defect could affect one or more of the specialized chaperone domains. The manifestations of a chaperonopathy depend on the domain or function that is impaired or abolished.35,36,37,38
The cost of a defective chaperone to the cell depends on the functional range of the affected chaperone. Some chaperones assist only a narrow range of clients (i.e., polypeptides in need of assistance for folding or refolding), whereas other chaperones serve a wide array of polypeptides. The proteins known as cofactors A through E, for example, are dedicated chaperones involved specifically in tubulin folding and assembly. In contrast, HSP70 and HSP90 participate in numerous activities involving various molecules. A functional impairment of such nonspecific chaperones can affect a multiplicity of cellular and organismic processes.
Acquired Chaperonopathies
Acquired chaperonopathies are associated with post-translational modifications of the chaperone and usually become clinically evident late in life. They may, however, start subclinically in early childhood or young adulthood and then progress with aging.
Mechanisms of post-translational modifications of proteins are diverse. They include: oxidation of amino acids, deamidation, glycation, phosphorylation, acetylation, nitrosylation, and truncation of the protein (cleavage or deletion of N-terminal or C-terminal residues).39 During senescence, these modifications damage many proteins, including chaperones. As a result, functionally incompetent chaperones are unable to deal with an excessive demand for the repair of proteins.37,40,41 In the chaperonopathies that are associated with aging or disease, chaperones can be structurally altered, increased or decreased, or distributed abnormally in tissues and cells (Table 2).
Table 2. Examples of Chaperonopathies Associated with Aging and Disease.
It is not certain how these changes occur. Quantitative changes could be due to up-regulation or down-regulation of chaperone genes, mutation of these genes, or post-translational modifications of the chaperone itself. Heat-shock factors (HSF), which are the proteins that regulate chaperone-gene transcription,57,58 could also be altered by mutation, post-translational modifications, or both, resulting in quantitative changes in the production of chaperones. Sequestration in inclusion bodies can decrease the amount of chaperone molecules in the fluid phase of a cell compartment. The presence of chaperones in body fluids is sometimes associated with disease or senescence and with antichaperone autoantibodies (Table 3).
Table 3. Occurrence of HSPs and Chaperones and Autoantibodies against HSPs and Chaperones in Biologic Fluids.
Acquired Chaperonopathies Associated with Aging and Disease
Hsp70 and Hsp90 in Aging
The amount and distribution of HSP70 in various cells and tissues in relation to disease and aging (Table 2) have been widely studied.2,22,44,49,50,62,65,66,67,68 Heat shock of rat hepatocytes increased the levels of the chaperones heat-shock cognate protein 70 (HSC70) and HSP70, an effect that was attributed to increased transcription of the respective genes relative to the levels in cells maintained at 37°C.42 The increase in the levels of HSP70 was less in hepatocytes from old rats than in those from young-adult rats.
Similar studies were performed with cultures of early-passage and late-passage human lung fibroblasts and epidermal melanocytes and of skin fibroblasts taken from younger donors (27 to 44 years of age) and older donors (78 to 92 years of age).43 The levels of HSP70 protein in lung fibroblasts were lower in late-passage than in early-passage cells, and the heat-induced increase of HSP70 and its mRNA was lower in late-passage cells (Table 2). These results were confirmed in cultured melanocytes.
The basal levels of HSP70 were lower in cultures of fibroblasts from the older donors than in similar cultures from the younger donors.43 Likewise, the increase in HSP70 after heat shock was lower in fibroblasts from older donors than in those from their younger counterparts. Heat-shock factor 1 (HSF-1) was present in fibroblasts from the younger and older donors, but the levels in the older donors were lower43 (Table 2). The levels of the transcription factor HSF-1, a regulator of chaperone-gene expression, decreased as the number of passages increased for both fibroblast populations, suggesting that the amount of the transcription factor decreases as a cell ages, even in a cell from a younger donor. Similar observations were made in retinal cells44 (Table 2). HSC70 and its parent mRNA were present in smaller amounts in retinal cells from an 80-year-old donor than in cells from a 4-year-old child.
HSP90 levels and the chaperoning capacity of hepatocyte cytosol from older rats were found to be lower than those in hepatocytes from younger rats.48 This finding is interesting because a functional assay was used to evaluate chaperoning capacity directly in cells from young and old animals, and it showed that the levels of HSC70 and HSP70 in cells from young and old rats did not differ. Thus, this report contradicts others that have found a decrease in these two chaperones occurring with age.
A decrease in serum HSP70 levels was observed in a population of Chinese persons 30 to 50 years of age, as compared with persons 25 to 30 years of age62 (Table 3). The highest levels were found in persons 25 to 30 years of age. Likewise, a decrease in the HSP70 levels was found in the lymphocytes of subjects older than 40 years, relative to younger controls. These findings indicate that serum chaperones are potential biomarkers of health status, particularly in the elderly. We can conjecture, considering the multiple roles of chaperones, that people with low levels of chaperones in serum will have a more pronounced decline in vital functions with age than people with higher levels of chaperones. However, it is not yet certain why, or by what mechanism, chaperones — currently considered typical intracellular proteins — appear in serum and other biologic fluids (Table 3).
Perhaps the levels of chaperones in serum reflect the degree of cell-membrane damage accompanied by leakage of intracellular proteins, such as that in vascular endothelial cells in old people. In this circumstance, high chaperone levels in serum indicate poor health rather than good health. Clinical studies are necessary to clarify this critical point. To understand chaperones in disease and aging, it will be useful to determine whether their presence in biologic fluids is always the result of leakage from sick cells or is due to an active mechanism that fulfills a need for circulating chaperones.
Crystallins in Neurodegenerative Diseases, Myositis, Cataracts, and Retinopathy
Crystallins comprise proteins from several groups (, , and ) and subgroups (e.g., A, B, A1, A2, A3, A4, B1, B2, and B3) and occur in various tissues, not just in the lens of the eye.69,70,71 Only the -crystallins have chaperoning abilities.71,72,73,74,75,76,77,78,79,80,81,82,83 The B-crystallin gene is expressed constitutively in muscle, heart, and the lens of the eye but not in lymphoid tissue. However, expression occurs in lymphocytes under stress (e.g., viral infection).
Neurodegenerative Disease
Abnormal tissue distribution of B-crystallin, possibly due to aberrant gene expression in different areas of the brain, has been detected in the brains of patients with Alzheimer's disease.51 Also, pathologic deposition of B-crystallin has been found in the glial inclusions of tauopathies (neurodegenerative diseases with tangled tau, a microtubule-associated protein)55 (Table 2). These abnormalities in the distribution and quantity of chaperones reflect attempts by the chaperoning systems to correct the folding defects inherent in the abnormal proteins in Alzheimer's disease and other neurodegenerative disorders.4,5,6,7,8 In these disorders, also called protein-misfolding diseases, the hallmark is the occurrence of molecules with a tendency to misfold and to precipitate — for example, -amyloid in Alzheimer's disease, -synuclein in Parkinson's disease, and huntingtin in Huntington's disease.4,5,6,7,8,22,84 These abnormal proteins are targeted by chaperones for refolding (Figure 1). If the tendency of the abnormal proteins to misfold and precipitate is overwhelming because of critical structural defects in them and because of the large quantity of these abnormal molecules, the chaperones, though capable of binding the defective proteins, are ineffective in assisting them to refold, and the chaperones precipitate together with the abnormal proteins.6,22,49,50,66
Inclusion-Body Myositis
In inclusion-body myositis, the commonest myopathy in persons older than 50 years of age,84 B-crystallin has been found to be increased in muscle fibers, whether inclusion bodies are present or absent52 (Table 2). These observations suggest that an increase in B-crystallin precedes the formation of the inclusion bodies and could be a result of overexpression of the B-crystallin gene in response to a stressor that is still to be identified.
Cataracts
The transparency and refractive capacity of the lens depend on the short-range interactions, concentration, and configuration of water-soluble proteins, the crystallins, within the fiber cells of the lens.69 Aging entails post-translational modifications of crystallins, including progressive loss of solubility and a tendency to aggregate that results in lens opacity.69 Although there is no evidence that the - and -crystallins function as chaperones, they are discussed together with the chaperones -crystallins because of their roles in maintaining transparency of the lens and because they all contribute to lens opacity if damaged.38,69,85,86,87
-Crystallins have two domains and variable N-terminal and C-terminal extensions.69,87 These extensions probably stabilize the -crystallin oligomers normally found in the lens, serve as spacers for preventing excessive polymerization, and regulate short-range, nonspecific protein interactions. During aging, the extensions undergo proteolysis, a likely cause of lens opacification.87 The chaperoning capacity of the -crystallins diminishes with aging (Table 2), just when they are most needed to stabilize the other aging crystallins.69,73 The extent of this acquired, age-related chaperoning deficiency is comparable to the deficiency caused by -crystallin mutations in hereditary cataracts.38,69,88
Retinopathy
An age-related decrease in the expression of A-crystallin and post-translational modifications of the protein (such as phosphorylation, acetylation or glycation of lysine, deamidation of asparagine or glutamine, and glutathionylation of cysteine) occur in the retina45 (Table 2). These modifications alter the charge distribution across the molecule, impair the chaperone function of A-crystallin, and promote aggregation of the chaperone; all these phenomena contribute to the development of age-related retinopathy.
Truncation is an additional modification of A-crystallin in the lens and retina associated with aging.45 In the lens, a truncation occurs at the C-terminal; 1 or more residues (up to 22) are deleted. In the retina, C-terminal truncations have been detected in young-adult and middle-aged rats, but not in old rats.45 In contrast, N-terminal truncations were present in the retinal A-crystallin from all three age groups and were most frequent in the oldest animals.
Other Pathologic Conditions Involving Chaperones
Von Hippel–Lindau Disease
One function of chaperones is the interaction with other chaperones to form multimolecular chaperoning machines; these, in turn, may assist in the assembly of other multimeric complexes with diverse functions. If the client molecule that the chaperone machine recognizes lacks a chaperone-recognition site (e.g., because of a mutation), the assembly process will not start, and the multimeric complex will not be built. An example is the chaperone complex TCP-1 that mediates the assembly of the von Hippel–Lindau (VHL) tumor suppressor protein with elongin B and elongin C.89 The assemblage VHL-elongin BC interacts with the suppressor of kinetochore protein 1 (Skp1)–Cullin 1 (Cul1)–F box (SCF) ubiquitin ligase complex to promote the destruction of a set of proteins necessary for vascularization and growth of tumors.89,90 If the entire multimolecular structure is not assembled correctly, the tumor-suppressor activity of VHL is lost, allowing the development and growth of tumors.
The VHL protein has a 55-amino-acid segment for CCT binding. A mutation in or deletion of this VHL segment is associated with von Hippel–Lindau disease, in which pheochromocytoma, renal carcinoma, and densely vascularized retinal and cerebellar tumors develop.90 Two mutations in the segment of the VHL protein that is the target of CCT recognition interfere with the binding of CCT and, consequently, the folding of the VHL protein.89
Autoantibodies against Chaperones
HSPs and molecular chaperones are immunogenic. Structural alterations in a chaperone caused by mutation or post-translational modification can transform it into an autoantigen, which can elicit an autoimmune response if the altered chaperone gains access to the immune system. It is possible that antichaperone antibodies cross-react with normal antigens. During senescence, a mechanism involving abnormal chaperones that leak into the extracellular space and circulation and evoke antichaperone autoantibodies that cause tissue damage is thought to develop and to contribute to the general functional decline accompanying aging59,60 (Table 3). Anti-HSP47 antibodies were found to be elevated in serum from patients with systemic lupus erythematosus and other autoimmune disorders (Table 3), but not in serum from patients with a nonautoimmune disease.61
These and other findings point to the need to determine the prevalence and pathogenetic roles of antichaperone autoantibodies. Antichaperone autoantibodies may be involved in the pathogenesis of autoimmune diseases by cross-reactions with other molecules61,63 and could contribute to the development of diseases that are not predominantly autoimmune but that have an autoimmune component, such as inflammatory bowel diseases.91 Such antibodies could also disrupt chaperoning mechanisms if they form a complex with a chaperone molecule and interfere with its functions.59,60,61,62,63,64,91 Interference with chaperone functions could also be caused by antibodies against a foreign (microbial) chaperone that cross-reacts with the human counterpart.92 Even though the likelihood of chaperone–antibody complex formation is low because chaperones function mainly intracellularly, exploring the potential of antichaperone autoantibodies as markers of disease progression and senescence would be worthwhile. After the pathogenetic role of antichaperone antibodies is established, it should become possible to develop strategies for treatment (e.g., immunosuppression).
Chaperonopathies and Malfunction of the Immune System
Chaperones are involved in various stages of the immune response: initiation of proinflammatory cytokine production, antigen recognition and processing, activation of antigen-presenting cells, and chaperoning of peptides during antigen presentation.91,92,93,94 The molecular mechanisms by which HSP70 participates in the immune response have not been elucidated. It is clear, however, that it binds viral, bacterial, and tumor peptides through its peptide-binding domain near the middle of the molecule and makes the peptides immunogenic; HSP70 has adjuvant properties.91,92,93,94 Other effects of HSP70 on the immune system, such as stimulation of interleukin 12, tumor necrosis factor , and nitric oxide production, are mediated by its C-terminal domain (consisting of 10 amino acids).94 It follows that if the peptide-binding domain or the C-terminal domain of the chaperone is impaired because of mutation or age-related post-translational modifications, immune responses will be defective.
In a rat model, exposure to stressors was shown to increase the extracellular levels of HSP72, presumably signaling the immune system to mount a response against a bacterial pathogen.95 How this signaling occurs was not ascertained in that study, but it was shown that stimulation of splenocytes and macrophages with HSP72 increased the levels of nitric oxide, tumor necrosis factor , interleukin-1, and interleukin-6. These observations suggested that extracellular HSP72 triggers the production of nitric oxide along with the immune response to bacterial antigens. Thus, if HSP72 is defective, the immune response to microbial pathogens will be adversely affected.
Chaperone-Gene Polymorphisms
Chaperone-gene polymorphisms are promising predictors of longevity because of the key roles of chaperones in cell physiology and antistress mechanisms. The frequency of allele (A ) –110 (the A/A genotype) in the HSP70-1 promoter region was decreased among long-lived women in southern Italy96 (Table 4). In that study, the A/A genotype was thought to contribute to a functional state unfavorable to longevity in women, perhaps in relation to female hormones. However, a similar study in another group, from Denmark, showed that the A/A genotype was more prevalent among persons with higher self-rated health scores, who are predicted to live longer, and no sex-specific association was observed97 (Table 4).
Table 4. Polymorphisms of Human HSP (Molecular Chaperone) Genes with Possible Effects on Longevity, the Risk of Disease, or Both.
There are three HSP70 genes within the HLA class III region.98 One of these, HSP70-HOM , is not heat-inducible, protects from oxidative stress, and exhibits a functional polymorphism (a methionine-to-threonine substitution at position 493 of the HSP70-Hom protein) (Table 4). A comparative study revealed that the T/T genotype (encoding methionine) was more frequent among older than among younger subjects, whereas the reverse held true for the T/C genotype (encoding threonine).98 These results show that methionine at HSP70-Hom position 493 is accompanied by an increase in the probability of living a long life. Position 493 is in the peptide-binding domain of the chaperone. Peptide binding is optimal when this position is occupied by methionine (a nonpolar and hydrophobic amino acid), but it is impaired when methionine is replaced by threonine (a polar and neutral amino acid), which results in chaperone malfunction. The adverse effect of this malfunction can be predicted to be widespread, affecting many cellular processes, such as the response to stressors, and allowing the accumulation of defective proteins, particularly those with post-translational modifications that increase with aging. These effects would be cumulative over a period of time and finally cause a shortening of the life span. This type of chaperonopathy is a good candidate for replacement therapy with, for example, recombinant HSP70-Hom with the optimal amino acid at position 493 or its gene (i.e., gene therapy).
The association of polymorphisms with Parkinson's disease was investigated by genotyping DNA samples obtained from peripheral-blood leukocytes99 (Table 4). No association with the disease was detected among the polymorphisms in the coding regions, with one possible exception. The frequencies of the HSP70-1 –110CC and +190CC genotypes were higher among the patients with Parkinson's disease than among the controls; in in vitro assays, –110A was a better HSP70-1 transcriptional inducer than –110C. The same patterns were observed in heat-shocked and nonstressed cells.
Genetic Chaperonopathies
At least 15 conditions have been reported that are associated with mutations in genes encoding chaperones or chaperone-like molecules (Table 5). These conditions have been reviewed elsewhere and can be considered "inborn errors of development,"38 a term coined to designate collectively human malformations associated with gene mutations.100
Table 5. Genetic Chaperonopathies.
Prospects for the Future
The way is open for basic, applied, and clinical investigations directed at discovering new chaperonopathies, expanding knowledge about those already identified, and elucidating the mechanisms by which defective chaperones contribute to disease. Chaperones are so universally distributed and perform so many different functions that the probability is high that they are involved in the initiation and progression of disease. Exciting challenges for the next few years will be to determine the prevalence and full extent of medical consequences of chaperonopathies, to standardize diagnostic tests, and to develop strategies for prevention and treatment.
We are indebted to Tara Garcia-Collins and Brian W. Meneghan for their help with the comparative and phylogenetic analyses of human genes, to Phil Grimley and Alex Macario for their critical reading of the manuscript.
Source Information
From the Wadsworth Center, Division of Molecular Medicine, New York State Department of Health (A.J.L.M., E.C.M.); and the Department of Biomedical Sciences, School of Public Health, State University of New York at Albany (A.J.L.M.) — both in Albany.
Address reprint requests to Dr. A.J.L. Macario at the Wadsworth Center, Room B-749, Division of Molecular Medicine, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, or at macario@wadsworth.org.
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