Protein Synthesis, Posttranslational Modifications, and Aging SURESH I. S. RATTAN,b ANASTASSIA DERVENTZI, AND BRIAN F. C. CLARK Laboratoly of CellularAgeing Department of Chemistry Aarhus University DK-8OOOAarhus-C,Denmark Age-related changes in the functioning of proteins can be due to both inefficient protein synthesis and an altered pattern of posttranslational modifications, including possible conformational alterations. The slowing down of bulk protein synthesis is a widely recognized biochemical change with age and has been observed in a variety of tissues, organs, and organisms.1.2At the same time, the accumulation of inactive, altered, and abnormal proteins during aging has been associated with both slower protein degradation and posttranslational modifications during agi11g.3,~However, the mechanisms of the regulation of synthesis, activity, and turnover of proteins during aging are currently poorly understood. For example, although total protein synthesis slows down during aging, the translational processes are never shut down completely. Furthermore, twodimensional gel electrophoretic analyses of the spectrum of proteins synthesized by young and old human cells, insects, and nematodes have shown that no major change occurs in the qualitative pattern of proteins during a g i ~ ~Similarly, g . ~ ~ ~ studies to elucidate age-related changes in the components of the protein synthetic machinery have also shown that altered activity and specificity, rather than quantities of various components such as ribosomes, tRNAs, tRNA-synthetases, and initiation and elongation factors, may be more important in the regulation of protein synthesis during aging.' These and several other studies on the biochemical basis of protein functioning and turnover during various biological processes indicate the crucial role of posttranslational modifications. According to one estimate, more than 140 types of posttranslational modifications of proteins have been described? Not all modifications have been studied in relation to aging. Most studies on protein modifications during aging have been directed towards altered protein turnover and the accumulation of abnormal proteins. However, recent studies have shown that age-related changes in the activity of various enzymes may also be due to an altered pattern of posttranslational modification. This article provides an overview of some of the major protein modifications that are known to have an important role in various biological processes and may be equally important .in the process of aging. These modifications include covalent modification reactions involving amino acid side chain residues (for example, phosphorylation, methylation, ADP-ribosylation, oxidation, acetylation, and glycation), "The Laboratory of Cellular Ageing is supported by a research grant from Velux Fund, Copenhagen. Some financial support from the EURAGE is also acknowledged. bAddress for correspondence: Dr. Suresh I. S. Rattan, Laboratory of Cellular Ageing, Department of Chemistry,Aarhus University, DK-8000 Aarhus-C, Denmark. 48

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deamidation, racemization, and noncovalent spontaneous changes in protein conformation. PHOSPHORYLATION

The reversible phosphorylation of serine, threonine, and tyrosine residues of proteins is one of the most common types of posttranslational modification. The coordinated activities of protein kinases, which catalyze phosphorylation, and protein phosphatases, which catalyze dephosphorylation, regulate several biological processes, including protein synthesis, cell division, signal transduction, cell growth and development:JO and, possibly, aging. TABLE 1 lists some of the major proteins whose activity is modulated by phosphorylation. It should be emphasized that more than 180 proteins of a total of about 3,000proteins are reported to be phosphorylated in human cells, as seen by two-dimensional gel electrophoresis.ll However, the identity of not more than 40 phosphorylated proteins is known at present. Among the

TABLE1. Phosphorylation as a Modulator of Protein Function A. Proteins whose activities increase afterphosphorylation DNA polymerase a Histones Vimentin Lamin S6 ribosomal protein Protein initiation factors, eIF-3,4B, 4F Aminoacyl-tRNA-synthetases Growth factor and hormone receptors Neurofilament proteins B. Proteins whose activities decrease afier phosphorylation Protein initiation factor eIF-2 Protein elongation factors EF-la, p, and EF-2 Products of cell division cycle (cdc) genes

phosphorylated proteins that may be of significance in the process of aging are DNA polymerase a, histones, protein initiation and elongation factors, S6 ribosomal protein, and various growth factor and hormone receptors. DNA synthesis: One of the major characteristics of cellular aging is the inhibition of DNA synthesis in senescent cells. Of various components involved in the synthesis of DNA is an age-related decline in the specific activity of DNA polymerase a as reported for nondividing senescent human f i b r o b l a ~ t s . ~This ~ - ~change ~ may be due to a decrease in the phosphorylation of DNA polymerase a as observed during cell-cycle-related changes in the activity of this enzyme in normal and leukemic human ~ e l l s . ~However, ~J~ direct studies on the phosphorylation status of DNA polymerase a during aging are yet to be performed. Similarly, it is not known whether the age-related decline in the fidelity of DNA polymerase a in aging human fibrobIastsl7is related to altered levels of phosphorylation. Cell-cycle-dependent alterations in phosphorylation have also been observed for histones and nonhistone proteins, vimentin and lamin.I8 There are no current data available for the age-related changes in phosphorylation of these proteins. However,

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an age-related decline of 50% in the phosphorylation of two acidic proteins and an increase of 300% in the phosphorylation of one basic protein in the microsomal and nuclear fraction of rat hepatocytes was reported.lg Recently, it was reported that senescent human fibroblasts fail to phosphorylate and inactivate the retinoblastoma gene product pllORb,which is a potent proliferation inhibitor, and this is suggested to be a reason for the age-related inability of cells to reinitiate DNA replication.m Protein synthetic components: Phosphorylation of several eukaryotic protein initiation factors (eIFs) can regulate their activities and hence the rates of protein synthesis. For example, phosphorylation of eIF-2 correlates with inhibition of initiation reactions and consequently with inhibition of protein synthesis by affecting the process of guanine nucleotide exchange.21Conditions such as starvation, heat shock, and viral infection, which inhibit the initiation of protein synthesis, induce the phosphorylation of eIF-2 in various cells." Similarly, stimuli such as insulin and phorbol esters modulate the phosphorylation of eIF-3, eIF-4B, and eIF-4F by activating various protein k i n a ~ e s . With 2 ~ ~ respect ~~ to aging, the activity of eIF-2 has been reported to decrease in old rat brain.25 However, it is not known if the phosphorylation status of eIF-2 also changes during aging. Similarly, no studies are available on age-related changes in other initiation factors. At the level of protein elongation, phosphorylation of elongation factors E F - l a and EF-2 appears to be involved in regulating their activities.2'jAs the activity and amounts of active EF-la and EF-2 are reported to decrease significantly during aging,' it will be interesting to see if this decline is accompanied by a parallel change in the extent of phosphorylation of these enzymes. Incidently, an increase in the levels of phosphorylated EF-1 and EF-2 has been reported during mitosis when minimal protein synthesis O C C U ~ S . ~ ~ ~ ~ Phosphorylation also occurs at other proteins that participate in the translational process. For example, phosphorylation of the S6 ribosomal protein correlates with the activation of protein synthesis. Failure of senescent human fibroblasts to phosphorylate S6 protein in response to serumm can be one reason for the decline in protein synthesis during aging. Similarly, the regulatory role of phosphorylation of aminoacyl-tRNA synthetase in protein synthesis has also been suggested.30However, to what extent the decline in the activity and the accumulation of heat-labile aminoacyl-tRNA synthetases reported in studies on various organs of aging mice3I and rats32is related to their phosphorylation is not known. Signal transduction: The role of protein phosphorylation and dephosphorylation in the pathways of intracellular signal transduction is a widely studied topic. All phosphorylation reactions result from the action of a single or multiple kinases. Phosphorylation can also be involved in the regulation of the activity of some of these kinases. The ratio between two interconvertible, active and inactive, forms of kinases acts as a control mechanism for many cellular functions?JO Typical examples of protein kinases regulating cellular mechanisms are the protein kinase C (PKC; signal transduction), growth factor receptors (cell growth), glucocorticoid receptors (hormonal action), and the glycogen phosphorylase (energy metabolism). For example, PKC is activated as a second messenger in response to extracellular receptor-mediated signals, such as growth factors, tumor promoters, and transformation factors. Proliferating cells exhibit higher activity and a different subcellular distribution of PKC than do quiescent cells, indicating an important role for PKC in cell r e p l i ~ a t i o n Studies .~~ performed on aging cells have not shown any deficiency in the amount, activity, or ability of PKC to elicit a signaling pathway.34Evidence also exists that senescent human fibroblasts retain their ability to phosphorylate proteins in the PKC signal transduction pathway.35 PKCs appear largely unaltered in fibro-

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blasts, although the body of information about phosphorylation mechanisms is still very limited. Growth factor receptors for EGF, FGF, PDGF, insulin, glucocorticoids, and several other hormones also possess protein kinase activity. Upon binding of a growth factor, the receptor gets autophosphorylated on the tyrosine residues of its catalytic subunit and becomes activated as tyrosine kinase on other intracellular proteins.36 The autophosphorylation is essential for the kinase activity and the translocation ability of the growth factor receptor.37Deficiencies in the phosphorylation process of receptors would be a logical explanation for the age-related decline of responsiveness to hormonal action and growth stimulation, but the available data are c o n f u ~ i n g .For ~ ~ .example, ~~ decreased autophosphorylation activity of EGF receptors has been reported in senescent human fibroblasts, but this observation could not be confirmed in later studies on other growth factor receptors (with the exception of the glucocorticoid receptor).a Another area in which protein phosphorylation plays a significant role is neuronal communication. The transfer of information conveyed by neurotransmitters and hormones is carried out by protein kinases, such as CAMP-kinase, PKC, and calmodulin-dependent kinases, which phosphorylate a series of proteins, leading to the activation of specific genes9 As one of the major characteristics of aging includes defective neuronal systems, it has been suggested that either a loss of activity of the mediating kinases or decreased substrate availability may be involved in this.41 Studies on aged rodents have demonstrated reduced CAMP-dependent phosphorylat i ~ n area-selective ,~~ modifications of PKC activity and translocation ability, and impairment of the adenylate cyclase system during aging.43,44 Energy metabolism: An interesting case of phosphorylation reciprocal control is the glycogen phosphorylase. The phosphorylation of two serines increases the affinity of glycogen phosphorylase by inducing conformational transition of the amino terminus, while the presence of glucagon and glucose-6-phosphate leads to its dephosphorylation and i n a ~ t i v a t i o n The . ~ ~ ratio between the active and inactive forms of the enzyme regulates the energy metabolism. Therefore, it will be interesting to find out if the age-related decline in the activity of glycogen phosphorylase reported for rat heart muscle and human aorta46is an example of allosteric control due to changes in the phosphorylation pattern of this enzyme. Thus, it can be concluded that phosphorylation of a wide variety of proteins has a significant influence in various biological processes, and it will be extremely useful to undertake detailed studies on this posttranslational modification in relation to the process of aging. METHYLATION

Methylation of nitrogens of arginine, lysine, and histidine and carboxyls of glutamate and aspartate residues is a widely observed posttranslational modification that is involved in many cellular functions.47 Specific enzymes, comprising three major groups of protein methyltransferases, have been identified on the basis of the amino acids that become methylated. Although most of our present understanding of the significance of protein methylation has come from studies on bacterial chemotaxis, muscle contraction, electron transport, processing of pituitary hormones, and gene expression, its role in aging is beginning to emerge. TABLE2 lists proteins whose activities are modulated by methylation. Of these, decreased methylation of histones in the liver and brain of aging rats has been reported.48On the other hand, no difference in the extent of methylation of newly

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TABLE2. Methylation As a Modulator of Protein Function A. Proteins whose activities increase aj?er methylation

Alcohol dehydrogenase Histones Ribosomal proteins Cytochrorne C Protein elongation factor EF-la Myosin Myelin Rhodopsin B. Proteins whose activities decrease afier methylation Calmodulin Erythrocyteband 3

synthesized histones was observed during cellular aging of human fibroblasts in c~lture!~.~~ Studies on the levels of methylated histidine, arginine, and lysine of myosin isolated from the leg muscles of aging rats, mice, and hamsters showed unchanged levels of histidine, decreased levels of arginine and trimethyllysine, and increased levels of mon~methyllysine.~~ During the aging of erythrocytes, an increase in the number of methyl groups per molecule of band 2.1 (ankyrin) and band 3 protein has been reported to correlate with increased membrane rigidity of erythrocytes during aging.50Similarly, a severalfold increase in the number of methyl acceptor proteins in the eye lenses of elderly persons and persons with cataracts has been reported?* The number of carboxylmethylatable sites of cerebral membrane-bound proteins also increases in rat brain during aging?3 An interesting role of protein methylation in modulating enzyme activity is that of elongation factor EF-la, which contains dimethyllysines at residues 55 and 165 and trimethyllysines at residues 36, 79, and 318.54 The increased activity of EF-la during morphogenesis of the fungus Mucor racernosus has been associated with its enhanced methylation levels.55Furthermore, the levels of methylated EF-la were reported to be significantly higher in rapidly growing SV40-transformed mouse 3T3 fibroblasts than in 3T3 cells without viral tran~formation.~~ As it had previously been reported that the activity and amounts of active EF-la declined in senescent human fibroblast^:^ attempts were made to correlate this decline with changes in the levels of methylated EF-la. However, no major differences in the levels of methylated and unmethylated EF-la during aging of human fibroblasts could be observed on two-dimensional gel electrophore~is.~~ At present, age-related changes in the methylation of other proteins such as ribosomal proteins, calmodulin, cytochrome C, and myosin have not been studied. However, protein methylation is clearly involved in diverse functions including protein synthesis and turnover, and it should be studied thoroughly in relation to the process of aging. ADP-RIBOSYLATION

ADP-ribosylation is the enzymic addition of an ADP-ribose derived from NAD+ to a ribosylatable protein. Although various proteins (except histones) that can be ribosylated have not been identified as yet, several ADP-ribosyltransferases that

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catalyze the reaction are present in both the nucleus and the cytoplasm of ~ e l l s . 5 ~ Generally, the cytoplasmic enzymes are known to catalyze the formation of a mono-ADP-ribosyl protein (mono-ADP-ribosyltransferases), while the nuclear enzyme catalyzes the addition of poly(ADP-ribose) to proteins (poly-ADP-ribosyltransferases). ADP-ribosylation has been indicated as a control mechanism in many cellular functions, such as DNA repair, protein synthesis, cell differentiation, and cell transformation.h0 As yet, no studies have been carried out on age-related changes in the involvement of ADP-ribosylation in DNA repair during aging.61A single report has been published showing that the activity of poly(ADP-ribose)polymerase in human fibroblasts decreases as a function of donor age.62The implications of this observation are not clear at present. One cytoplasmic protein that can be specifically ribosylated by at least two bacterial toxins, namely, diphtheria toxin and exotoxin A, is the protein elongation factor EF-2. ADP-ribosylation of the diphthamide (modified histidine 715) residue of EF-2 results in the complete abolition of its catalytic activity.s4 With regard to aging, some evidence indicates that increased ADP-ribosylation of EF-2 is correlated with cellular aging. For example, the amount of EF-2 that can be ADP-ribosylated in the presence of diphtheria toxin in cell-free extracts is reported to decrease significantly during the aging of human fibroblasts in culture.63However, no decline in the amount of ADP-ribosylatable EF-2 was observed in rat livers during aging.h4Therefore, it is not clear to what extent the ADP-ribosylation-mediated decline in the activity of EF-2 correlates with the age-related decline in the rate of protein synthesis. OXIDATION Even under normal physiological conditions, proteins in an organism are continuously exposed to endogenous and exogenous oxidation factors. Oxidation and inactivation of proteins can be provoked by oxygen radicals generated from intracelMar processes,65and can also be catalyzed by the so-called mixed-function oxidation (MFO) systems that involve different reductases and oxidases.6h MFO-systemmediated oxidation of proteins can lead to the formation of asparaginyl, aspartyl, and 3 lists enzymes that are considered to become inacticarbonyl derivativesh’ TABLE vated by oxidation. With the amount of carbonyl groups as a measure of the extent of MFO-catalyzed protein oxidation, increased levels of oxidatively modified proteins have been TABLE3. Oxidation As a Modulator of Protein Function Proteins that become inactive or denatured after oxidation Glyceraldehyde-3-phosphate dehydrogenase 6-Phosphogluconate dehydrogenase Glutamine synthetase Glucose-&phosphate dehydrogenase Fructose-1,6-diphosphatase Lactate dehydrogenase Ornithine decarboxylase Liver malic enzyme Superoxide dismutase Lens crystallin Collagen

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reported in human erythrocytes of higher density (considered as old) and cultured human fibroblasts from normal old donors and from individuals with progeria and Werner’s syndrome.6s With the same method, a twofold increase in the protein carbonyl content of the brain proteins of retired breeder Mongolian gerbils has been reported.69 Furthermore, chronic treatment of gerbils with a spin-trapping compound, N-tea-butyl-a-phenylnitrone, caused a decrease in the level of oxidized protein, signifying the role of oxygen-free radicals in the oxidation of proteins during aging.69 It has also been suggested that the loss of 11 lysine residues, presumed to be due to oxidation, may be the cause of the inactivation of 6-phosphogluconate dehydrogenase in the rat livers7nand in human erythrocytes during aging.71Similarly, the loss of activity of rat liver malic enzyme during aging is related to the loss of histidine residues by oxidation.72Furthermore, oxidation of a cysteine residue in glyceraldehyde-3-phosphate dehydrogenase may be responsible for its inactivation during aging in rat muscles.73 It has also been reported that the concentration of the oxidation products of human lens proteins and skin collagen increases during aging.74 Related to these findings, the accumulation of oxidative forms of a-crystallin has been reported in patients with age-related ~ataract.’~ Structural alterations introduced into proteins by oxidation can lead to the aggregation, fragmentation, denaturation, and distortion of secondary and tertiary structure, increasing thereby the proteolytic susceptibility of oxidized p r o t e i n ~ . ~ f j ~ - ~ ~ Thus, the accumulation of abnormal proteins during aging may be due to impairment of the protein degradation processes or to defective protection from oxidative damage, or both.76-77 If better methods can be developed for the accurate estimation of the levels of oxidation products of proteins, this figure can be used as a biomarker of aging.4 GLYCATION Glycation is the nonenzymatic reaction of free amino groups of proteins with glucose, leading to the formation of a ketoamine called Amadori product followed by a sequence of further reactions and rearrangements producing the so-called ad~,~~ long-lived structural provanced glycosylation endproducts ( A G E S ) . ~Generally, teins such as lens crystallins, collagen, and basement membrane proteins are more susceptible to glycation. The glycated proteins are then more prone to form crosslinks with other proteins, leading to structural and functional alteration^.^^ A n increase in the levels of glycated proteins during aging is observed in almost all species tested so far. For example, an increase in the level of glycated lysine residues of rat sciatic nerve, aorta, and skin collagen during aging has been reported.8ns81Similarly, the age-related increase in glycation of human c ~ l l a g e n ~ ~ . ~ ~ and o ~ t e o c a l c i nhas ~ ~ been reported. The glycation of lens crystallins shows an age-related increase in bovine78but not in humans and rats.8s-R6 On the other hand, an increase in the levels of glycation of collagen, hemoglobin, and human lens crystallin occurs in patients with d i a b e t e ~ Calorie-restricted . ~ ~ ~ ~ ~ ~ ~ ~rodents whose aging is generally slowed down have reduced levels of glycated hemoglobin compared with those of freely fed animaksY The formation and accumulation of AGES are implicated in the physiology and pathology of For example, it has been observed that pentosidine (cross-linked glycated lysine and arginine) and carboxylmethyllysine (glycated and oxidated proteins) increase with age in human^.^^.^^ Pyrroline, another AGE protein, increases in diabeticsy3However, many more studies are still required to understand

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differences in the rates of formation and removal of glycated proteins in different species with different lifespans and rates of aging. DEAMIDATION For many long-lived proteins, the change in catalytic activity, heat stability, affinity for substrate, and other physical characteristics may also be due to the charge change introduced by conversion of a neutral amide group to an acidic group by d e a m i d a t i ~ n .Slow ~ ? ~ ~and spontaneous deamidation of asparaginyl and glutaminyl residues of several proteins has been related to the observed accumulation of their inactive and heat-labile isoforms during aging. For example, the sequential deamidation of two asparagine residues (Asn 15 and Asn 71) of triphosphate isomerase is responsible for differences in the isoenzymes present in aging cells and tissues, such as bovine eye lens,y4and human skin fibroblasts from old donors and patients with progeria and Werner’s syndrome.y5 Similarly, deamidation of glucose-6-phosphate isomerase produces the variant of the enzyme that accumulates in aging bovine lenses.y6 The biological function of deamidation is not yet clear. It has been suggested that structural alterations in the deamidated proteins increase their susceptibility to proteolytic action, a hypothesis based on the observed inverse relation between the amide content and the half-life of proteins.76However, no further evidence has been presented in support of this hypothesis. RACEMIZATION AND ISOMERIZATION The interconversion of optical isoforms of amino acids, called racemization, has been reported to increase during aging. For example, the concentration of D-aspartate in protein hydrolysates from human teeth, erythrocytes, and eye lens increases with age.4-y7Similarly, the spontaneous prolyl cis-trans isomerization in proteins that may cause some of the so-called spontaneous conformational changes has been implicated in the age-related decline in the activity of certain enzyme^.^ However, no definitive examples of enzymes undergoing this kind of posttranslational modification during aging are available. Also, to what extent the conformational changes associated with old rat muscle phosphoglycerate kinase,y8 enolase, and other enz y m e ~are ~ associated with racemization and isomerization is not known. SOME OTHER MODIFICATIONS In addition to these types of posttranslational modifications observed during aging, there are some other modifications that determine the structure and function of various proteins and may have a role to play during aging. For example, the incorporation of ethanolamine into protein elongation factor EF-la may be involved in determining its stability and interaction with intracellular membrane^.^^ Whether this modification has any role in regulating the activity of EF-la and hence regulating protein synthesis is not known at present. Similarly, the protein initiation factor eIF-4D contains an unusual amino acid, hypusine, which is synthesized posttranslationally as a result of a series of enzymatically catalyzed altcrations of a lysine residue.9y Hypusine is suggested to regulate the activity of eIF-4D, because its

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absence blocks the initiation of protein synthesis.'" Therefore, it will be interesting to investigate whether this modification is involved in the regulation of eIF-4D activity during aging when total protein synthesis slows down. Other posttranslational modifications that may have significance in protein alterations during aging are, for example, protein tyrosine sulfation involved in determining the biological activity of neuropeptides and the intracellular transportation of a secretory proteinlo'; and prenylation, the covalent attachment of isoprenoid lipids on cysteine-rich proteins in the regulation of the activity of some protooncogenic ras proteins and the nuclear lamins A and B.102J03 These studies have indicated a critical role for prenylation in the regulation of oncogenesis, nuclear structure, signal transduction, and cell cycle progression,1w functions very much related with the causative aspects of aging. Similarly, detyrosination of microtubuleslo5 affecting the cytoskeletal organization, and hence many other cellular functions, may be important during aging. Furthermore, the roles of chaperones in protein folding and conformational organizationlo6 and of ubiquitin in protein degradationlo7are yet to be studied in relation to the aging process.

CONCLUSIONS

The significance of posttranslational modifications in regulating the structure, stability, and function of proteins is well established. Various lines of research discussed briefly show that protein modifications have a significant role during aging with respect to several biological processes. These include: 1. Regulation of the pathways of genetic information transfer including DNA, RNA, and protein synthesis by determining the structure, stability, and activities of various components involved in these processes, such as DNA synthesizing enzymes, ribosomal proteins, initiation factors, and elongation factors; 2. Determining the efficiency of receptor-mediated pathways of signal transduction and cellular responsiveness; 3. Maintaining the intracellular structural organization and cytoskeleton; 4. Regulating the events of cell cycle progression including chromatin reorganization; 5. Determining the efficiency of various enzymes involved in the pathways of protein degradation; 6. Marking abnormal and inactive proteins for degradation by increasing their susceptibility and accessibility to proteolytic enzymes, thereby determining the extent of accumulation of abnormal proteins and other protein-conjugated products; 7. Determining the extent of intra- and extracellular cross-links between proteins and other macromolecules; and 8. Altering the structural and functional characteristics of long-lived proteins, such as lens crystallins.

The possibility remains that several other processes that have not been studied are also affected by posttranslational modifications and are crucial in the aging process. However, what has become clear is that posttranslational modification of proteins is an integral part of the transfer of genetic information from genes to gene products. Without a thorough understanding of the mechanisms, role, and signifi-

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cance of posttranslational modifications, our knowledge of the genetic and molecular basis of aging is incomplete and insufficient.

SUMMARY Posttranslational modifications of proteins are involved in determining their activities, stability, and specificity of interaction. More than 140 major and minor modifications of proteins have been reported. Of these, only a few have been studied in relation to the aging of cells, tissues, and organisms. These include phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and deamidation. Several of these modifications occur on proteins involved in crucial cellular processes, such as D N A synthesis, protein synthesis, protein degradation, signal transduction, cytoskeletal organization, and the components of extracellular matrix. Some of the modifications are the markers of abnormal and altered proteins for rapid degradation. Others make them less susceptible to degradation by normal proteolytic enzymes, and hence these accumulate during aging. REFERENCES 1. MAKRIDES, S. C. 1983. Protein synthesis and degradation during aging and senescence. Biol. Rev. 5 8 343422. A. & 1. SEMSEI. 1987. Effect of aging on translation and transcription. Rev. 2. RICHARDSON, Biol. Res. Aging 3: 467483. M. 1985. The alteration of enzymes in aging. In Modification of Proteins 3. ROTHSTEIN, during Aging. R. C. Adelman & E. E. Dekker, eds. 53-67. Alan R. Liss. New York. E. R. 1988. Protein modification in aging. J. Gerontol. 43: B112-Bl20. 4. STADTMAN, S. J. FEY,P. MOSELARSEN & J. E. CELIS.1983. Expression of 5. BRAVO,R., J. BELLATIN, cellular proteins in normal and transformed human cultured cells. In Gene Expression in Normal and Transformed Cells. J. E. Celis & R. Bravo, eds.: 263-290. Plenum Press. New York. J. E., E. QUATTROCKI, G. LATTER,J. MIQUEL,R. MARCUSON, E. ZUCKER6. FLEMING, KANDL & K. G. BENSCH.1986. Age-dependent changes in proteins of Drosophila melanogaster. Nature 231: 1157-1159. 7. RATTAN,S. I. S. 1991. Protein synthesis and the components of protein synthetic machinery during cellular aging. Mutat. Res. 2 5 6 115-125. 8. ALIX,J. H. & D. HAYES.1983. Why are macromolecules modified post-synthetically? Biol. Cell 47: 139-160. 9. STEELE,R. E. 1990. Protein-tyrosine phosphorylation: A glimmer of light in the darkness. Trends Biochem. Sci. 15: 124-126. 10. ROACH,P. J. 1991. Multisite and hierarchal protein phosphorylation. J. Biol. Chem. 266 14139-14142. P. MADSEN, H. LEFFERS,K. DEJGAARD, B. 11. CELIS,J. E., B. GESSER,H. H. RASMUSSEN, HONORE,E. OLSEN,G. RATZ, J. B. LAURIDSEN, B. BASSE,S. MOURITZEN, M. HELLERUP, A. ANDERSEN, E. WALBUM, A. CtLis, G. BAUW,M. PUYPE, J. VANDAMME & J. VANDEKERCKHOVE. 1990. Comprehensive two dimensional gel protein databases offer a global approach to the analysis of human cells: The transformed amnion cells (AMA) master database and its link to the genome DNA sequence data. Electrophoresis 11: 989-1071. 12. KRAUSS,S. W. & S. LINN.1982. Changes in DNA polymerase a, !3, and y during the replicative life span of cultured human fibroblasts. Biochemistry 21: 1002-1009. 13. KRAUSS,S. W. & S. LINN.1986. Studies of DNA polymerase alpha and beta from cultured human cells in various replicative states. J. Cell. Physiol. 126 99-106.

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