6.3. POST-TRANSLATIONAL MODIFICATIONS
Almost all proteins undergo post-translational modifications, which is the final step in the transfer of genetic information from a gene into a functional gene product. The term post-translational modification covers: (i) covalent modifications that yield derivatives of individual amino acid residues, for example, phosphorylation, methylation, ADP-ribosylation, oxidation and glycation; (ii) proteolytic processing through reactions involving the polypeptide backbone; and (iii) nonenzymic modifications, e.g. deamidation, racemization and spontaneous changes in protein conformation.
The coordinated activities of protein kinases, which catalyze phosphorylation, and protein phosphatases which catalyze dephosphorylation, regulate numerous biological processes, including transcription, translation, cell division, signal transduction, cell growth and development (35). Several putative inhibitors of DNA synthesis have been identified in senescent cells, and the activity of several of these inhibitors is regulated by phosphorylation. A decrease in phosphorylated cyclin E and Cdk2, and failure to phosphorylate RB1 gene product p110Rb, and cdc2 product p34cdc2 during cellular aging have been reported (36). In contrast, approximately 3-5-fold increased phosphorylation of serum response factor (SRF), which binds the serum response element, has been reported in senescent human skin fibroblasts (37).
Components of the protein synthetic apparatus also undergo phosphorylation and dephosphorylation and thus regulate the rates of protein synthesis (38). Phosphorylation of eIF-2 correlates with inhibition of initiation reactions and, consequently, with the inhibition of protein synthesis. Conditions like starvation, heat shock and viral infection, which inhibit the initiation of protein synthesis, induce the phosphorylation of eIF-2 in various cells. Stimuli such as insulin and phorbol esters modulate the phosphorylation of eIF-3, eIF-4B and eIF-4F by activating various protein kinases.
At the level of protein elongation, increased phosphorylation of EF-2 correlates with a reduction in the rate of protein synthesis in mammalian cells (39). There is indirect evidence that alterations in the phosphorylation and dephosphorylation of EF-2 due to changes in the activities of EF-2-specific protein kinase III (40), and PP2A phosphatase (41) may affect the rates of protein synthesis during aging. Furthermore, since the phosphorylation of the S6 ribosomal protein correlates with the activation of protein synthesis, failure to phosphorylate S6 protein in senescent human fibroblasts in response to serum (42) can be one of the reasons for the decline in the rate of protein synthesis observed during aging. Pathways of intracellular signal transduction depend on sequential phosphorylation and dephosphorylation of a wide variety of proteins. However, studies performed on aging cells have not shown any deficiency in the amount, activity or ability of PKC to elicit a signaling pathway (43).
The structure and function of many proteins such as nuclear proteins topoisomerase I, DNA ligase II, endonuclease, histones HI, H2B and H4, DNA polymerases α and β, and cytoplasmic proteins adenyl cyclase and elongation factor EF-2 is modulated by ADP-ribosylation (44). Indirect evidence suggests that poly-ADP-ribosylation of proteins may decrease during aging because the activity of poly-(ADP)ribose polymerase (PARP) decreases in aging human fibroblasts both as a function of donor age and during serial passaging in vitro (45). Similarly, the direct relationship observed between maximum lifespan of a species and the activity of PARP in mononuclear leukocytes of 13 mammalian species indicates its important role in aging and longevity (46). One cytoplasmic protein that can be specifically ribosylated by diphtheria toxin and exotoxin A is the protein elongation factor EF-2.
The amount of EF-2 that can be ADP-ribosylated in the presence of diphtheria toxin in cell-free extracts decreases significantly during aging of human fibroblasts in culture (33). Another protein, which appears to be mono-ADP-ribosylated, is the antiproliferative protein prohibitin, located primarily in the mitochondria (47). However, further studies are required to establish the role of ADP-ribosylation on the activity of various proteins during aging.
Methylation of nitrogens of arginine, lysine and histidine, and carboxyls of glutamate and aspartate residues is a widely observed post-translational modification that is involved in many cellular functions. Proteins whose activities are increased by methylation include alcohol dehydrogenase, histones, ribosomal proteins, cytochrome C, elongation factor EF-1α, myosin, myelin and rhodopsin. Of these, decreased methylation of histones in livers and brains of aging rats has been reported. On the other hand, there is no difference in the extent of methylation of newly synthesized histones during cellular aging of human fibroblasts in culture. 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 monomethyllysine (for details, see 48).
One of the main reasons for the inactivation of enzymes during aging may be their oxidative modification by oxygen free radicals and by mixed-function oxidation (MFO) systems or metal catalyzed oxidation (MCO) systems. Since some amino acid residues, particularly proline, arginine and lysine, are oxidized to carbonyl derivatives, the amount of carbonyl content of proteins has been used as an estimate of protein oxidation during aging (49). Increased levels of oxidatively modified proteins have been reported in old human erythrocytes of higher density, and in cultured human fibroblasts (49). Similarly, the reduced motor coordination of old mice has been related to high levels of oxidized proteins in the cerebellum (50). Structural alterations introduced into proteins by oxidation can lead to the aggregation, fragmentation, denaturation, and distortion of secondary and tertiary structure (49).
Glycation is one of the most prevalent covalent modifications in which the free amino groups of proteins react with glucose, forming a ketoamine called Amadori product. This is followed by a sequence of further reactions and rearrangements, producing the so-called AGE, or advanced glycosylation end products (51). Most commonly, it is the long-lived structural proteins such as lens crystallins, collagen and basement membrane proteins, which are more susceptible to glycation. The glycated proteins are then more prone to form crosslinks with other proteins, leading to structural and functional alterations.
An increase in the levels of glycated proteins during aging has been observed in a wide variety of systems, such as rat sciatic nerve, aorta and skin collagen (52), and human collagen and osteocalcin (53). An age-related increase in collagen pentosidine has been reported in eight mammalian species, and the rate of increase was inversely related with the maximum lifespan of the species (54). Pyrroline, another AGE protein, has been shown to increase in diabetics (51). By using AGE-specific antibodies, an AGE-modified form of human hemoglobin has been identified whose levels increase during aging and in patients with diabetes-induced hyperglycemia (55).
6.3.6. DEAMIDATION, RACEMIZATION AND ISOMERIZATION
Age-related changes in the catalytic activity, heat stability, affinity for substrate and other physical characteristics, such as the conformation of proteins, may also be due to the charge change introduced by conversion of a neutral amide group to an acidic group by deamidation. Spontaneous deamidation of asparaginyl and glutaminyl residues of several proteins has been related with the observed accumulation of their inactive and heat labile isoforms during aging (56). The interconversion of optical isoforms of amino acids, called racemization, has been reported to increase during aging. The concentration of D-aspartate in protein hydrolysates from human teeth, erythrocytes and eye lens increases with age (57).
Racemization of tyrosine has been reported to occur in the aging brunescent human cataract lenses (58).
6.3.7. OTHER MODIFICATIONS
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-1α may be involved in determining its stability and interaction with intracellular membranes (59). However, no studies with respect to its age-related changes have been performed as yet.
The protein initiation factor eIF-5A contains an unusual amino acid, hypusine, which is synthesized post-translationally as a result of a series of enzymically catalyzed alterations of a lysine residue (60). A dramatic attenuation of hypusine formation in senescent human fibroblasts has been reported (11). Protein tyrosine sulfation is another post-translational modification that may have significance in protein alteration during aging because it is involved in determining the biological activity of neuropeptides and the intracellular transportation of a secretory protein (61). Prenylation, the covalent attachment of isoprenoid lipids on cysteine-rich proteins, is involved in the regulation of the activity of some proto-oncogenic ras proteins and the nuclear lamins A and B (62).
Recent evidence shows an age-dependent decrease in the activity of prenyltransferases in the rat liver, which may account for the changes in the synthesis and turnover of mevalonate pathways lipids including cholesterol, ubiquinone and dolichol (63). Altered acetylation of histones results in premature growth arrest and a senescence-like state in human fibroblasts (64).
The roles of chaperones in protein folding and conformational organization are poorly understood in relation to the aging process. There is some evidence that both the pentose-mediated protein crosslinking (65) and transglutaminase-mediated crosslinking (66) of proteins is involved in aging. There is a high correlation between pentosidine protein crosslinks and pigmentation in senescent and cataract-affected human lens (67). Similarly, an increase in transglutaminase activity during cellular apoptosis, differentiation and aging of human keratinocytes (68, 69) indicates an important role of this modification in the process of aging.
Finally, intracellular translation and post-translational modifications of proteins are closely related processes that practically set a limit on their functional efficiency and stability. It is therefore crucial that in order to elucidate the mechanisms of aging, various enzymic and non-enzymic protein components of the networks of repair and maintenance processes are analyzed in detail in terms of their synthesis, modifications and turnover.