Crystallin modifications associated with cataracts The lens crystallins are a major potential target for accumulating damage associated with age-related cataracts, although there are certainly others. Thus, as the crystal-lins accumulate modifications and damage over the lifetime of an individual, their ability to participate in appropriate intermolecular interactions, and even to remain in solution, decreases. Whether proteins in age-related cataracts become insoluble as a result of complete or partial denaturation, or whether they simply become less soluble due to modifications that leave their protein folds largely intact or both, is not currently known. However, it seems clear that modifications to crystallin proteins accumulate with aging and accelerate during cataractogenesis, and the combination of crystallin modification, disulfide-crosslinking, denaturation, and aggregation results in loss of lens transparency and cataract formation (Hanson et al., 2000). The protein modifications involved in this process include, but are not limited to, proteolysis, racemation, oxidative changes, and glyca-tion. The many factors believed to induce these modifications include free radicals and superoxides, along with a loss of the lens' reducing state causing oxidation and disulfide-crosslinking, sugar accumulation causing glyca-tion, and cyanate causing carbamylation.
Protein modifications in age-related cataracts are believed to arise from a combination of environmental and endogenous factors. For instance, considerable evidence suggests that oxidative modifications are a hallmark of age-related cataracts and oxidation of crystallins and other lens proteins likely results from reactive oxygen species that are produced by both UV-light exposure and are also a byproduct of mitochondrial respiration during which as much as 2% of respiratory oxygen is converted to reactive oxygen species. A major result of oxidation is conversion of methionine to methionine sulfoxide, which increases with age in the human lens and reaches levels as high as 60% in age-related cataracts relative to clear lenses.
Multiple identified and yet unidentified proteases are present in the lens and proteolyzed crystallins are a predominate feature of age-related cataracts. Among multiple lens proteases that have been identified to act on crystallin proteins, calcium-activated proteases are believed to play major roles. Proteolysis of specific crystallins is believed to result in protein aggregation and cataracts.
Proteins in age-related cataracts become insoluble as a result of complete or partial denaturation or by becoming less soluble due to modifications that leave their protein folds largely intact, or perhaps by a combination of these processes. Many highly studied Mendelian congenital cataract models support both denaturation, as is seen in the association of some severe crystallin mutations with cataracts, and simple insolubility with maintained protein folds as is seen in other cataracts. Many classical studies have demonstrated that lens proteins become insoluble because they are denatured as the lens ages. Insoluble protein in the aged cataractous lens not only is denatured and crosslinked, but a fraction exists as relatively short peptides cleaved from larger proteins. It seems likely that the presence of large amounts of unstable or precipitated crystallin, or other protein, does damage to the lens cell and its proteins and eventually contributes to cataracts not only directly through light scattering by protein aggregates but eventually also through disruption of cellular metabolism and damage to the cellular architecture. This is clear from numerous mouse models of cataracts resulting from crystallin mutations (Graw and Loster, 2003).
Gene expression changes in cataract In addition to crystallin modifications, age-related cataracts are also associated with changes in gene expression detected at the level of increased or decreased mRNA in the lens epithelium (Hejtmancik and Kantorow, 2004). Since the lens epithelial cells cover the anterior surface of the lens, whereas in age-related cataracts the opacities tend to occur in the nuclear or cortical fiber cells, these gene expression changes likely reflect responses of lens epithelial cells to the presence of underlying cataracts and/or altered epithelial function in the presence of cataracts. These gene expression changes nevertheless point to altered lens pathways associated with this disease. For instance, the mRNAs encoding metallothio-nein and osteonectin (also known as SPARC, secreted acidic protein rich in cysteines) are increased in cataracts, whereas those for protein phosphatase 2A regulatory subunit and some ribosomal proteins including L21, L15, L13a, and L7a are decreased. These alterations suggest that increased binding of toxic metals and Ca++ with a concomitant decrease in growth pathways and protein synthesis are features of cataract.
In addition to the identification of individual alterations in gene expression, more recent studies have sought to identify the full range of gene expression changes that occur in the lens epithelium upon cataract formation using DNA microarrays. Although literally thousands of genes whose expression is altered in cataract have been identified in these studies, some specific examples of genes increased in cataract include SP1 required cofactor for transcriptional regulation, osteomodulin, chloride channel 3, Na+K+ transporting polypeptide beta 1, and Ca++ transporting ATPase, whereas genes decreased include aA-crystallin, multiple glutathione peroxidases, multiple ribosomal subunits, HSP 27, Na+/K+ ATPase and transketolase. The majority of the identified genes are decreased in cataract, suggesting loss of gene expression as a consequence of lens damage. Functional clustering of the identified genes suggests that the genes increased in cataract tend to be associated with transcriptional control, ionic and cytoplasmic transport, protein salvaging pathways, and extracellular matrix components; transcripts decreased in cataract tend to be associated with protein synthesis, defense against oxidative stress, heat shock/chaperone activity, structural components of the lens, and cell cycle control (Hejtmancik and Kantorow, 2004).
Enzyme changes associated with cataracts In addition to the protein modification and gene expression changes noted earlier, numerous metabolic and enzyme activity changes are also associated with age-related cataracts. These changes include decreased reduced glutathione content, decreased NADPH levels, increased free Ca++ levels, increased activity of specific proteases, and decreased ionic balance, among others. Considerable evidence suggests that many of these changes, other metabolic changes, and loss of lens protein function results from loss of the activities of specific lens protective and repair enzymes and other homeostatic systems. Although the evidence for these changes has been almost exclusively derived from animal, cell, and organ culture experimental systems, loss of the activities of multiple protective systems including a-crystallins, MnSOD, catalase, glutathione peroxidase, and y-glutamylcysteine synthetase among many others are believed to contribute to loss of lens function and ultimately cataract formation. In addition to the loss of lens protective and homeostatic systems, the loss of key repair systems including thioltransferase and methionine sulfoxide reductases are also believed to be key events in cataract formation.
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