SummaryPresbyopia, the inability to focus up close, affects everyone by age 50 and is the most common eye condition. It is thought to result from changes to the lens over time making it less flexible. We present evidence that presbyopia may be the result of age-related changes to the proteins of the lens fibre cells. Specifically, we show that there is a progressive decrease in the concentration of the chaperone, α α α α -crystallin, in human lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and insoluble protein. This is accompanied by a large increase in lens stiffness. Stiffness increases even more dramatically after middle age following the disappearance of free soluble α α α α -crystallin from the centre of the lens. These alterations in α α α α -crystallin and aggregated protein in human lenses can be reproduced simply by exposing intact pig lenses to elevated temperatures, for example, 50 °°°° C. In this model system, the same protein changes are also associated with a progressive increase in lens stiffness. These data suggest a functional role for α α α α -crystallin in the human lens acting as a small heat shock protein and helping to maintain lens flexibility. Presbyopia may be the result of a loss of α α α α -crystallin coupled with progressive heat-induced denaturation of structural proteins in the lens during the first five decades of life.

It has only recently been appreciated that the human body contains many long-lived proteins. Their gradual degradation over time contributes to human aging and probably also to a range of age-related disorders. Indeed progressive damage of proteins may be implicated in the fact that many neurological diseases do not appear until after middle age. A major factor responsible for the deterioration of old proteins is the spontaneous breakdown of susceptible amino acid residues resulting in racemisation, truncation, deamidation, and cross-linking. When proteins decompose in this way, their structures and functions may be altered and novel epitopes can be formed that can induce an autoimmune response.

Background It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification. Scope of review Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. Major conclusions The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein-protein interactions are disrupted and lens opacification ensues. General Significance Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases.

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characterizing these can have clinical relevance for understanding the molecular basis of ocular conditions such as presbyopia ( 2 ) and nuclear cataract ( 3, 4 ).It is not known which are the critical factors predisposing the human lens to age-related nuclear (ARN) cataract. Research from one of us, over many years, suggests that the formation of an internal barrier to the diffusion of small molecules may be a key event in the onset of this pathology ( 5, 6 ). The barrier forms in the normal lens at middle age and uncouples the center of the lens from the metabolically active outer region. Because the lens grows continuously throughout life, the outmost part of the lens is the region that was formed most recently and has the highest concentration of active enzymes ( 7,8 ). It is where the major antioxidant glutathione (GSH) is synthesized and the oxidized form of glutathione rereduced ( 9, 10 ). Since GSH is essential for maintaining a reducing environment in the lens center and for protection of protein structure, formation of the barrier allows oxidative modifi cation of nuclear proteins that is the hallmark of nuclear cataract. The dimensions of the barrier region (7 mm equatorial × 3 mm axial) corresponds to the part of the adult lens that was synthesized immediately after birth ( 11,12 ).Recent data implicate the binding of denatured proteins to the fi ber cell membrane as the mechanism responsible for the barrier ( 13 ), possibly by occluding the membrane pores that normally facilitate the movement of GSH and water from cell to cell via connexon ( 14 ) and aquaporin 0 ( 15 ) channels, respectively. Moreover, aquaporin 0 requires interaction with distinct membrane lipids to form its correct functional conformation ( 15 ). If interactions with fi ber cell Abstract The formation of an internal barrier to the diffusion of small molecules in the lens during middle age is hypothesized to be a key event in the development of age-related nuclear (ARN) cataract. Changes in membrane lipids with age may be responsible. In this study, we investigated the effect of age on the distribution of sphingomyelins, the most abundant lens phospholipids. Human lens Due to a lack of turnover ( 1 ), the lens is an ideal tissue for examining age-related changes to biomolecules, and

As the human lens ages, bound water is progressively changed to free water. Advanced nuclear cataract may be associated with lower total hydration of the lens nucleus.

Changes in lens proteins and membranes can be detected in each decade of life; however, major changes to the lens crystallins of the nucleus take place between age 40 and 50, after the loss of free soluble alpha crystallin. These alterations are consistent with large-scale binding of crystallin aggregates to fiber cell membranes after middle age.

SummaryThe centre of the human lens, which is composed of proteins that were synthesized prior to birth, is an ideal model for the evaluation of long-term protein stability and processes responsible for the degradation of macromolecules. By analysing the sequences of peptides present in human lens nuclei, characteristic features of intrinsic protein instability were determined. Prominent was the cleavage on the N-terminal side of serine residues. Despite accounting for just 9% of the amino acid composition of crystallins, peptides with N-terminal Ser represented one-quarter of all peptides. Nonenzymatic cleavage at Ser could be reproduced by incubating peptides at elevated temperatures. Serine residues may thus represent susceptible sites for autolysis in polypeptides exposed to physiological conditions over a period of years. Once these sites are cleaved, other chemical processes result in progressive removal or 'laddering' of amino acid residues from newly exposed N-and C-termini. As N-terminal Ser peptides originated from several crystallins with unrelated sequences, this may represent a general feature of long-lived proteins.Key words: human lens; long-lived proteins; protein instability; proteolysis; spontaneous cleavage.Long-lived polypeptides are present in heart, lung, brain, lens (Lynnerup et al., 2008), teeth, blood vessels, skin and connective tissue (Ritz-Timme & Collins, 2002). Most, like elastin and collagen, are extracellular, although some, for example, crystallins are cytosolic. Over decades at body temperature, persistent proteins undergo modifications that may affect their structure and function. These include racemization, deamidation, modification by reactive metabolites and truncation (Cloos & Fledelius, 2000;Truscott, 2011), although little is known about which processes are most significant. Enzymatic cleavage of long-lived polypeptides is a significant route for decomposition; however, the centre of the adult human lens is devoid of active proteases. In their absence, it is possible to identify amino acids that are prone to spontaneous cleavage under physiological conditions. One well-characterized reaction at Asn residues (Geiger & Clarke, 1987) involves intramolecular attack, followed by ring opening and peptide bond cleavage with the formation of a new C-terminal Asp residue. To uncover cleavage sites of long-lived proteins, we examined the terminal residues of peptides present in the centre of aged human lenses.A total of 211 unique peptides were identified in the nuclei of four human lenses aged 16, 44, 75 and 83 years (Table S1, Supporting information), with a false discovery rate of 0.09. Peptides from aA-and aB-crystallin were most numerous (Fig. S1, Supporting information), while b-and cS-crystallin peptides were also present (Fig. S2). Interestingly, peptides originating from cC-and cD-crystallins were not detected. The diversity of peptides suggested that either extensive chemically mediated hydrolysis was taking place or proteases remain active in the mature nucleus. As no pr...