Carbon turnover in the water-soluble protein of the adult human lens (original) (raw)
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eLife
The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in diseases such as age-related cataracts. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using nitrogen-15 labeling of mice and mass spectrometry. We measured the 14N/15N-peptide ratios of 248 lens proteins, including Crystallin, Aquaporin, Collagen and enzymes that catalyze glycolysis and oxidation/reduction reactions. Direct comparison of lens cortex versus nucleus revealed little or no 15N-protein contents in most nuclear proteins, while there were a broad range of 14N/15N ratios in cortex proteins. Unexpectedly, like Crystallins, many enzymes with relatively high abundance in nucleus were also exceedingly long-lived. The slow replacement of these enzymes in spite of young age of mice suggests their p...
Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging
2021
ABSTRACTCataract disease, a clouding of the eye lens due to precipitation of lens proteins, affects millions of people every year worldwide. The proteins that comprise the lens, the crystallins, show extensive post-translational modifications (PTMs) in aged and cataractous lenses, most commonly deamidation and oxidation. Although surface-exposed glutamines and asparagines show the highest rates of deamidation, multiple modifications can accumulate over time in these long-lived proteins, even for buried residues. Both deamidation and oxidation have been shown to promote crystallin aggregation in vitro; however, it is not clear precisely how these modified crystallins contribute to insolubilization. Here, we report six novel crystal structures of a major human lens protein, γS-crystallin (γS): one of the wild-type in a monomeric state, and five of deamidated γS variants, ranging from three to nine deamidation sites, after varying degrees of sample aging. Consistent with previous work ...
Molecular vision, 2013
To compare levels of S-glutathiolation and S-cysteinylation occurring at more than 60 cysteine residues of 12 different guinea pig lens water-soluble nuclear crystallins following treatment of the animals with hyperbaric oxygen (HBO). Guinea pigs (initially 18 months old) were treated 30X (3X per week for 10 weeks) with HBO (2.5 atm 100% O(2) for 2.5 h) as a model to study the formation of nuclear cataract. This treatment produces a moderate increase in lens nuclear light scatter (compared to denser scatter occurring after 80 HBO treatments), with five- to sixfold increases in levels of protein-bound glutathione (PSSG) and protein-bound cysteine (PSSC). Trypsin digests of lens nuclear water-soluble proteins were analyzed with two-dimensional liquid chromatography and mass spectrometry to identify specific cysteine residues binding either glutathione or cysteine. Lens nuclei of age-matched untreated animals were used as controls. All major crystallins, except αB, were modified to som...
Kinetic Stability of Long-Lived Human Lens γ-Crystallins and Their Isolated Double Greek Key Domains
Biophysical Journal, 2019
The g-crystallins of the eye lens nucleus are among the longest-lived proteins in the human body. Synthesized in utero, they must remain folded and soluble throughout adulthood to maintain lens transparency and avoid cataracts. gDand gS-crystallin are two major monomeric crystallins of the human lens. gD-crystallin is concentrated in the oldest lens fiber cells, the lens nucleus, whereas gS-crystallin is concentrated in the younger cells of the lens cortex. The kinetic stability parameters of these two-domain proteins and their isolated domains were determined and compared. Kinetic unfolding experiments monitored by fluorescence spectroscopy in varying concentrations of guanidinium chloride were used to extrapolate unfolding rate constants and half-lives of the crystallins in the absence of the denaturant. Consistent with their long lifespans in the lens, extrapolated half-lives for the initial unfolding step were on the timescale of years. Both proteins' isolated N-terminal domains were less kinetically stable than their respective C-terminal domains at denaturant concentrations predicted to disrupt the domain interface, but at low denaturant concentrations, the relative kinetic stabilities were reversed. Cataract-associated aggregation has been shown to proceed from partially unfolded intermediates in these proteins; their extreme kinetic stability likely evolved to protect the lens from the initiation of aggregation reactions. Our findings indicate that the domain interface is the source of significant kinetic stability. The gene duplication and fusion event that produced the modern two-domain architecture of vertebrate lens crystallins may be the origin of their high kinetic as well as thermodynamic stability.
new method for studying lens protein changes
Human lenses can be separated into concentric layers by dissolution and the fates of various lens constituents in such layers can be studied with appropriately sensitive techniques. These techniques have been applied in a study of the ageing of lens proteins. It was found that insoluble protein increases with progression from periphery to lens centre. This increase is more marked in older lenses. Analysis of soluble protein using High Performance Liquid Chromatography shows that the proportion of a-crystallin decreases towards the centre of the lens, and that this decrease becomes greater with age. j3-Crystallins maintain a constant proportion except in inner layers of older lenses. y-Crystallins show a slight decrease in content from periphery to centre. With cataract formation ageing changes are exaggerated.
Experimental Eye Research, 2000
This investigation of the water-insoluble crystallins from human lenses has used multiple chromatographic separations to obtain proteins of suf®cient purity for mass spectrometric analysis. Each fraction was analysed to determine the molecular masses of the constituent proteins as well as peptides in tryptic digests of these proteins. The major components of the water-insoluble crystallins were identi®ed as aAand aB-crystallins. In addition, gS-, bB1-, gD-, bA3/A1-and bB2-crystallins were found, in order of decreasing abundance. Although there was evidence of some backbone cleavage, the predominant forms of aA-, aB, bB2-, gS-and gD-crystallins were the intact polypeptide chains. The major modi®cations distinguishing the water-soluble crystallins were increased disul®de bonding, oxidation of Met, deamidation of Gln and Asn and backbone cleavage. Of the many reactions hypothesized to lead to crystallin insolubility and cataract, these results most strongly support metal-catalysed oxidation, deamidation and truncation as initiators of conformational changes that favor aggregation.
Experimental Eye Research, 1984
The aL of cortical and nuclear fiber cells have been studied using hydrodynamical and physicochemical techniques. From the sedimentation and the diffusion coefficients in identical conditions, it can be concluded that aL.n is appreciably larger than aL.c but both have a similar structure in solution: a spherical particle with a high hydration. The aL N not only contains several degraded aA-and aB-peptides but also a typical pattern of j%peptides. The fluorescence spectrum indicates a shift of the hydrophobic tryptophan residues from a hydrophobic environment in aLc to a more solvent-exposed and polar neighbourhood for aL N. Also solubility studies on aLc and aL,N in different solvent conditions and temperatures, indicate more apolar interactions between the peptides of the nuclear aL, than its cortical counterpart. The more hydrophobic interaction pattern of the peptides in aL,N can also be reconciled with a lower mean hydration potential, indicative of a higher hydrophobicity of the degraded aA-peptides.