Degradation of an Old Human Protein

Michael G Friedrich,Roger J.W Truscott,Jackson Lam

doi:10.1074/jbc.m112.391565

Michael G Friedrich, Roger J.W Truscott + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m112.391565

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Abstract

Long-lived proteins exist in a number of tissues in the human body; however, little is known about the reactions involved in their degradation over time. Lens proteins, which do not turn over, provide a useful system to examine such processes. Using a combination of Western blotting and proteomic methodology, age-related changes to a major protein, γS-crystallin, were studied. By teenage years, insoluble intact γS-crystallin was detected, indicative of protein denaturation. This was not the only change, however, because blots revealed evidence of significant cross-linking as well as cleavage of γS-crystallin in all adult lenses. Cleavage at a serine residue near the C terminus was a major reaction that caused the release of a 12-residue peptide, SPAVQSFRRIVE, which bound tightly to lens cell membranes. Several other crystallin-derived peptides with double basic residues also lodged in the cell membrane fraction. Model studies showed that once cleaved from γS-crystallin, SPAVQSFRRIVE adopts a markedly different shape from that in the intact protein. Further, the acquired helical conformation may explain why the peptide seems to affect water permeability. This observation may help explain the changes to cell membranes known to be associated with aging in human lenses. Age-related cleavage of long-lived proteins may therefore yield peptides with untoward biological activity.

Highlights

IntroductionConclusion: The peptide released from ␥S-crystallin can adopt an ␣-helical conformation and may alter permeability by interacting with cell membranes
Western Blots—In order to visualize the extent of age-related modification of ␥S-crystallin, samples of both water-soluble protein (WSP) and waterinsoluble proteins, which were solubilized with 8 M urea to generate ureasoluble protein (USP), from individual human lenses across the age range were examined by gel electrophoresis and Western blotting
Each antibody was first examined with fetal WSP because these proteins should show no age-related post-translational modifications (PTMs)

Summary

Introduction

Conclusion: The peptide released from ␥S-crystallin can adopt an ␣-helical conformation and may alter permeability by interacting with cell membranes. Significance: Peptide binding may explain why human lens membranes change with age. Insoluble intact ␥S-crystallin was detected, indicative of protein denaturation This was not the only change, because blots revealed evidence of significant cross-linking as well as cleavage of ␥S-crystallin in all adult lenses. Cleavage at a serine residue near the C terminus was a major reaction that caused the release of a 12-residue peptide, SPAVQSFRRIVE, which bound tightly to lens cell membranes. The acquired helical conformation may explain why the peptide seems to affect water permeability This observation may help explain the changes to cell membranes known to be associated with aging in human lenses. Age-related cleavage of long-lived proteins may yield peptides with untoward biological activity

Results

Discussion

Conclusion