May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Effects of Post-Synthetic Modifications to Proteins on Their Degradation by the Ubiquitin Proteasome Pathway
Author Affiliations & Notes
  • W. Jiao
    Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
  • E. Dudek
    Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
  • F. Shang
    Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
  • X. Zhang
    Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
  • K. Lampi
    Department of Integrative Biosciences, Oregon Health and Sciences University, Portland, Oregon
  • R. H. Nagaraj
    Departments of Opthalmology and Pharmacology, Case Western Reserve University, Cleveland, Ohio
  • A. Taylor
    Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts
  • Footnotes
    Commercial Relationships W. Jiao, None; E. Dudek, None; F. Shang, None; X. Zhang, None; K. Lampi, None; R.H. Nagaraj, None; A. Taylor, None.
  • Footnotes
    Support NEI RO1 13250, RO1 11717, USDA 58-950-4-401, Johnson and Johnson Focused Giving
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4425. doi:
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      W. Jiao, E. Dudek, F. Shang, X. Zhang, K. Lampi, R. H. Nagaraj, A. Taylor; Effects of Post-Synthetic Modifications to Proteins on Their Degradation by the Ubiquitin Proteasome Pathway. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4425.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: During normal aging, proteins undergo extensive post-synthetic modifications including oxidation, truncation, deamidation and glycation, as well as the sequela to these primary modifications. These modified proteins, if not removed from cells, can form intracellular aggregates and contribute to age-related diseases including cataract, AMD, dementia, diabetes, etc. In this study we determined the susceptibility of modified ßB and αA-crystallins to degradation by the ubiquitin proteasome pathway (UPP).

Methods:: Deamidated mutants of ßB crystallins were produced using site-directed mutagenesis of recombinant Wt proteins. Glycation of Wt αA-crystallin was achieved using methylglyoxal (MGO) treatment at 37oC for 24 h. Human lens cell lysate and rabbit reticulocyte lysate were used as the source of UPP components.

Results:: Wt ßB1-crystallin and the corresponding Q157E and Q204E deamidated mutants demonstrated rapid UPP-dependent degradation as indicated by an absolute requirement for ATP and the proteasome. In contrast, Wt ßB2-crystallin was not degraded at all. In comparison deamidated ßB2 Q162E was degraded primarily by an ATP-dependent (UPP) pathway and ubiquitin conjugates were observed. Wt αA and αA162 (the C-terminally truncated crystallin which is found in the diabetic lens) demonstrated only ATP-dependent degradation, degradation of the latter being specifically stimulated by ubiquitin conjugating enzyme 4. Modification of both with increasing concentrations of MGO, a reactive glycation agent, demonstrated a dose-dependent decrease in ATP-dependent degradation, and increase in aggregation and a corresponding decrease in mono-ubiquitinated conjugates, consistent with failure of the UPP to recognize the glycated αA-crystallins.

Conclusions:: The ubiquitin pathway is a critical regulator of protein levels. The susceptibility of modified crystallin proteins to degradation by the UPP is dictated by the specific modification. In turn, this selectivity is probably related to the ability of the UPP to maintain cellular function and tissue viability.

Keywords: proteolysis • crystallins • protein modifications-post translational 
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