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