Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

A role for the ubiquitin-proteasome (Ub-Prot) pathway in the degradation of newly synthesized apolipoprotein B100 (apoB) in hepatocytes was studied. In vitro the apoB degradation assay included rabbit reticulocyte lysate (RRL) as the source of the Ub conjugation system and Prot. [3H]-labeled apoB was immunoprecipitated from [3H]-leucine labeled Hep G2 cells, a hepatoma cell line. Isolated [3H]-apoB was stable for 2 hr at 37C in the presence of buffer plus RRL alone, but was degraded when exogenous ATP plus ATP regenerating system was added to the reaction mixture. Degradation of apoB was inhibited about 50% by addition of potent proteasome inhibitors, lactacystin beta-lactone (10uM) and MG-132 (50uM), but was not inhibited by a cysteine protease inhibitor, E-64, or a serine protease inhibitor, PMSF. Ubiquitin aldehyde, an inhibitor of Ub-protein isopeptidases, inhibited apoB degradation up to 83% in a dose dependent manner. The current results suggest that extremely large apoB polypeptide is ubiquitinated and degraded by the Prot. This in vitro assay can be utilized to identify intermediate steps in proteasomal degradation of apoB.

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Poster Number PAsakata0627
Keywords: apolipoprotein B, Ubiquitin, Proteasome