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S Endicott

Publications and source records attributed to S Endicott.

4 recordsLinked to original sources
PubMed1999

Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers.

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer. When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits. Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present. On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.

Cross-Linking Reagents↗
PubMed1998

Proteasome activation by REG molecules lacking homolog-specific inserts.

The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein. REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai. At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert. These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.

Amino Acid Sequence↗