Regulation of Proteasome Activity

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PROJECT SUMMARY / ABSTRACT The focus of my laboratory’s efforts is the proteasome, the protease that degrades ubiquitin-protein conjugates. That the proteasome is the central activity within the ubiquitin-proteasome system (UPS) was known from the time the UPS was charted, but its significance was not fully recognized at the time because the proteasome was presumed, incorrectly, to be unregulated and passive–essentially a dumb enzyme. But this perception has been overturned by the progressive identification of diverse mechanisms that finely regulate proteasome synthesis, turnover, localization, substrate specificity, and specific activity. These mechanisms are of special interest because they provide the means for global control of UPS output. In parallel, evidence of the importance of proteasome activity in disease has accumulated. For example, even a modest elevation of proteasome levels substantially increases the lifespan of D. melanogaster and C. elegans, as well as their ability to withstand stresses such as the expression of toxic polyQ proteins. A fascinating mode of proteasome regulation is that involving the dynamic reconfiguration of ubiquitin chains on a substrate at the proteasome. Two such chain-editing factors are highly active as well as conserved across eukaryotes: Ubp6/USP14 and Hul5/UBE3C. Ubp6 is a deubiquitinating enzyme and Hul5 is a ubiquitin ligase, and they work in opposition to one another; Ubp6 will remove ubiquitin groups added to the substrate by Hul5. Both are recruited to proteasomes when the UPS is impaired or challenged. The specificity of Ubp6 is remarkable in that it acts only on substrates that carry multiple ubiquitin chains. We will characterize this specificity further and investigate its mechanistic basis. We have reported that Hul5 functions as an E4 on the proteasome–it ubiquitinates proteins that are already ubiquitinated. We will focus now on how this E4 activity promotes the processivity of the proteasome, as Hul5 appears to be the main regulator of processivity. We will reconstitute the processivity effect in a purified system and use highly specific mutants in Hul5 in single-molecule analysis. We will also investigate the mechanisms by which Ubp6 and Hul5 are controlled by stress. The third major factor recruited to proteasomes under proteostasis stress is Ecm29, which has the unique feature of binding both the RP and CP. Ecm29 regulates both the activity and assembly of the proteasome, most likely by bridging these complexes. Our studies will focus on how Ecm29 is recruited to faulty proteasomes and how it affects their structure and stability. Finally, substrate recognition by the proteasome is mediated by six distinct ubiquitin receptors. However, our detailed genetic analysis in yeast indicates the existence of at least one additional, unknown ubiquitin receptor within the proteasome. We will attempt to identify this receptor, and once we generate suitably precise mutants we will explore this receptor’s role in substrate recognition and processing. In summary, we propose, by applying the methods of genetics, enzymology, structural biology, and quantitative global proteomics, to elucidate major pathways of proteasome function and regulation.