Mechanisms of Chaperone-Mediated Control in the Assembly of the Proteasome - Project Summary In the current paradigm of protein degradation by the ubiquitin-proteasome system, ubiquitination of protein substrates has been considered as a major regulatory step. Hundreds of ubiquitinating enzymes regulate polyubiquitination, to target proteins to the proteasome for degradation. This paradigm has led to remarkable discoveries on how protein degradation is required for fundamental cellular processes, and also for destroying misfolded or aberrant proteins, which can lead to cancers and neurodegenerative conditions. However, a significant knowledge gap exists in the current paradigm, as to how the cell ensures the proper number of functional proteasomes. Our research goal is to overcome this gap, by elucidating mechanisms of proteasome assembly. We discovered multiple evolutionarily conserved chaperones that are dedicated to proteasome assembly. In our model of proteasome assembly, chaperones hinder subunit addition until a given step of assembly occurs correctly. This model suggests a new concept, that assembly is not simply a series of subunit additions—it is also integrated with extensive quality control (QC). We will pursue this new direction of research—unraveling mechanisms of chaperone-mediated QC during proteasome assembly. For QC, the assembly process is a “moving target” as one step progresses to the next. How can these moving targets be monitored? Chaperones can “mark” all of them, using a fundamental mechanism—specifically binding to assembly intermediates. We will investigate how chaperones serve as components (e.g. adaptors) of extensive QC of the proteasome, via three aims. First, we will examine chaperones’ new connections to proteasomal nuclear localization signals (NLSs), which have been known for ~20 years to exist on proteasome subunits, but their functions are poorly characterized. We hypothesize that proteasomal NLSs drive chaperones into the nucleus, as a QC mechanism, to prevent defective proteasomes from forming in the cytoplasm. Second, we will investigate the non-canonical role of ubiquitination via an E3 ligase, Not4, during proteasome assembly. Bulky polyubiquitin sterically prevents the addition of the next subunits, blocking proteasome assembly, for QC. We hypothesize that this non-canonical role of polyubiquitin depends on a specific chaperone, Nas6 (an oncoprotein, Gankyrin in humans). Third, we will interrogate how chaperones help form proteasome storage granules, which preserve functional proteasomes during nutritional stress. We hypothesize that chaperones help distinguish functional proteasomes—via binding to sub-complexes—but in this case, resulting from proteasome disassembly during nutritional stress. We will interrogate these hypotheses using our well- established biochemical, cell biological, and proteomics strategies, together with our powerful yeast model to assess proteasome functions in vivo. Proteasome assembly chaperones are dysregulated in many cancers, altering proteasomal activities. Since the proteasome is a proven therapeutic target, we envision exploiting these chaperones to modulate cellular protein degradation, to broaden the application of the proteasome as a target.
