Proteasome homeostasis and substrate prioritization

PROJECT SUMMARY During optimal growth, cells have sufficient proteasome capacity to degrade proteins that have been marked for degradation by ubiquitination. However, in response to stress, like heat stress or oxidative stress, proteasome capacity is adjusted to meet cellular demand. It is important to understand how cells regulate this adjustment as many stress conditions occur upon aging and in human diseases, like neurodegenerative diseases and cancer. Diverse cellular responses have been observed depending on the type of stress, and the overarching goal of this proposal is to gain mechanistic insight into different proteasome responses following stress. The upregulation of proteasomes is one such response, which not only involves transcriptional regulation, but also the assembly of newly synthesized subunits in proteasome complexes. A surprising complexity exists in early steps of assembly where it is unclear how various proteasome associated factors, like Pba1-Pba2/PAC1-PAC2, Fub1/PI31, and Blm10/PA200, contribute to sequential steps or parallel pathways of the assembly process. Understanding these pathways is a focus of this proposal because it impacts the amount and type of proteasomes that form and thereby the cells' degradative capacity. The relocalization of proteasomes is another stress response which involves either cytosolic condensate formation or autophagic targeting (proteaphagy). Condensate formation occurs via liquid-liquid phase separation and functions to store proteasomes, protect them from proteaphagy, or concentrate them with substrates in degradation centers. The formation of such condensates generally depends on the formation of multivalent interactions. The interactions that drive this process for the proteasome, however, are not well understood. To gain new insights into the process, the proposed research will utilize newly identified conditions and factors that regulate proteasome condensates in yeast. Finally, the proposal will test the hypothesis that under conditions of limited proteasome capacity, proteasomes prioritize the degradation of certain substrates over others. This is an original and important concept that will be analyzed using in vivo degradation assays. The goal is to determine how proteasome- associated proteins contribute to this prioritization. The proteasome responses to stress have been described in yeast as well as human cells, indicating evolutionary conserved responses that are intricately connected to various cellular processes and changing conditions. Using both budding yeast and mammalian tissue culture, the biochemical and cell biological assays proposed here will provide original and new insights into the processes that determine the levels, composition, and substrate prioritization of proteasomes in the cell. Such insights will provide fundamental knowledge about these important cellular complexes and create a foundation for efforts to identify targets and drugs that increase or decrease proteasome levels and localized activity in cells. The ability to manipulate proteasome in vivo has been shown to be of therapeutic value.