Regulatory mechanisms linking protein synthesis and degradation

Project Summary Properly functioning cells must tightly regulate protein synthesis, folding and degradation: es- sential processes for gene expression collectively known as proteostasis. Proteostasis is a balanc- ing act between the activities of the ribosome and the proteasome, which respectively synthesize and degrade proteins. Dysregulated proteostasis may lead to or promote tumorigenesis, and cor- relates with age-related diseases like Parkinson’s and Alzheimer’s. The clinical use of proteasome inhibitors, particularly to treat multiple myeloma, further underscores the need to determine what stress responses and regulatory mechanisms they activate, and how they do so. Cells are known to respond to proteasome inhibition by invoking mechanisms of translational control that lead to the phosphorylation of translation initiation factor eIF2α, which attenuates protein synthesis. However, it is unclear which kinases phosphorylate eIF2α upon proteasome inhibition, and the mechanisms that regulate this translational response are not well understood. I propose to close gaps in our knowledge of the regulatory landscape between protein synthesis and protein degradation, by pursuing two primary objectives. First, I will investigate the factors that modulate eIF2α phosphorylation in response to proteasome inhibition, including determining which eIF2alpha kinases are activated by proteasome inhibition. I have also conducted pooled, RNA- linked CRISPR knockout and polysome profiling experiments that led to intriguing preliminary data. These data suggest that an E3 ligase, UBR1, plays a significant role in attenuating translation upon proteasome inhibition. Accordingly, my second objective is to determine how UBR1, and its substrates, are influenced by proteasome inhibition to regulate the ensuing translational response. Through a combination of innovative sequencing techniques and classic biochemical ap- proaches, my research will further elucidate the regulatory networks linking protein synthesis and degradation. The outcomes of my work could enhance our understanding of how proteasome inhibitors function as chemotherapies, and inform new therapeutic strategies for use against age- related diseases and cancers.