Regulatory Dynamics of the Proteostasis Network

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PROJECT SUMMARY / ABSTRACT Cancer and neurodegeneration are often thought to be on opposing ends of the disease spectrum – the former characterized by unchecked cell growth and the latter by premature cell death. However, they are linked by dysregulation of a common underlying cellular state: protein homeostasis (proteostasis). Proteostasis refers to the dynamic balance of protein synthesis, folding and degradation, the maintenance of which is required for the vast majority of cellular processes. Defects in proteostasis lead to protein aggregation, a hallmark of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s and ALS. Conversely, many cancer cells overexpress proteostasis machinery to counteract high mutational loads. The long-term goal of my research program is to define the mechanisms that healthy cells employ to maintain proteostasis and to establish how this regulation breaks down and is hijacked in disease. The central regulatory axis of the proteostasis network (PN) is the heat shock response (HSR), a universally conserved gene expression program under the control of transcription factor Hsf1 in eukaryotes. Despite its identification 30 year ago, the mechanisms that control Hsf1 activity and thereby expression of the HSR have remained elusive. Recent work from my laboratory using budding yeast revealed a key feedback loop that governs Hsf1 activity. We found that the chaperone Hsp70 represses Hsf1 in unstressed cells and that Hsf1-mediated induction of Hsp70 is requied to deactivate Hsf1. Thus, Hsf1 and Hsp70 constitute a feedback loop that promotes homeostatic adaptation to stress This proposal builds upon this discovery and the tools and assays we have developed to answer major outstanding questions concerning Hsf1 regulation. In Aim 1 we will define the upstream signaling events that activate Hsf1. We hypothesize that ribosome-nascent chain complexes with C-terminal alanine/threonine extensions (“CAT-tails”) and newly synthesized “orphan” ribosomal proteins (oRPs) trigger the HSR by sequestering the Hsp70 co-chaperone Sis1 away from Hsf1. In Aim 2 we will define the molecular mechanisms by which Hsp70 represses Hsf1. We will test the hypothesis that Hsp70 represses Hsf1 both by inhibiting Hsf1 DNA binding and by blocking Hsf1-mediated transactivation. In Aim 3 we will reveal novel feedback loops that control Hsf1. We will systematically determine the contribution of all Hsf1 targets to wild-type Hsf1 activation dynamics. By undertaking a multi-faceted analysis of the HSR – combining cell biology, molecular genetics, biochemistry and systems biology – we will establish the signals, regulatory mechanisms and feedback loops that govern this central adaptive circuit that lies at the heart of human diseases as diverse as cancer and neurodegeneration.