Title: Structural and mechanistic insights into the regulation of protein degradation by the p97/Cdc48 chaperone
Funder: Cancer Research UK

Abstract: 1) Background _x000D_ The regulation of protein turnover by the Ubiquitin Processing System is fundamental to the viability of eukaryotic cells. Part of this process involves detecting and regulating the turnover of misfolded proteins via endoplasmic reticulum associated protein degradation (ERAD). ERAD plays an important role in ER homeostasis to ensure optimal cell function and survival. One key regulator of ERAD is the AAA-type ATPase p97 (also known as VCP or Cdc48 in yeast), which together with several protein co-factors mediates a number of key biochemical events. Ongoing work suggest two modes of action for p97, namely translocation of partially unfolded substrates such as in ERAD and mitochondria protein regulation or disassembly of multi-protein or protein-DNA complexes such as HSP90-HSF1 or the a2 repressor promoter DNA complex._x000D_ _x000D_ 2) Specific aims of the research_x000D_ This proposal has two main aims: _x000D_ (1) To decipher the structural and mechanistic details of the p97 ‘unfoldase’ and ‘segregase’ activities _x000D_ (2) To determine the structures and mechanisms of p97-cofactor complexes directly implicated in cancer pathways. _x000D_ The detailed structural, biophysical and biochemical analyses of p97-mediated processes will underpin future opportunities for therapeutic intervention._x000D_ _x000D_ More specifically, we will _x000D_ (i) Study the organisation, architecture and conformational changes of the p97-core machinery (p97-Ufd1/Npl4 and Cdc48-Npl4/Vms1 complexes) with ubiquitylated substrates_x000D_ (ii) Decipher the biochemical mechanisms of p97 by developing an in vitro unfolding/disassembly assay _x000D_ (iii) Determine the structures and mechanisms of the cancer-relevant p97-FAF1, p97-UBXD7 cofactor complexes _x000D_ _x000D_ 3) Methods_x000D_ To achieve our aims we will:_x000D_ (i) Reproduce and characterize in vitro specific p97-cofactor-substrate complexes using recombinant proteins and biophysical characterization tools (e.g. ITC, SAXS, native mass spectrometry)_x000D_ (ii) Determine the structures of p97-cofactor-substrates by combining X-ray crystallography and single particle cryo-EM approaches_x000D_ (iii) Develop an in vitro unfolding/disassembly assay using physiologically relevant ubiquitylated substrates_x000D_ (iv) Test mechanistic hypothesis for p97-cofactor function using in vivo assays (through collaboration)_x000D_ _x000D_ 4) How the results of this research will be used_x000D_ The results of this research will provide a fundamental understanding of how mammalian cells regulate the turnover of unfolded proteins as well as important multi-protein and protein-DNA complexes. They will also provide mechanistic insights into key cellular processes such as cell cycle regulation, ERAD and the DNA damage response and will underpin parallel studies aimed at developing specific p97-cofactor inhibitors for cancer therapeutic development. _x000D_ _x000D_ _x000D_


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS