Dissecting neomorphic functions mediated by mutant-specific structures of SPOP

St. Jude Children'S Research Hospital, Memphis TN

Investigators

Abstract

Summary Mutations in the ubiquitin-proteasome system (UPS), including in ubiquitin-ligase components such as VHL, BRCA1, FBW7, APC and SPOP, lead to deregulation of protein levels and can drive tumorigenesis. Therapeutics against such UPS components are therefore promising, but toxic on-target effects on the wild-type protein in normal cells can severely limit tolerated doses and the therapeutic window. Mutant-specific therapeutics could overcome these limitations. Hence, the characterization of cancer mutants that adopt different structures than WT proteins is of importance for making progress in the direction of mutant-specific therapies. Here we propose to capitalize on recent breakthroughs from my laboratory, in which we solved the first structure of full-length SPOP (the Speckle-type POZ protein), which is a substrate receptor of the Cullin3-RING ubiquitin ligase (CRL3) and recruits substrates to the ligase for their ubiquitination and subsequent degradation. SPOP is frequently mutated in prostate, endometrial, and other solid tumors. Our structure of SPOP reports new protein-protein interfaces that have not been observed in previous structures of partial SPOP constructs. These interfaces are lined with mutations whose underlying molecular mechanism was so far not understood because the mutated residues seemed to be solvent exposed. Furthermore, the structures of two SPOP endometrial cancer mutants and other preliminary data show that these activating cancer mutations have effects on the higher-order structure of SPOP that differentiate them decisively from the WT structure: (i) They shift the equilibrium between a linear filament and a circular donut state, the latter of which is likely inactive based on several lines of preliminary data. Or (ii) they radically alter the higher-order assembly structures. We will capitalize on our recent progress and integrate structural characterization with our previously developed biophysical, biochemical and cell biological tools and reagents to answer the following questions: (1) How does self-association into different states, including linear and circular oligomers, regulate SPOP function? (2) Do the structure-altered SPOP mutants function through their altered structural states? (3) And what is the potential of SPOP mutants to form altered quaternary assemblies? The expected structures of donut states and active complexes will be interrogated by mutagenesis and supplemented with functional assays in vitro and in cells to dissect the molecular mechanisms underlying their super-physiological function. We expect to gain decisive insight into the question whether the structurally altered forms are functionally important, with implications for targeting specifically the mutant forms. We will also gain understanding of how higher-order assemblies can drive hyper-morphic or neo-morphic disease phenotypes. Using machine learning models, we will make our results transferable to other cancer-associated proteins whose function is intrinsic to their ability to form higher-order oligomers.