Poster abstracts

Poster number 143 submitted by Jingxuan Tang

Neurodegenerative VCP Mutant and Wild type Co-assembly and Stoichiometry Assessment by Single Molecule Assays

Jingxuan Tang (Department of Chemistry, University of Michigan ), Ben Dodd (Department of Human Genetics, University of Michigan ), Andreas Schimidt (Department of Chemistry, University of Michigan ), Stephanie Moon (Department of Human Genetics, Center for RNA Biomedicine, University of Michigan *co-corresponding), Nils Walter (Department of Chemistry, Center for RNA Biomedicine, University of Michigan *co-corresponding)

Abstract:
The ATPase valosin-containing protein (VCP), with its hexametric structure, is crucial for unfolding ubiquitinated protein substrates to facilitate proteasomal degradation. A single amino acid mutation within VCP can cause amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). The mechanism of pathogenesis remains undefined. Studies have revealed persistent ubiquitin-positive aggregates in patient tissues with the VCP mutation, indicating that the ubiquitinated misfolded proteins may not be properly unfolded and degraded. This may be due to a loss of function caused by the mutation. Surprisingly, in vitro studies have demonstrated enhanced ATPase activity and unfoldase rate in mutated VCP homomers when compared to the wildtype. Given that these studies were conducted only with homomeric mutant VCP, but most patients have heterozygous VCP mutations, our hypothesis is that mutant VCP monomers can co-assemble with wildtype, leading to compromised motor protein function. To test whether wildtype and mutant VCP co-assemble, we co-expressed SNAP- and Halo-tagged, distinctly fluorophore-labeled wildtype and mutant VCP, respectively, in HeLa cell lysate and visualized the assembly stoichiometry using single molecule pull-down (SiMPull) combined with total internal reflection fluorescence (TIRF) microscopy-based single molecule photobleaching (SMPB). Unfolding assays will further be employed to ask whether co-assembled heteromeric VCP shows loss of function.

Keywords: ALSFTD, Motor proteins, Single molecule pull-down assay