Date of Award
Doctor of Philosophy (PhD)
Kelly L. Jordan-Sciutto
Multisystem proteinopathy (MSP) is a degenerative syndrome incorporating features of inclusion body myopathy (IBM), Paget’s disease of bone (PDB), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) that is currently incurable and ultimately fatal. Missense mutations in the prion-like domains (PrLDs) of the genes encoding the RNA-binding proteins (RBPs) heterogeneous nuclear ribonucleoprotein (hnRNP) A1 (D262V) and hnRNPA2 (D290V) cause MSP. These MSP-linked mutations introduce a potent steric zipper into the PrLD and accelerate spontaneous hnRNPA1 and hnRNPA2 fibrillogenesis. However, the mechanism by which these variants of hnRNPA1 and hnRNPA2 cause disease is unknown. Here, we employ Saccharomyces cerevisiae as a model system to recapitulate the cellular phenotype seen in MSP patients and map the determinants of hnRNPA1 and hnRNPA2 toxicity and misfolding. We have also utilized a candidate gene approach and an unbiased gene deletion screen to identify genetic modifiers of hnRNPA1 and hnRNPA2 toxicity. Using a series of deletion and truncation constructs, we have determined that hnRNPA1 and hnRNPA2 require at least one intact RNA-recognition motif and a portion of the low complexity PrLD to confer toxicity. Thus, we propose a mechanism of toxicity that requires RNA binding and formation of cytoplasmic inclusions by hnRNPA1 or hnRNPA2. hnRNPA1 and hnRNPA2 form self-templating fibrils in vitro, which cannot occur in the absence of the PrLD. We identified forty gene deletions that suppressed the toxicity of hnRNPA1 and hnRNPA2, including RNP (ribonucleoprotein)-granule components (Sbp1, Lsm6, and Lsm7), molecular chaperones (Hsc82, Sti1, Sse1, and Ydj1), and spliceosome proteins (Lsm6, Lsm7, Prp18, and Bud31). In all cases, genetic suppressors of hnRNPA1 toxicity also suppressed hnRNPA2 toxicity, indicating mechanistic convergence. Importantly, only five genes from this list are known modifiers of FUS or TDP-43 toxicity in yeast. TDP-43 and FUS are also RBPs with PrLDs implicated in the pathogenesis of neurodegenerative disease. This lack of overlap in genetic modifiers suggests important mechanistic differences in the underpinnings of cellular toxicity mediated by hnRNPA1 and hnRNPA2 versus TDP-43 or FUS. By contrast, engineered variants of a protein disaggregase, Hsp104, that possess potentiated disaggregase activity suppressed the toxicity of TDP-43 and FUS in addition to hnRNPA1, hnRNPA2, and both MSP-linked mutant hnRNPs. Potentiated Hsp104 variants, therefore, represent a possibly broadly efficacious therapeutic that could be developed to combat a range of neurodegenerative phenotypes caused by RBPs with PrLDs. The toxicity suppressors that we have identified may ultimately have therapeutic implications for not only MSP patients, but also patients with sporadic ALS, FTD, IBM, and PDB. Future work should include investigation of existing small-molecule inhibitors, for example Hsp90 or Hsp70 inhibitors, that mimic the genetic deletions we have uncovered for potential therapeutic use.
Ford, Alice Flynn, "Mechanisms And Modifiers Of Protein Misfolding And Toxicity Associated With Multisystem Proteinopathy" (2017). Publicly Accessible Penn Dissertations. 2285.
Available for download on Saturday, August 15, 2020