Torsins are essential ATPases that localize within the endoplasmic reticulum (ER)/nuclear envelope (NE) membrane system where they carry out poorly defined functions. Mutations in TorsinA, one of the four human Torsins, cause the debilitating neurological movement disorder DYT1 dystonia, which has no cure. Torsins are unusual ATPases because they alone are inactive. To hydrolyze ATP, Torsins must interact with one of two known transmembrane cofactors that reside within the ER/NE system, lamina associated polypeptide 1 (LAP1) and luminal domain like LAP1 (LULL1). As DYT1 dystonia associated TorsinA mutations disrupt its interactions with LAP1/LULL1, this active complex is critical for normal neural development. Of the four human Torsins, three are stimulated by LAP1 and/or LULL1 but Torsin2A has an unknown activation mechanism. The hallmark phenotype of Torsin loss-of-function is deformations of the inner nuclear membrane (INM) referred to as blebs that sequester unique protein quality control (PQC) foci enriched for K48-linked polyubiquitinated proteins. The combined loss of TorsinA, TorsinB, and Torsin3A in HeLa cells results in a moderate number of blebs, however, the additional loss of Torsin2A has the strongest effect on blebs and causes the most to form. This suggests that an additional, unknown activator for Torsin2A exists. While recent studies demonstrate that blebs are disrupted, immature nuclear pore complexes (NPCs), many questions remain about the PQC foci within blebs. The source, identity, and recruitment mechanism of the K48-ubiqutinated protein substrates remain unknown; however, my preliminary studies suggest a role for the nucleoporin NDC1 in targeting PQC substrates to the INM. This unusual PQC defect that arises upon Torsin deficiency likely contributes to DYT1 dystonia etiology as all models of this disease demonstrate this phenotype. The overarching hypothesis of this proposal is that Torsins participate in a specific PQC pathway that affects the stability of proteins require for normal NPC biogenesis. I will determine the contribution to Torsin?s PQC pathway of specific chaperones I have identified to localize within blebs. I will accomplish this by using a truncated viral protein that localizes specifically to blebs in a ubiquitin-dependent manner. I will investigate the connection between NPC biogenesis and PQC by exploring the role for NDC1 in targeting Torsin PQC substrates to the INM. To discover yet-unidentified Torsin activators and pathways redundant to the Torsin/activator system, I will perform a genome wide CRISPR screen in cells devoid of LAP1 and LULL1. These data will be compared to our previously conducted screens in 4 Torsin knockout and wildtype cells. Discovering redundant pathways is a largely unexplored strategy that I expect will have clinical relevance as modulating these proteins instead of TorsinA may represent a therapeutic opportunity for DYT1 dystonia.
The aims described herein will uncover molecular details of Torsin?s PQC pathway and identify additional Torsin activators. These results will facilitate achieving the long-term goal of this research?to discover the precise molecular function of Torsins so that targeted DYT1 dystonia therapies can be developed.
Protein quality control is a central feature of normal cellular homeostasis and its perturbation is implicated in a variety of diseases. One such neurological disease, DYT1 dystonia, arises upon mutation of the ATPase TorsinA, which localizes within the endoplasmic reticulum/nuclear envelope system where I hypothesize it contributes to nuclear pore biogenesis by participating in an elusive protein quality control pathway. Because Torsin?s precise function remains to be defined, DYT1 has no cure and therefore, this proposal aims to describe the molecular details of Torsin?s protein quality control pathway so that targeted DYT1 dystonia therapies can be developed.
