Architecture and function of condensates formed by ubiquitin-binding shuttle proteins and protein quality control components - PROJECT SUMMARY/ABSTRACT: Efficient and effective protein quality control (PQC) is essential to proper cell function. Despite extensive research, mechanisms underlying PQC remain incompletely understood. Consequently, development of new therapies to treat conditions with dysregulated PQC such as proteinopathies and neurodegenerative disorders is impeded. This MIRA research program targets the Ubiquitin (Ub)-mediated PQC pathways, and specifically Ub-containing biomolecular condensates. These condensates are membraneless assemblies comprising Ub-binding shuttle proteins (e.g., UBQLNs, Rad23s) that preferentially condense with specific types of polyUb chains and PQC components; the condensates are involved in a wide range of physiological processes including stress response, proteasomal degradation, autophagy, and immune system activation. My lab’s work is grounded in the hypothesis that dysregulation of these condensates leads to Ub-containing inclusions characteristic of proteinopathies and neurodegenerative disorders, and specifically amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We recently determined that ALS-linked UBQLN2 forms stress-induced biomolecular condensates in cells and that the full-length UBQLNs (1/2/4) all undergo phase separation in vitro to form condensates under physiological conditions. Important to the premise guiding our projects is the observation that interactions with polyUb chains (in length- and linkage- dependent manners) tune conditions for condensate assembly and disassembly. We predict that other PQC components further tune the assembly/disassembly and structure of these condensates. In this five-year MIRA program, my lab will use molecular biophysics and cell biology-based approaches to achieve a mechanistic understanding of how Ub-binding shuttle proteins (UBQLNs, Rad23s, DDIs) regulate function of biomolecular condensates in stress response and PQC. Our preliminary data from reconstitution experiments suggest that condensates drive distinct PQC outcomes (e.g., protecting substrates from degradation or driving substrate degradation). We surmise that these different outcomes stem from different conditions driving condensate formation and/or the varied molecular architectures of condensates composed of differing compositions of shuttle proteins and ubiquitinated substrates. Our projects will determine (1) the physiological functions of UBQLN condensate formation in cells under a myriad of cell stress conditions, (2) the molecular rules by which polyUb chains modulate condensation of shuttle proteins and PQC components (e.g., full proteasomes, deubiquitinases, ligases), and (3) the emergent functions of shuttle protein PQC condensates in regulating substrate degradation, protection from degradation, and substrate ubiquitination/deubiquitination. The results from these projects will uncover the role of condensates in mediating PQC under physiological and stress conditions.
