Sunday, December 22, 2024
12/22/2024
PROJECT SUMMARY/ABSTRACT UBR1 is a RING-family ubiquitin ligase that regulates protein quality control through the N-end rule pathway. Human disease variants in UBR1 contribute to the development of Johanson-Blizzard Syndrome. JBS is a rare but serious disorder with symptoms including pancreatic insufficiency, mental retardation, and psychomotor developmental delay. Due to a lack of suitable animal models, in vivo functions of UBR1 in the nervous system have not been thoroughly examined. In this proposal, we use C. elegans as a model organism to study UBR1 function. UBR-1 in C. elegans is the sole ortholog of mammalian UBR1/UBR2. In preliminary studies, we have identified locomotor defects in ubr-1 deletion mutants. We have also observed expression of UBR-1 in head neurons using a novel CRISPR-tagged GFP::UBR-1 construct. These preliminary data suggest that UBR-1 functions in the nervous system to regulate locomotor behavior. Additionally, we have performed the first CRISPR-based in vivo AP proteomics in C. elegans, and have identified four glutamate metabolic enzymes: GDH-1, GOT-2.2, GLN-3, and GFAT-1. We hypothesize that these four enzymes represent a regulatory network for glutamate synthesis, with UBR-1 as the master regulator. In order to test our hypothesis, we will 1) characterize UBR-1 function in the nervous system through genetic, behavioral, and protein expression studies and 2) determine biochemical mechanisms of UBR-1 regulation of glutamate enzymes through expanded proteomics, biochemical validation, and genetic suppression experiments. This proposal makes use of a highly innovative approach to set CRISPR-based AP proteomics as the next-generation standard for in vivo proteomics in C. elegans. We also make extensive use of computational and automated behavioral tracking to assess locomotor behavior in an unbiased manner. Finally, we have generated numerous CRISPR-edited and engineered strains that: 1) assess UBR-1 and glutamate metabolic enzyme function in the nervous system, 2) distinguish between UBR-1 substrates and non-substrate binding partners, 3) can be used to identify UBR-1 expression in specific cell populations, and 4) test function and pathogenicity of human variants of UBR-1. The novel strategies and reagents that we will use to complete our goals will lay the groundwork to carry out comprehensive studies to determine physical and genetic interactions between UBR-1 and glutamate metabolic enzymes in nervous system function.
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