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Investigating Novel Turnover Mechanisms of Huntingtin in Yeast
Author(s) -
Warner Amanda,
Buchan Ross
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00571
Subject(s) - huntingtin , endocytosis , microbiology and biotechnology , cytoplasm , endocytic cycle , biology , huntingtin protein , autophagy , protein aggregation , vacuole , chemistry , mutant , apoptosis , biochemistry , cell , gene
Huntington’s disease is a devastating neurodegenerative disease which leads to symptoms of involuntary movements, memory loss, and ultimately death. The disease is characterized by a CAG repeat expansion mutation in the huntingtin (HTT) protein which leads to its aggregation inside the cytoplasm of striatum neurons. It is unknown whether these aggregates are directly toxic or if they reflect a cellular protection mechanism. Recently, endocytosis activity has been shown to reduce the toxicity, protein levels and propensity to aggregate of TDP‐43, a protein that forms potentially toxic aggregates in amyotrophic lateral sclerosis (ALS) patient motor‐neurons. Conversely, high expression of aggregation‐prone TDP‐43 can impair endocytosis function. Thus, there is a possibility that HTT aggregates, similar to TDP‐43 in ALS, will also inhibit endocytosis and be cleared by the endocytosis pathway. Using a Huntington’s disease yeast model, wild type and mutant HTT localize to the cytoplasm as well as the vacuole, an acidic organelle where proteins are trafficked by endocytosis for degradation. “WT” HTT with 25 CAG repeats is expressed diffusely throughout the cytoplasm while “pathological” HTT with 103 CAG repeats forms cytoplasmic aggregates, in addition to exhibiting diffuse cytoplasmic localization. Pathological HTT exhibits slight growth defects in vps34Δ and vps15Δ strains; these PI3K subunit genes are key to normal endocytic (and autophagic) function. Protein half‐life assays indicate that WT HTT has a slower turnover rate compared to the pathological HTT. Proteasome inhibition also slows WT HTT turnover but does not impact pathological HTT turnover rate. A genetic screen using the tet‐off regulatable promoter library will be conducted to determine for the first time what essential genes genetically interact with mutant HTT. Further microscopy and turnover assays will be conducted to determine if enhancement or suppression of endocytosis leads to clearance or accumulation of HTT in the vacuole respectively, and whether endocytosis activity represents a viable therapeutic target for Huntington’s disease. Support or Funding Information This project was funded by the University of Arizona Undergraduate Biology Research Program with funds from the Arnold and Mabel Beckman Foundation.