The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding.
Misfolded proteins are aggregation prone and protein aggregation are associated with many human diseases, such as neurodegenerative disorders
and cancer. However, the molecular basis underlying the proteotoxicity and the mechanisms to combat this toxicity remain poorly understood. We
used S. cerevisiae, budding yeast, as a model organism to address these questions as much of the protein homeostasis machinery is conserved form
yeast to humans. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein
(Htt), which is responsible for the development of Huntington's disease, a neurodegenerative disorder. Heat shock proteins (HSPs) function as
molecular chaperones that aid in protein folding and degradation of misfolded proteins. However, the role of heat shock proteins in the
clearance of mutated Htt remains poorly understood. Our previous data indicate that the degradation of mutated Htt with a 103 polyQ expansion
(Htt103QP) depends on both the ubiquitin proteasome system and autophagy in budding yeast. Extended induction of Htt103QP-GFP leads to the
formation of a single inclusion body in wild-type yeast cells. We showed that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors
(Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via
autophagy. In addition, mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Notably, we detected more aggregated
forms of Htt103QP in sse1∆ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in
the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance but are less critical for
proteasome-dependent degradation. These findings uncover the role of HSPs in the inclusion body formation and autophagy-mediated clearance of
mutated Huntingtin. Using budding yeast as a model system, we further asked why misfolded proteins are toxic, and how eukaryotic cells combat
this toxicity. Our results support the notion that ubiquitinated misfolded protein aggregates drain free ubiquitin and compromise
ubiquitin-dependent protein degradation, but the AAA+ ATPase Cdc48 counteracts the toxicity by segregating these protein aggregates. Using
Htt103QP as model misfolded protein, we found that Cdc48 and its two predominant cofactors, Npl4 and Ufd1, are required for the segregation and
degradation of Htt103QP in yeast cells. We also identified the E3 ubiquitin ligase San1 that catalyzes Htt103QP ubiquitination and facilitates
its proteasome-dependent degradation. Unexpectedly, deletion of San1 and another ubiquitin ligase, Ubr1, suppressed the growth defects and
accumulation of ubiquitinated substrates in cells lacking functional Cdc48Ufd1/Npl4. We further show compromised ubiquitin-proteasome system in
cdc48 mutants, as well as the suppression of these defects by san1∆ ubr1∆, indicating that ubiquitination of misfolded proteins contributes to
the growth defect in cdc48 mutants. Importantly, we found that overexpression of ubiquitin partially rescued the growth defects in cdc48
mutants. Finally, we showed that blocking ubiquitination of misfolded proteins by san1∆ ubr1∆ increases the resistance of yeast cells to some
proteotoxic stressors. Our results reveal the basis for the cytotoxicity of misfolded proteins and highlight the role of Cdc48 in alleviating
this toxicity. Lastly, we showed the ubiquitin ligase Rsp5 is necessary for the inclusion body formation and autohagic degradation of Htt103QP.
Also, cells with defective Rsp5 exhibit compromised K63-linked ubiquitination of Htt103QP indicating K63-linked ubiquitination could facilitate
autophagic clearance of Htt103QP. Supporting this notion, cells expressing a mutant form of ubiquitin (K63R) that are unable to promote
K63-linked ubiquitination exhibit Htt103QP IB defect. Rsp5 has also been implicated in ubiquitinating the autophagy adapter protein Cue5. We
showed yeast cells lacking Cue5 exhibit Htt103QP autophagy defect which is consistent with published data that used a different mutated
Huntingtin protein. Taken together, our research work identified several components in the cellular response to misfolded proteins. We
identified a several factors required for mutated Huntingtin inclusion body formation and autophagic degradation. Also, we uncovered a mechanism
that can explain why misfolded proteins are cytotoxic and how cells combat this toxicity. These findings may provide novel targets in developing
strategies to combat protein misfolding diseases and cancer. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements
for the degree of Doctor of Philosophy. / Fall Semester 2018. / November 16, 2018. / Includes bibliographical references. / Yanchang Wang, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Timothy Megraw,
Committee Member; Yi Zhou, Committee Member; Akash Gunjan, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_661147 |
| Contributors | Higgins, Ryan K. (author), Wang, Yanchang (professor directing dissertation), Yu, Hong-Guo (university representative), Megraw, Timothy L. (committee member), Zhou, Yi (committee member), Gunjan, Akash (committee member), Florida State University (degree granting institution), College of Medicine (degree granting college), Department of Biomedical Sciences (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, doctoral thesis |
| Format | 1 online resource (118 pages), computer, application/pdf |
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