Deciphering The Role Of Hsp110 Chaperones In Diseases Of Protein Misfolding

Author

Unekwu M. Yakubu, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

0000-0003-2230-0066

Date of Graduation

12-2021

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Kevin A. Morano, Ph.D.

Committee Member

Nayun Kim, Ph.D.

Committee Member

Theresa M. Koehler, Ph.D.

Committee Member

Ambro van Hoof, Ph.D.

Committee Member

Sheng Zhang, Ph.D.

Abstract

Molecular chaperones maintain protein homeostasis (proteostasis) by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to the onset of numerous neurodegenerative disorders including Alzheimer’s, Parkinson’s, and Huntington’s disease. Hsp110 is a member of the Hsp70 class of molecular chaperones and acts as a nucleotide exchange factor (NEF) for Hsp70, the preeminent Hsp70-family protein folding chaperone. Hsp110 promotes rapid cycling of ADP for ATP, allowing Hsp70 to properly fold nascent or unfolded polypeptides in iterative cycles. In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate binding domain (SBD) whose biological roles are undefined. Previous work in Drosophila melanogaster has shown that loss of the sole Hsp110 gene (Hsc70Cb) accelerates the aggregation of polyglutamine-expanded human huntingtin (HTT), the causative agent of Huntington’s disease; while its overexpression protects against polyQ-mediated neuronal cell death. I hypothesize that in addition to its role as an Hsp70 NEF, Drosophila Hsp110 (Hsc70Cb) may function as a protective protein “holdase”, preventing the aggregation of unfolded polypeptides via the SBD-β subdomain. In the process of generating deletion mutants in Hsc70Cb to dissect the role of the SBD-β subdomain in holdase activity, I uncovered a redundant and heretofore unknown potent holdase capacity in a 138-amino acid region carboxyl-terminal to both SBD-β and SBD-α (henceforth called the C-terminal extension). This sequence is highly conserved in animal Hsp110 genes and completely absent from fungal representatives, including Saccharomyces cerevisiaeSSE1. Furthermore, the human Hsp110s, Apg-1 and Hsp105α, also contain a C-terminal extension substrate binding region, indicating this site may be conserved among some metazoans. Upon further analysis, I determined C-terminal extension chaperoning is mediated by a predicted short intrinsically disordered region (IDR) in both fly and human Hsp110s. I demonstrate for the first time the carboxy-terminal IDRs in fly and human Hsp110s effectively prevent aggregation of numerous substrates, including amyloidogenic peptides Aβ_1-42 and α-synuclein, in vitro. Additionally, I use the Hsc70Cb^ΔSBD-β construct to show the C-terminal extension is essential for fly embryonic development, and can prevent HTT aggregation in an in vivo disease model. These data indicate Hsc70Cb modulates neurodegeneration by blocking aggregation via a combination of its holdase and nucleotide exchange activities. Through my work I have attributed a biological role to Hsp110 substrate binding, while bestowing molecular and biological significance to a previously undiscovered chaperoning domain contained within the C-terminal extension.

Keywords

Hsp110, Drosophila melanogaster, Hsc70Cb, neurodegenerative diseases, molecular chaperones, substrate binding, huntingtin, amyloid beta, alpha synuclein, metazoan