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Date of Award
Sokol V. Todi
Protein quality control (PQC) is indispensable for normal cellular functions, ensuring proteins are properly folded and removing those proteins that are functioning aberrantly. Perturbations in PQC can lead to various malignancies, neurodegeneration and neurological diseases. The Ubiquitin-Proteasome Pathway (UPP) is one important pathway in PQC, it relies on ubiquitination-dependent post-translational modification to selectively degrade misfolded or short-lived protein. Ubiquitination is reversed by the action of proteases known as deubiquitinating enzymes (DUBs). By hydrolyzing ubiquitin linkages, DUBs are responsible for cleaving and activating newly produced ubiquitin molecules, editing poly-Ub chains, removing ubiquitin from a substrate protein and recycling mono-Ub. Here, I investigated two proteins critically involved in the UPP: one that is important for normal cellular function (USP5), and another whose mutations cause neurodegeneration (ataxin-3).
Ubiquitin Specific Protease 5 (USP5) is a member of the USP class of DUBs. Based on structural and in vitro results, it was long thought to function as a recycler of mono-Ub in the cell, by hydrolyzing unanchored poly-Ub chains. We studied the function of USP5 in vivo in Drosophila melanogaster. Knockdown of DmUSP5 in the whole fly results in death during developmental stages and in an increase in poly-Ub chains, but does not result in a depletion of mono-Ub in the fly, contrary to long-held opinion. Based on our data, lethality from USP5 knockdown is not due to depletion of mono-Ub, but likely resulted due to accumulation of unanchored poly-Ub chains and disruption of proteasomal degradation. Our results also suggest that USP5 has specific substrates.
The other DUB investigated, ataxin-3, is the disease causing protein in the age-related, poly-glutamine (polyQ) dependent disease Spinocerebellar Ataxia Type-3 (SCA3). Through its different interacting partners, ataxin-3 is involved in protein quality control pathways. One such protein is the AAA ATPase Valosin Containing Protein (VCP). Ataxin-3 interacts directly with VCP through a VCP Binding Motif (VBM). Through biochemistry, Drosophila genetics and physiological assays, we showed that pathogenic ataxin-3 with a mutated VBM is markedly less toxic than its intact pathogenic counterpart. Mutating the VBM of ataxin-3 caused a delay in the aggregation of ataxin-3 in vivo, in various tissues tested. We propose that VCP acts as a nucleation center to increase local concentrations of ataxin-3 proteins and increase their likelihood of interacting and fibrilization. Our work suggest this interaction as a potential therapeutic target for SCA3.
Ristic, Gorica, "Deubiquitinases In Ubiquitin Homeostasis And Disease" (2017). Wayne State University Dissertations. 1863.