Dynamics of Ubiquitin-Conjugate Population in Response to Inhibition of Ubiquitin-Proteasome Pathway

Abstract

The goal of this research was to evaluate the dynamics of ubiquitin-protein conjugate populations in response to treatment with inhibitors of the ubiquitin-proteasome pathway. The 26S proteasome is a large multimolecular complex found in all eukaryotic cells that is responsible for maintaining protein homeostasis through degradation of aberrant or excess proteins. Ubiquitin-activating enzyme (UAE) lies upstream of the proteasome and is responsible for labelling proteins for degradation by the proteasome. Chemical or genetic inhibition of UAE activity leads to an accumulation of proteins that are normally turned over by the proteasome. Little is known about the population-level response of ubiquitinated proteins to inhibition of UAE and the proteasome. In the work described in this thesis, the subpopulations of ubiquitin-linkage type were assessed by cellular colocalization assays and quantitative mass spectrometry in bortezomib and TAK-243 treated cells in culture. Immunofluorescence localization data suggests the ubiquitin conjugates that accumulate in perinuclear aggregates are the only conjugates that persist in response to TAK-243 treatment (i.e. are resistant to DUBs). Furthermore, the data suggests that all linkage types of ubiquitin conjugates stochastically accumulate in response to bortezomib and then a fraction of these aggregate in perinuclear bodies in a form that is resistant to degradation or deubiquitination.