Date of Award

January 2016

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Chittaranjan Das

Committee Member 1

Mathew C Tantama

Committee Member 2

Mark A Lipton

Abstract

Chlamydia Trachomatis (C. trachomatis), a gram negative, obligate intracellular bacterium, is implicated in a variety of diseases including sexually transmitted urogenital infection, as well as ocular infection. Reliance on the host cell for its survival and propagation causes the bacterium to release various bacterial effector proteins to manipulate its host cell processes. Two of these factors which may aid in the bacterium’s survival, C. trachomatis deubiquitinase 1 (ChlaDub1) and C. trachomatis deubiquitinase 2 (ChlaDub2) are cysteine proteases which have been previously shown to exhibit deubiquitinating and deneddylating activity. In this study, constructs of ChlaDub2 were cloned, purified, and subjected to enzymatic assays to probe its activity. Using diubiquitin of defined linkage type, it was demonstrated that ChlaDub2 shows a preference for Lys-63 linked diubiquitin over Lys-48 linked diubiquitin, albeit at a reduced rate with an increased enzyme concentration. Additional biochemical assays were carried out with polyubiquitinated GFP substrate that demonstrated ChlaDub2’s activity was more enhanced at a lower enzyme concentration and reaction time. As the polyubiquitinated GFP would contain a heterogenous array of different linked polyubiquitin chains with mainly a population of Lys-63 linked chains, insight is gained that ChlaDub2 may potentially prefer longer length polyubiquitin. These results potentially point towards the physiological role ChlaDub2 plays in the cell by protecting the C. trachomatis vacuole from Lys-63 induced lysosomal degradation. Additionally, crystallographic studies were performed and diffraction and a dataset were obtained; however, at the time, the structure could not be phased.

Download

Share

COinS