Date of Award

1-1-2015

Document Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Scott D. Pegan

Second Advisor

Martin Margittai

Keywords

Deubiquitinating protease, ISG15, Ubiquitin, vOTU

Abstract

Proteases from the Ovarian Tumor domain (OTU) superfamily of deubiquitinating enzymes (DUBs) are expressed by a range of RNA viruses. Viral OTUs (vOTUs) are found in nairoviruses such as Crimean Congo Hemorrhagic Fever virus (CCHFV), Nairobi sheep disease virus (NSDV), the Erve virus (ERVEV), and the Dugbe virus (DUGV), as well as in the areterivirus Porcine Reproductive and Respiratory Syndrome virus (PRRSV), among others. vOTUs, which interfere with host innate immune response through editing of host Ub and Ub-like molecules such as interferon stimulated gene 15 (ISG15), have been identified as a potential virulence factor through their role in evading the innate immune response. Study and characterization of vOTUs involves evaluating their activity and preferences for Ub, ISG15, as well as the variety of poly-Ub linkages that can form. While previous studies have compared vOTUs among different viruses, it was not known how vOTUs from differing strains of the same virus vary. To investigate vOTUs from differing strains of the same virus, vOTUs from two strains of PRRSV were selected. The vOTU of a highly pathogenic PRRSV strain, JXwn06, and a strain used in vaccination, MLV, were enzymatically characterized. Intriguingly, the JXwn06 vOTU displayed a strong preference for K63 linked di-Ub, with over 40-fold greater activity than the MLV vOTU. Additionally, neither strain showed significant activity towards ISG15, which is intriguing, as, at one point, it was believed that deISGylating activity was a property of all vOTUs. In contrast to these PRRS vOTUs, a strong deISGylating enzyme, the ERVE vOTU, was selected for enzymatic and structural investigation. ERVE vOTU was found to not only prefer ISG15 over Ub, but recognized both mouse and human ISG15. In order to gain insight into this ability to recognize more than one species of ISG15, the X-ray crystal structure of the ERVE vOTU in complex with the C-terminal domain of mouse ISG15 (CmISG15) was elucidated to 2.47 Å. This structure not only revealed the structural configuration of the ERVE vOTU in complex with its substrate, but also provides the first structural information for mISG15. Key areas of binding were identified upon comparison of the manner in which ERVE vOTU recognizes the mouse analog of ISG15 with CCHF vOTU substrate binding. These key regions were explored through site-directed mutagenesis to determine if mutation at key binding areas would allow CCHF vOTU to more tightly bind mISG15. Based on CCHF vOTUs apparent lack of recognition of mISG15, special ISG15 constructs were designed to allow for testing against a panel of ISG15s from varying species.

In another area of interest, adenylosuccinate lyase (ADSL) was structurally and enzymatically investigated to build understanding of ADSL deficiency, a rare autosomal recessive disorder, which causes a defect in purine metabolism resulting in neurological and physiological symptoms. To better understand the causation of disease due to the R303C mutation, the wild type (WT) and the R303C mutant of ADSL were investigated enzymatically and thermodynamically. Additionally, the X-ray structures of ADSL in its apo form as well as with the R303C mutation were elucidated, providing insight into ADSL's cooperativity.

Comments

Copyright is held by the author.

Provenance

Recieved from ProQuest

Rights holder

Michelle Kay Deaton

File size

90 p.

File format

application/pdf

Language

en

Discipline

Biochemistry

Included in

Biochemistry Commons

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