Author ORCID Identifier

https://orcid.org/0000-0002-1615-1992

Date of Award

Fall 12-16-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Parjit Kaur

Second Advisor

Dr. Margo A. Brinton

Third Advisor

Dr. Zehava Eichenbaum

Fourth Advisor

Dr. Eric Gilbert

Abstract

Mammalian ATP-binding cassette subfamily F member 3 (ABCF3) is a class 2 ABC protein that has previously been identified as a partner of the mouse flavivirus resistance protein 2′,5′-oligoadenylate synthetase 1B (OAS1B). The functions and natural substrates of ABCF3 are currently not known. In Chapter 1 of this study, we show that purified ABCF3 is an active ATPase. Binding analyses with a fluorescent ATP analog TNP-ATP suggested unequal contributions by the two nucleotide-binding domains. We further showed that ABCF3 activity is increased by lipids, including sphingosine, sphingomyelin, platelet-activating factor, and lysophosphatidylcholine. However, cholesterol inhibited ABCF3 activity, whereas alkyl ether lipids either inhibited or resulted in a biphasic response, suggesting small changes in lipid structure differentially affect ABCF3 activity. Point mutations in the two nucleotide-binding domains of ABCF3 affected basal and sphingosine-stimulated ATPase activity differently, further supporting different roles for the two catalytic pockets. We propose a model in which pocket 1 is the site of basal catalysis, whereas pocket 2 engages in ligand-stimulated ATP hydrolysis. Co-localization of the ABCF3–OAS1B complex to the virus-remodeled endoplasmic reticulum membrane has been shown before. We show that co-expression of ABCF3 and OAS1B in bacteria alleviated growth inhibition caused by expression of OAS1B alone, and significantly enhanced OAS1B levels, indirectly showing interaction between these two proteins in bacterial cells. As viral RNA synthesis requires large amounts of ATP, we conclude that lipid-stimulated ATP hydrolysis may contribute to the reduction in viral RNA production characteristic of the flavivirus resistance phenotype.

Chapter 2 of this dissertation provides a comprehensive review of the major known antibiotic resistance mechanisms, including the function of ABC proteins, found in producer soil bacteria and discusses different horizontal gene transfer mechanisms that may play a role in the dissemination of resistance genes from producer and non-producer environmental bacteria to pathogenic bacteria in clinical settings. Many bacterial and eukaryotic ABC proteins are polyspecific in nature and are capable of transporting structurally diverse compounds, including drugs and lipids. These proteins are responsible for intrinsic or acquired multidrug resistance, which can also spread to pathogenic organisms through the horizontal transfer mechanisms discussed in this review.

DOI

https://doi.org/10.57709/15726231

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