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Characterization of the C. elegans Ortholog of SAMHD1

Studdard, Lydia (2017)
Master's Thesis (48 pages)
Committee Chair / Thesis Adviser: Kim, Baek
Committee Members: Anderson, Larry J ; Kelly, William G ; Schinazi, Raymond F ; Steel, John
Research Fields: Biochemistry; Molecular biology; Microbiology
Keywords: SAMHD1; C. elegans; dNTP
Program: Laney Graduate School, Biological and Biomedical Sciences (Microbiology & Molecular Genetics)
Permanent url: http://pid.emory.edu/ark:/25593/s31vj

Abstract

Aicardi-Goutières Syndrome (AGS) is a genetic disorder that mimics in-utero viral infection. The defining characteristic of AGS is the presence of increased levels of interferon alpha in the cerebral spinal fluid in the absence of any infection. It is thought that AGS is a result of defects in responses to aberrant nucleic acids. Mutations thought to lead to AGS are found in genes coding for proteins involved in regulating cellular responses to these nucleic acids. One such protein is sterile alpha motif and HD domain containing protein 1 (SAMHD1) which is responsible for degrading dNTPs in non-cycling cells. The dNTPase activity of SAMHD1 allows it to restrict HIV-1 infection in nondividing cells. Currently a mouse model is used to study SAMHD1 linked AGS, however this model has not been shown to reproduce the clinical phenotype of AGS. In this thesis, C. elegans and its ortholog of SAMHD1 are studied as a possible alternative animal model.

Total RNA derived from C. elegans was used to generate a cDNA clone of the C. elegans ortholog of SAMHD1 (ceSAMHD1). This cDNA was cloned into a bacterial expression plasmid and a lentiviral expression plasmid. The protein was produced and purified from bacteria and used in several biochemical assays. ceSAMHD1 was also expressed in a monocytic cell line lacking human SAMHD1 and its effect on dNTP levels as well as HIV-1 restriction was assayed. Finally, ceSAMHD1 was knocked down in C. elegans and the animals were observed for developmental defects.

This work determined that ceSAMHD1 is capable of degrading dNTPs and is regulated in a similar manner as human SAMHD1in biochemical assays. In human cells, ceSAMHD1 was not capable of degrading dNTPs or restricting HIV-1 infection. Finally, worms treated with ceSAMHD1 RNAi did not show any developmental defects. In conclusion, C. elegans does not provide a more clinically relevant animal model than the mouse model used for studying SAMHD1 related AGS.

Table of Contents

Table of Contents

Chapter I: Introduction

A. Aicardi-GoutiΓ¨res Syndrome…………….……………………………………pg. 1

a. Clinical Phenotype

b. Implicated Genes

c. Mouse Model of SAMHD1 associated AGS

B. SAMHD1……………………………………………………………………...pg. 2

a. Effect of SAMHD1 on HIV-1

b. dNTPase Function and Regulation

c. Other Proposed Functions

d. C. elegans as a model to study SAMHD1

Chapter II: Biochemical Characterization of the SAMHD1 Ortholog in Caenorhabditis elegans, ZK177.8

A. Abstract………………………………………………………………………..pg. 7

B. Introduction…………………………………………………..………………..pg. 8

C. Results………………………………………………………………………..pg. 11

D. Discussion………………..…………………………………………………..pg. 15

E. Acknowledgements…………………………………………………………..pg. 18

F. Figures………………………………………………………………………..pg. 19

G. Experimental Procedures…………………………………………………….pg. 26

Chapter III: Conclusion……………………………………………...…………….pg. 30

References…………………………………………………………………………...pg. 32


Table of Figures

Figure 1………………………………………………………………………………pg. 19

Figure 2………………………………………………………………………………pg. 20

Figure 3………………………………………………………………………………pg. 21

Figure 4………………………………………………………………………………pg. 23

Figure 5………………………………………………………………………………pg. 24


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