Purification and phosphatidylcholine specificity of StAR related lipid transfer domain containing 7 Open Access

Sharma, Ayushi (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/cj82k7327?locale=en
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Abstract

 

STAR related lipid transport domain containing 7, or StarD7, is a lipid binding protein that binds and shuttles phosphatidylcholine (PC) from the endoplasmic reticulum and Golgi apparatus to the mitochondria. StarD7 is important in maintaining the mitochondria’s membrane composition, which is important for oxidative phosphorylation and cellular homeostasis, as knockdown of StarD7 greatly attenuated activity of enzymes involved in oxidative phosphorylation. Dysregulation of StarD7 is associated with cancer, and leads to many disorders such as pulmonary inflammation, mucous cell metaplasia, and airway hyperresponsiveness. We set out to determine the first structure of StarD7 using X-ray crystallography to gain insights into certain proteins necessary for cellular metabolism and homeostasis. We hypothesize that StarD7 has evolved to bind specific PCs, in order to regulate mitochondrial PC composition; the inner mitochondrial membrane has a higher concentration of PCs that the outer mitochondrial membrane. Determining the structure will help us better understand the mechanism by while StarD7 bind PCs and allow us to create specific mutations to probe StarD7 biology. The purification and isolation of StarD7 proved to be incredibly difficult in both bacterial and mammalian cell systems, despite the use of several solubility tags and different StarD7 truncations. In the future, we plan to create a chimera of StarD7 with an added N-terminal helix from a homolog that was previously crystallized to help shield hydrophobic regions of StarD7 to aid in protein solubility and purification.

 

Table of Contents

 

 

1 Chapter 1 Introduction

9 Chapter 2 Isolating StarD7 – pET SUMO Vector

17 Chapter 3 Isolating StarD7 – pET15b Vector

22 Chapter 4 Isolating StarD7 – Truncated Transcript

23 Chapter 5 Crystallizing Truncated StarD7 – Two Tags

28 Chapter 6 StarD7 in a Mammalian Cell Line System

29 Chapter 7 Fixing the Truncated StarD7 with a Chimera

32 Chapter 8 Future Directions and Conclusions

33 Bibliography

 

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