Student Theses and Dissertations

Date of Award

2004

Document Type

Thesis

RU Laboratory

Breslow Laboratory

Keywords

cholesterol, StarD4, cholesterol metabolism, lipid transfer proteins

Abstract

thesis describes the discovery, cloning, and initial characterization of StarD4, sterol-regulated gene encoding a StAR-related lipid transfer (START) protein, and its close homologues, StarD5 and StarD6. StarD4 was identified using cDNA microarrays, as liver StarD4 expression decreased three-fold in mice fed a high cholesterol diet. StarD4 also sterol-regulated in cultured cells, and a functional sterol regulatory element (SRE) identified in its promoter. StarD4 was preferentially activated in mouse liver by SREBP-rather than SREBP-1, supporting a role in cholesterol rather than fatty acid metabolism. X-ray crystal structure of StarD4 was solved, revealing a hydrophobic lipid binding cavity described for other START domains. StarD5 and StarD6 were identified by homology to StarD4, and these three genes constituted a novel subfamily most similar to the cholesterolbinding START domains of StAR and MLN64. StarD4 and StarD5 were ubiquitously expressed with highest mRNA levels in liver, while StarD6 expression was limited to male germ cells of the testis. StarD5 was not activated by SREBP or LXR transcription factors, well-characterized regulators of cholesterol metabolism, but rather by the ER stress response, a recently-described means by which cholesterol regulates gene expression. The nematode C. elegans has one StarD4 subfamily protein, K02D3.2. Reporter studies indicated that gene was not regulated by cholesterol or ER stress, and it was only expressed in hypodermal seam cells of embryos and larvae. Overexpression of StarD4 and StarD5 revealed functional activity in three cell culture assays: (1) StAR-like activation of steroidogenesis by mitochondrial P450 side chain cleavage enzyme, (2) repression of an SREBP-regulated reporter, and (3) activation of an LXR-regulated reporter. The START domains of StAR and MLN64 were active in these assays, while the related phosphatidylcholine transfer protein (PCTP) was an inactive negative control. Based on these results, the novel StarD4 subfamily is likely to play roles in the intracellular transport and metabolism of cholesterol.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/207

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