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NPC1-mediated trafficking of intracellular cholesterol.

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Call No.	QP752.C5 S84 2003
Author	Sugii, Shigeki.
Title	NPC1-mediated trafficking of intracellular cholesterol.
Date	2003
Abstract	Niemann-Pick type C (NPC) is a neurovisceral disease that causes intracellular accumulation of cholesterol and other lipids. The exact mechanism how the defects in cholesterol trafficking pathways result in the progressive neurological damage is largely unknown at present. By cloning the hamster NPC1 gene, we report that mutations in two independently isolated Chinese hamster ovary (CHO) cell mutants, CT60 and CT43, lead to premature translational terminations of the NPC1 protein. We find that BC theta, a new probe for detecting cholesterol-rich microdomains at the cell surface, could also be used to monitor the distribution of intracellular cholesterol-rich domains in normal and NPC1 cells by microscopy. This tool will be valuable for studying cholesterol trafficking processes in the future. Next, we revise our previous model of low-density lipoprotein (LDL)-derived cholesterol transport by combining an improved kinetic assay involving cyclodextrin, a compound that extracts cholesterol from the cell, biochemical fractionation, and microscopic approaches. The results indicate that there are two distinct endocytic pools: an acid lipase-containing compartment that hydrolyzes the cholesteryl ester of LDL and an NPC1-containing late endosomal compartment. These results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment, then moves to the NPC1-containing late endosome/lysosome before reaching the plasma membrane (PM) and the endoplasmic reticulum. In order to further delineate specific endocytic steps of the cholesterol transport pathway, we biochemically characterize CHO cell mutants defective in the endocytic pathway. We find that a defect in lysosome biogenesis does not significantly affect the trafficking of LDL-derived, PM-labeled, and endogenously synthesized cholesterol. In contrast, a defect in maturation of early endocytic compartments into the late endosome has a great impact on the trafficking processes of cholesterol. Importantly, it was previously shown that the deficiency of a cholesterol biosynthetic enzyme is responsible for this phenotype; we find that inhibition of general cholesterol synthesis partially mimics this phenomenon. The repletion of cholesterol by expressing this enzyme rescues the cholesterol trafficking defects. These results lead us to speculate that cholesterol itself regulates the biogenesis of mature late endosomal compartments.
Description	xvi, 181 leaves : ill. (some col.) 28 cm.
Format	application/pdf
Advisor	Gustav E. Lienhard.
Subject	Cholesterol -- Physiological transport. Cholesterol -- Genetic aspects. Cholesterol -- Metabolism. Genetic disorders -- Molecular aspects.
College	Dartmouth Medical School -- Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.7

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	NPCl-Mediated Trafficking of Intracellular Cholesterol A Thesis Submitted to the Faculty In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Shigeki Sugii DARTMOUTH COLLEGE Hanover, New Hampshire March 3, 2003 Examining Committee: G~av E. Lienhard»h.D. (Chair) Ta-tuan Chang, Ph.D. ; ~ Char~. Ba~lowe, PJJ..B; Paul A. Dawson, Ph.D. Carol L. Folt, Ph.D. Dean of Graduate Studies

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NPC1-mediated trafficking of intracellular cholesterol.

NPC1-mediated trafficking of intracellular cholesterol.