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Characterization of the Proteins and the Mouse Models Associated with Niemann-Pick Type C Disease, A Lysosomal Cholesterol Storage Disorder

Sayali Dixit
M.Sc., 2002
Indian Institute of Technology
Mumbai, India

Thesis Advisors:
Ann Stock, PhD and Peter Lobel, PhD
Graduate Program in Biochemistry and Molecular Biology

CABM 010

Monday, March 17, 2008
10:00 am


Niemann-Pick Type C is a genetic disorder caused by a defect in the intracellular trafficking of cholesterol which leads to an accumulation of unesterified cholesterol in endo/lysosomes. There are two complementation groups associated with this disorder a) NPC1, resulting from defects in NPC1 protein, a 1278 residue-long transmembrane glycoprotein. b) NPC2, resulting from defects in NPC2 protein, a 132 residue-long soluble glycoprotein. Both the NPC proteins reside in endo/lysosomes and are intimately linked to egress of cholesterol out of endo/lysosomes.
My project was focused on characterizing NPC2 protein and NPC1- and NPC2-deficient mice. The previous biochemical characterization of NPC2 had demonstrated existence of six glycoforms of recombinantly-expressed human NPC2; hence we tested functionality of the glycoforms in a cell-culture based assay and demonstrated that all of them are functionally active. We also developed in vitro and in vivo photocrosslinking-based and affinity-purification based methods to screen for binding partners of NPC2.
In order to understand manifestations of the deficiency of the NPC proteins, we analyzed subcellular fractions of the brain and the liver of the wild-type and the NPC-mutant mice. Our data demonstrated that there are massive perturbations in the hydrolase-containing compartments (lysosomes) the NPC-mutant mice. The perturbations include a significant decrease in the buoyant density of lysosomes and an upregulation of the lysosomal enzymes; the extents of perturbations are similar in both the deficiencies.
We also investigated the role of NPC1 and NPC2 in the intestinal cholesterol absorption by measuring efficiencies of cholesterol uptake and absorption in the wild-type and the NPC-mutant mice. Our data indicate that deficiencies in either protein do not have an effect on cholesterol uptake or absorption. This contrasts with results in Drosophila melanogaster which indicated that NPC1 deficiency leads to activation of a cholesterol uptake pathway, underscoring fundamental differences in mammalian and non-mammalian cholesterol metabolism.

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