Dysfunctional cholesterol homeostasis is involved in the etiology of cardiovascular and other diseases. While much is known about extracellular cholesterol transport in plasma lipoproteins, the molecular mechanisms of intracellular cholesterol trafficking are not as well understood. A major remaining question in cellular cholesterol homeostasis concerns its transit through the endosomal vesicle recycling system. The lysosomal storage disease Niemann-Pick C (NPC) provides a window into this critical step. NPC disease is characterized by the accumulation of LDL-derived cholesterol in the late endosomal/lysosomal compartment (LE/LY). Caused by a deficiency in either of two LE/LY proteins, NPC1 or NPC2, the similarities in cellular and clinical phenotypes of NPC1 and NPC2 diseases suggest that they act in a common pathway. Studies from our laboratory have shown that NPC2 is a cholesterol transporter, and that the unique lysosomal phospholipid lyso-bis phosphatidic acid (LBPA), which is localized to the internal membranes of the LE/LY, causes order of magnitude increases in the rate of sterol transfer by NPC2. For cholesterol to exit across the limiting membrane of the LE/LY, it has been proposed that NPC2 transfers its cholesterol directly to the N-terminal domain of NPC1, although such protein-protein interactions have not yet been experimentally demonstrated. Our recent structure-function studies indicate that multiple domains on the surface of NPC2 are necessary for its cholesterol transport activity. This suggested to us that NPC2 may promote membrane-membrane interactions, and recent in vitro studies show that NPC2, indeed, causes vesicle-vesicle interactions. Thus, our working hypothesis is that NPC2 acts at LBPA-rich membrane contact sites, clearing cholesterol by functioning as a bridge between multivesicular (inner) LE/LY membranes, and that it then delivers sterol to NPC1 in the limiting (outer) LE/LY membrane, by an as-yet unresolved mechanism. The present proposal will therefore focus on defining the mechanisms of cholesterol transport by the NPC2 and NPC1 proteins. Studies in Aim 1 will use targeted mutagenesis to establish the structure-function relationships for NPC2 in cholesterol transport, and a kinetics approach to resolve the mechanism of sterol transfer from NPC2 to NPC1.
Aim 2 will explore the molecular mechanisms underlying the dramatic effects of LBPA on sterol transport. The proposed studies will use fluorescence and infrared spectroscopy, as well as biochemical and microscopic approaches, to examine sterol transfer kinetics, protein-membrane interactions, protein-protein interactions, and cholesterol clearance in cells from NPC patients. Overall, the present studies will help enable a molecular level understanding of how the NPC proteins functions in normal cholesterol clearance from the LE/LY, and how NPC2 and LBPA function together in endolysosomal cholesterol transport, thus providing new information about the mechanisms of intracellular cholesterol trafficking.

Public Health Relevance

Mechanisms of cholesterol transport by Niemann-Pick C proteins Niemann-Pick type C (NPC) disease is an inherited disorder that leads to neurological impairment and is usually fatal. It is characterized by excessive buildup of cholesterol inside cells, specifically in the endosome-lysosome compartment. Abnormal function of either of two proteins, NPC1 or NPC2, has been shown to be the cause of the cholesterol accumulation. In the proposed research, we will investigate the exact mechanism by which NPC2 and NPC1 proteins can effectively clear cellular endo-lysosomes of cholesterol, in an effort to understand why cholesterol 'trafficking' is abnormal in NPC disease. In addition, using artificial membranes and cell culture models of NPC disease, we will study how a lipid that is unique to the endo- lysosomal compartment of the cell, called lyso-bis phosphatidic acid or LBPA, dramatically stimulates cholesterol transport by the NPC2 protein. These studies will help to explain the basic processes by which cholesterol is transported inside cells, which could then aid development of therapies for NPC disease and other disorders of cholesterol accumulation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Maas, Stefan
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Rutgers University
Earth Sciences/Resources
United States
Zip Code
Davidson, Cristin D; Fishman, Yonatan I; Puskás, István et al. (2016) Efficacy and ototoxicity of different cyclodextrins in Niemann-Pick C disease. Ann Clin Transl Neurol 3:366-80
McCauliff, Leslie A; Xu, Zhi; Li, Ran et al. (2015) Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport. J Biol Chem 290:27321-31