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theses

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by mutations in either of two lysosomal proteins, NPC1 or NPC2. NPC2 is a 16kDa soluble protein that binds cholesterol. Previous work showed that NPC2 rapidly transports cholesterol to/from vesicles via direct interaction with membranes. In the present site-directed mutagenesis studies, results suggest that the NPC2 surface may have two membrane interacting domains necessary for its cholesterol transport properties. Using a light scattering assay, it was found that NPC2 promotes vesicle-vesicle interactions, supporting the hypothesis for two membrane interaction domains. Results from kinetic sterol transfer assays also indicate that lysobisphosphatidic acid (LBPA), found uniquely in endo/lysosomes, dramatically enhances cholesterol transfer rates by wt NPC2. Sterol transfer and protein-lipid binding studies of NPC2 mutant proteins indicate that NPC2 directly interacts with LBPA, and suggest that the ‘hydrophobic knob’ region of NPC2 is the LBPA-sensitive domain. Cyclodextrins (CD) have been shown to reduce symptoms and extend lifespan in murine models of NPC disease. In the present studies the mechanism of sterol transport by CD was investigated. Results indicate that CD directly interacts with membranes to transfer sterol, similar to NPC2. Absolute transfer rates were slower for CD than NPC2, however, and unlike NPC2, LBPA did not enhance transfer rates by CD. CD also promoted vesicle-vesicle interactions, similar to NPC2. Thus, the efficacy observed in NPC disease models is likely the result of CD enhancement of cholesterol transport between membranes, with rapid sterol transfer occurring during CD−membrane interactions. Unfortunately, continuous administration of CD is required for therapeutic benefit, due to rapid renal elimination. We tested a library of novel CD polyrotaxanes (PRTx), which should have increased circulation time in plasma, in npc2-/- fibroblasts. These polymers have environmentally sensitive endcaps, theoretically allowing for controlled release of CD monomers at high concentrations within the endo/lysosomal compartment. Indeed, the PRTx compounds rapidly reduced accumulated intracellular cholesterol in npc2-/- fibroblasts, suggesting that they may prove useful in therapeutic application to NPC disease.  

ETD_5253

Ph.D.

Includes bibliographical references

by Leslie Ann McCauliff

doi:10.7282/T30C4SWK

ETD doctoral

The author owns the copyright to this work.