The Hydrophobic Handoff Between NPC2 and the N-Terminal Domain of NPC1 in the Export of Cholesterol from Lysosomes




Wang, Michael Leechun

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Low density lipoproteins (LDL) and related plasma lipoproteins deliver cholesterol to cells by receptor-mediated endocytosis. The lipoprotein is degraded in late endosomes and lysosomes where its cholesterol is released. Egress of cholesterol from late endosomes and lysosomes (hereafter referred to as lysosomes) requires two proteins: Niemann-Pick C2 (NPC2), a soluble protein of 132 amino acids; and NPC1, an intrinsic membrane protein of 1278 amino acids and 13 postulated membrane-spanning helices that span the lysosomal membrane. Recessive loss-of-function mutations in either NPC2 or NPC1 produce NPC disease, which causes death in childhood owing to cholesterol accumulation in lysosomes of liver, brain, and lung. Consistent with their cholesterol export role, NPC2 and NPC1 both bind cholesterol. The cholesterol binding site on NPC1 is located in the NH2-terminal domain (NTD), which projects into the lysosomal lumen. This domain, designated NPC1(NTD), can be expressed in vitro as a soluble protein of 240 amino acids that retains cholesterol binding activity. This thesis studies NPC2 and NPC1(NTD) in detail as summarized below. Two major differences exist between the cholesterol binding of NPC2 and NPC1(NTD). 1) Competitive binding studies and crystal structures indicate that the two proteins bind cholesterol in opposite orientations. NPC2 binds the iso-octyl side chain, leaving the 3ß hydroxyl exposed, whereas NPC1 binds the 3ß-hydroxyl, leaving the side chain partially exposed. 2) Kinetic studies of cholesterol binding reveal that NPC2 binds and releases cholesterol rapidly (half-time < 2 min at 4oC), while NPC1(NTD) binds cholesterol very slowly (half-time > 2 hr at 4oC).
Its rapid cholesterol binding allows NPC2 to transfer cholesterol to and from liposomes. Unlike NPC2, NPC1(NTD) cannot rapidly transfer its bound cholesterol to liposomes. However, NPC1(NTD) can accomplish this delivery when NPC2 is present. Furthermore, cholesterol binding to NPC1(NTD) is accelerated by >15-fold when the sterol is first bound to NPC2 and then transferred to NPC1(NTD). These data led us to advance a model in which NPC2 can mediate bi-directional transfer of cholesterol to or from NPC1(NTD). In cells, we envision that NPC2 accepts cholesterol in the lysosomal lumen and transports it to membrane-bound NPC1, thus accounting for the requirement for both proteins for lysosomal cholesterol export. Amino acid residues important or binding or transfer of cholesterol on NPC2 and NPC1(NTD) were identified through alanine scan mutagenesis. For both NPC2 and NPC1(NTD), residues that decreased binding mapped to areas surrounding the binding pockets on the crystal structures; residues that decreased transfer, but not binding, mapped to discrete surface patches near the opening of the binding pockets. These surface patches may be sites where the two proteins interact to transfer cholesterol. The most severe mutations disrupting binding were P120S for NPC2 and P202A/F203A for NPC1(NTD); and those that disrupted transfer were V81D for NPC2 and L175Q/L176Q for NPC1(NTD). Furthermore, the functional significance of both the binding and transfer of cholesterol by NPC2 and NPC1(NTD) in the egress of cholesterol from lysosomes was confirmed. The above binding- or transfer-defective mutants of NPC2 and NPC1 were unable to rescue LDL-stimulated cholesteryl ester synthesis in NPC2 or NPC1-deficient cells, respectively, in contrast to wild-type NPC2 and NPC1. With these data, we envision that NPC2 binds cholesterol the instant that it is released from LDL, either as the free sterol or after cleavage of lipoprotein-derived cholesteryl esters by lysosomal acid lipase. This binding would prevent cholesterol from crystallizing in the lysosomal lumen. According to the model, NPC2 can transfer its bound cholesterol to NPC1(NTD) directly, thus avoiding the necessity for the insoluble cholesterol to transit the water phase. This transfer of cholesterol from NPC2 to NPC1(NTD) has a special functional relevance in light of the near-absolute insolubility of cholesterol in water, and we have named this process a "hydrophobic handoff."

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