Determinants of NPC1 expression and action: Key promoter regions, posttranscriptional control, and the importance of a "cysteine-rich" loop

Mutations in the NPC1 gene cause Niemann-Pick type C disease, which is char acterized by the accumulation of free cholesterol and other lipids in lysos omes, The NPC1 glycoprotein is located in a late endosomal compartment that transiently interacts with lysosomes. To identify factors regulating NPC1 expression and action, we analyzed the function of the human NPC1 promoter in human-derived ovarian, hepatic, and neuronal cells. A fragment containin g the first 208 base pairs upstream from the major transcription initiation site was sufficient to drive near maximal NPC1 promoter activity. Deletion analysis revealed that sequences between base pairs -111 and -37 play an i mportant role in controlling NPCI transcription. Treatment of proliferating granulosa cells with 30 mu M progesterone, which induces a reversible phen ocopy of the cholesterol trafficking defect of Niemann-Pick type C disease, increased NPC1 mRNA levels threefold. The protein synthesis inhibitor, cyc loheximide, also increased NPC1 mRNA levels, augmenting the progesterone-in duced increase in NPC1 mRNA abundance. Progesterone treatment was shown to increase the mRNA half-life, but did not affect NPCI promoter activity. Cys teine residues in a "cysteine-rich" loop predicted to reside in the intralu menal compartment of vesicles containing NPCI were mutated, resulting in pr oteins that were incapable of correcting the cholesterol trafficking defect in CT60 cells, a Chinese hamster cell line in which the endogenous NPCI ge ne is inactivated. Converting isoleucine 1061, also predicted to lie within the cysteine-rich loop, to a threonine residue inactivated the protein as well. The I1061T mutation is one of the most common mutations in Niemann-Pi ck type C disease. All of the cysteine-rich loop mutants were localized to cholesterol-engorged lysosomes in a pattern mimicking the distribution of N PCI in progesterone-treated cells. A recombinant protein representing the c ysteine-rich loop was shown to bind to a zinc-NTA agarose column. We conclu de:(1) that cis elements residing in the first 111 base pairs upstream from the transcription start site are critical for transcription of the NPCI ge ne;(2) that NPCI expression is subject to posttranscriptional regulation in response to treatments that disrupt NPC1 function; and (3) that an intralu menal cysteinerich loop with zinc-binding activity is critical to NPC1's ab ility to unload lysosomal cargo. (C) 2000 Academic Press.