The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca 2؉ -independent and that within the zymogen autocatalytic processing site SSVFAQ2SIP Val at P4 and Pro at P3 are critical. The S127R and D374Y mutations result in ϳ50 -60% and >98% decrease in zymogen processing, respectively. In contrast, the double [D374Y ؉ N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mM ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal ϳ9-fold increase in circulating LDL cholesterol, while in LDLR(؊/؊) mice a delayed ϳ2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.The mammalian proprotein convertases constitute a family of 9 serine proteinases related to bacterial subtilisin. These include the 7 basic amino acid-specific convertases known as PC1/PC3, PC2, furin, PC4, PACE4, PC5/PC6, PC7/LPS (1, 2) and the two enzymes cleaving at nonbasic residues SKI-1/S1P (3, 4) and NARC-1/PCSK9 (5). These proteases are implicated in the limited proteolysis of precursors of secretory proteins that regulate a variety of cellular functions, including cellular growth, adhesion, differentiation, cell to cell communications, and endocrine/paracrine functions (6, 7). Published gene knockout analyses (reviewed in Ref. 8) revealed that only furin (9) and SKI-1/S1P (10) are embryonic lethal. So far, nothing is known about the phenotype consequences of NARC-1 1 knockout in mice. The cDNA of the enzyme NARC-1 was cloned during pharmaceutical screening of mRNAs up-regulated following induction of neural apoptosis by serum withdrawal, and the encoded protein was called "neural apoptosis regulated convertase 1" (NARC-1) (11). We characterized this enzyme, and we showed that it is highly expressed in liver and small intestine and that specific mutations in the prosegment of NARC-1 completely abrogated its autocatalytic processing (5). We further showed that overexpression of NARC-1 enhances neurogenesis of progenitor brain telencephalic cells. The sustained expression of NARC-1 in liver and small intestine and its transient expression in telencephalon, kidney, and cerebellum beg for the identification of its physiological substrates, which are still unknown.Human genetic point mutations resulting in pathology have been rep...

IntroductionThe LDL receptor plays a critical role in the regulation of plasma LDL levels by mediating approximately two thirds of LDL clearance (1-3). Loss of LDL receptor function leads to decreased LDL catabolism and elevated LDL levels (4). LDL receptor levels are affected by diet, hormones, and most dramatically, by mutations in the LDL receptor locus that lead to familial hypercholesterolemia (FH).Early studies of LDL metabolism in patients with FH revealed that in addition to the LDL clearance defect, they overproduce LDL (5, 6) and relatively small VLDL particles (7). VLDL is the metabolic precursor of LDL and is converted to LDL through the action of lipoprotein lipase, a triacylglycerol lipase that acts upon VLDL while it circulates in the bloodstream (8). Increased production of VLDL can lead to increased LDL simply by providing more precursor. In addition, impaired clearance of VLDL remnants can lead to LDL overproduction (9).A long-standing paradox in the lipoprotein field is posed by the cholesterol-lowering drugs known as statins. These drugs inhibit 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, a tightly regulated step in the cholesterol biosynthetic pathway (10). Cells respond to the dearth of cholesterol by upregulating transcription of cholesterol-regulated genes, including the LDL receptor (11). Statins are ineffective in patients homozygous for null alleles of the LDL receptor (12). It has therefore been inferred that statins act by increasing LDL catabolism via upregulation of the LDL receptor. Paradoxically, statins do not always affect the LDL clearance rate. Rather, in many clinical studies (13)(14)(15) and animal studies (16,17), statins decrease VLDL and/or LDL production (reviewed in ref. 18).The post-translational fate of apoB, the major protein component of VLDL, can be explained by multiple mechanisms. In human and rat hepatoma cell lines, a large proportion of newly synthesized apoB is degraded within the secretory pathway (19). Thus, the rate of apoB secretion, and hence, VLDL secretion, from the liver is determined by the proportion of apoB that escapes coor post-translational degradation (20,21). In addition, reuptake of newly secreted lipoproteins has also been proposed to regulate the net output of apoB (22).How can the presence or absence of a functional LDL receptor affect the production of lipoproteins? We addressed this question by studying apoB secretion in cultured hepatocytes isolated from wild-type mice and mice lacking a functional LDL receptor (Ldlr -/-). Similar to FH, previous studies with Ldlr -/-mice revealed a decrease in LDL clearance (23) and a marked increase in plasma apoB levels (23-26). Our results with primary hepatocytes from these animals indicate that the LDL receptor is involved Jaap Twisk and Donald L. Gillian-Daniel contributed equally to this work.Received for publication October 6, 1999, and accepted in revised form December 28, 1999. Familial hypercholesterolemia is caused by mutations in the LDL receptor gene (Ldlr). Elevated plasma LDL ...

The ATP-binding cassette transporter A1 (ABCA1) participates in the efflux of cholesterol from cells. It remains unclear whether ABCA1 functions to efflux cholesterol across the basolateral or apical membrane of the intestine. We used a chicken model of ABCA1 dysfunction, the Wisconsin hypoalpha mutant (WHAM) chicken, to address this issue. After an oral gavage of radioactive cholesterol, the percentage appearing in the bloodstream was reduced by 79% in the WHAM chicken along with a 97% reduction in the amount of tracer in high density lipoprotein. In contrast, the percentage of radioactive cholesterol absorbed from the lumen into the intestine was not affected by the ABCA1 mutation. Liver X receptor (LXR) agonists have been inferred to decrease cholesterol absorption through activation of ABCA1 expression. However, the LXR agonist T0901317 decreased cholesterol absorption equally in both wild type and WHAM chickens, indicating that the effect of LXR activation on cholesterol absorption is independent of ABCA1. The ABCA1 mutation resulted in accumulation of radioactive cholesterol ester in the intestine and the liver of the WHAM chicken (5.0-and 4.4-fold, respectively), whereas biliary lipid concentrations were unaltered by the WHAM mutation. In summary, ABCA1 regulates the efflux of cholesterol from the basolateral but not apical membrane in the intestine and the liver.

Mutations in the low density lipoprotein (LDL) receptor (LDLR)cause hypercholesterolemia because of inefficient LDL clearance from the circulation. In addition, there is a paradoxical oversecretion of the metabolic precursor of LDL, very low density lipoprotein (VLDL). We recently demonstrated that the LDLR mediates presecretory degradation of the major VLDL protein, apolipoprotein B (apoB). Kinetic studies suggested that the degradation process is initiated in the secretory pathway. Here, we evaluated the ability of several LDLR variants that are stalled within the secretory pathway to regulate apoB secretion. Both a naturally occurring mutant LDLR and an LDLR consisting of only the ligand-binding domains and a C-terminal endoplasmic reticulum (ER) retention sequence were localized to the ER and not at the cell surface. In the presence of either of the ER-localized LDLRs, apoB secretion was essentially abolished. When the ligand-binding domain of the truncated receptor was mutated the receptor was unable to block apoB secretion, indicating that the inhibition of apoB secretion depends on the ability of the LDLR to bind to its ligand. These findings establish LDLR-mediated pre-secretory apoB degradation as a pathway distinct from reuptake of nascent lipoproteins at the cell surface. The LDLR provides an example of a receptor that modulates export of its ligand from the ER.M olecular defects in the low density lipoprotein (LDL) receptor (LDLR) cause Familial Hypercholesterolemia (FH), a condition associated with elevated plasma LDL cholesterol levels (1). Reduced expression, altered ligand binding, or defective transport to the cell surface all lead to a reduction in the functionally effective population of LDLRs at the cell surface.LDL is produced in the circulation from its precursor, very low density lipoprotein (VLDL). Apolipoprotein B (apoB) is the major protein component of VLDL and LDL. Two observations have suggested that the LDLR might be involved in apoB secretion. First, overproduction of apoB-containing lipoproteins occurs in some cases of FH (2-4). Second, drugs that lower LDL levels by increasing the expression of the LDLR (statins) in many instances have been shown to lower LDL without increasing LDL clearance; i.e., they lower LDL and͞or VLDL production (5).The proportion of apoB that escapes degradation within the secretory pathway primarily determines the rate of VLDL secretion. We recently demonstrated that the presence of the LDLR greatly increases the proportion of apoB subject to presecretory degradation. Our results directly link VLDL overproduction in FH with the loss of the LDLR (6).Several additional studies support a role for the LDLR in modulating apoB secretion. Increased secretion of VLDL is observed in vivo from both Ldlr Ϫ/Ϫ and transgenic Ldlr Ϫ/Ϫ mice that overexpress the nuclear form of sterol regulatory element binding protein-1a (SREBP-1a) and in vitro in hepatocytes from these animals (7). In contrast, transgenic SREBP-1a animals with a wild-type LDLR accumulate cholesterol a...

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