Y K Ho

Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and inlivers of parabiotic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cell-surface LDLRs in a dose- and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermates. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.

Identification of Complexes between the COOH-terminal Domains of Sterol Regulatory Element-binding Proteins (SREBPs) and SREBP Cleavage-Activating Protein

SREBP cleavage-activating protein (SCAP) stimulates the proteolytic cleavage of membrane-bound SREBPs, thereby initiating the release of NH2-terminal fragments from cell membranes. The liberated fragments enter the nucleus and stimulate transcription of genes involved in synthesis and uptake of cholesterol and fatty acids. Sterols repress cleavage of SREBPs, apparently by interacting with the membrane attachment domain of SCAP. In the present studies we show that SCAP, like the SREBPs, is located in membranes of the endoplasmic reticulum and nuclear envelope. The COOH-terminal domain of SCAP, like that of the SREBPs, is located on the cytosolic face of the membranes. Co-immunoprecipitation experiments show that SCAP and SREBP-2 form a complex that can be precipitated with antibodies to either component. Complex formation occurs when cells express only the COOH-terminal domain of either SREBP-2 or SCAP, indicating that the complex forms between the two COOH-terminal domains. Truncation of SREBP-2 at its COOH terminus prevents the formation of complexes with SCAP and simultaneously reduces proteolytic cleavage. We conclude that proteolytic cleavage of SREBPs requires the formation of a complex with the COOH-terminal domain of SCAP and that SCAP is therefore a required element in the regulation of sterol and fatty acid metabolism in animal cells.

The SREBP Pathway in Drosophila: Regulation by Palmitate, Not Sterols

In mammals, synthesis of cholesterol and unsaturated fatty acids is controlled by SREBPs, a family of membrane-bound transcription factors. Here, we show that the Drosophila genome encodes all components of the SREBP pathway, including a single SREBP (dSREBP), SREBP cleavage-activating protein (dSCAP), and the two proteases that process SREBP at sites 1 and 2 to release the nuclear fragment. In cultured Drosophila S2 cells, dSREBP is processed at sites 1 and 2, and the liberated fragment increases mRNAs encoding enzymes of fatty acid biosynthesis, but not sterol or isoprenoid biosynthesis. Processing requires dSCAP, but is not inhibited by sterols as in mammals. Instead, dSREBP processing is blocked by palmitic acid. These findings suggest that the ancestral SREBP pathway functions to maintain membrane integrity rather than to control cholesterol homeostasis.

Genetics of the low density lipoprotein receptor. Diminished receptor activity in lymphocytes from heterozygotes with familial hypercholesterolemia.

Using circulating mononuclear cells as a readily available tissue and using the rate of high affinity degradation of 125-I-labeled low density lipoprotein (LDL) as an index of cell surface LDL receptor activity, we have measured receptor activity in cells from 53 individuals. This group includes 32 healthy subjects, 15 subjects with the heterozygous form of familial hypercholesterolemia, and 6 subjects with hyperlipidemic disorders other than familial hypercholesterolemia. 7 of the healthy subjects and 10 of the heterozygotes were members of a single large kindred with five-generation transmission of the mutant familial hypercholesterolemia gene. LDL receptor activity was assayed in blood mononuclear cells under two sets of conditions. First, 125I-LDL degradation was measured in purified lymphocytes that had been incubated for 3 days in the absence of lipoproteins so as to induce a high level of LDL receptor activity. Phase-contrast autoradiograms of cells incubated with 125I-LDL and electron micrographs of cells incubated with ferritin-labeled LDL confirmed the existence of LDL receptors on lymphocytes. Second, 125I-LDL degradation was measured in mixed mononuclear cells (85-90% lymphocytes and 5-15% monocytes) immediately after their isolation from the bloodstream. This assay represented an attempt to assess the number of receptors actually expressed on the cells when they were in the circulation. Under both sets of conditions, cells from the familial hypercholesterolemia heterozygotes expressed an average of about one-half the normal number of LDL receptors. The current findings are consistent with the conclusion that heterozygotes with familial hypercholesterolemia possess only one functional allele at the LDL receptor locus and that the consequent deficiency of LDL receptors produces the clinical syndrome of heterozygous familial hypercholesterolemia.

Stimulation of cholesteryl ester synthesis in macrophages by extracts of atherosclerotic human aortas and complexes of albumin/cholesteryl esters.

Cholesteryl ester-rich particles extracted from human atherosclerotic plaques were shown to increase the rate of incorporation of [14C]oleate into cholesteryl [14C]oleate and to cause massive accumulation of cholesteryl esters in monolayers of mouse peritoneal macrophages. This stimulation showed saturation kinetics and susceptibility to competition by polyanions (polylnosinic acid, fucoidin, dextran sulfate), suggesting that cell surface binding was required. Cellular uptake and lysosomal hydrolysis of the cholesteryl esters were also required, as Indicated by the finding that stimulation of cholesteryl ester formation was prevented by the lysosomal inhibitor, chloroqulne. The cholesterol esterlflcation-stlmulatlng activity of the aortic extracts was excluded on a 2% agarose column and floated In the density range of 1.006 to 1.063 g/ml. Cholesterol-rich extracts from human adrenal glands and liver did not stimulate cholesteryl ester formation In macrophages. The aortic extracts did not stimulate cholesteryl ester synthesis In human fibroblasts. Complexes of 126l-labeled albumin and cholesteryl llnoleate formed In vitro were taken up and degraded in macrophages, but not in fibroblasts, by a process resembling the uptake of the aortic extracts. The current data suggest that macrophages express mechanisms for internalizing certain types of cholesteryl ester-rich lipid/protein complexes, Including those present In atherosclerotic plaques.

Use of monoclonal anti-receptor antibodies to probe the expression of the low density lipoprotein receptor in tissues of normal and Watanabe heritable hyperlipidemic rabbits.

Watanabe Heritable Hyperlipidemic (WHHL) rabbits, like humans with familial hypercholesterolemia, have a genetic defect in the low density lipoprotein (LDL) receptor. WHHL fibroblasts produce a low molecular weight precursor form of the receptor that is not glycosylated normally and is not transported to the cell surface at a normal rate. In the current studies, we have used a monoclonal antibody that reacts with the rabbit LDL receptor to extend these findings to intact rabbits. We have made the following observations: (a) In normal rabbits the liver and adrenal glands synthesize high molecular weight mature LDL receptors like those in fibroblasts. (b) In WHHL rabbits the adrenals express only the low molecular weight receptor precursor, and the liver expresses no detectable receptors. (c) When injected intravenously, the radioiodinated anti-LDL receptor monoclonal antibody is cleared from plasma 6-10-fold faster in normal than in WHHL rabbits, with specific uptake demonstrable in livers and adrenals of normal but not WHHL rabbits. The latter finding raises the general possibility that the total number of cell surface receptors expressed by an animal or human in vivo can be estimated by measuring the rate of clearance of an intravenously injected monoclonal antibody directed against the receptor of interest.