Biomedical subjects

Jonathan C Cohen

Publications and source records attributed to Jonathan C Cohen.

41 records · Page 3Linked to original sources

PubMed2002

ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2.

Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K(D), approximately 44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the beta(2)-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of beta(2)-adaptin that is required for interaction with the adapter protein beta-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery.

Adaptor Protein Complex 2↗

PubMed2002

Sources of variability in genetic association studies: insights from the analysis of hepatic lipase (LIPC).

Genetic association studies have been widely used to identify loci that influence plasma lipoprotein concentrations, but few of the associations reported have proved consistently reproducible across different study populations. This lack of consistency is a widely recognized limitation of association studies, and is often ascribed to inadequate statistical power, population substructure, and population-specific linkage disequilibrium. However, few studies have assessed the causes of variability underlying a given genotype-phenotype association. We have examined two possible sources of variability in the association between the -514 polymorphism in hepatic lipase (LIPC) and plasma HDL-C concentrations. First, we compared the association between this polymorphism and hepatic lipase activity in four populations. A single copy of the -514C allele was associated with a 10 mmol.hr(-1).l(-1) increase in hepatic lipase activity in white American and Turkish men but only approximately 5 mmol.hr(-1).l(-1) in Chinese and African-American men. Second, we tested the effects of a stanozolol-induced increase in hepatic lipase activity on plasma HDL-C concentrations in men with normal (< 150mg/dl) or elevated (150-300mg/dl) levels of plasma triglyceride. The increase in hepatic lipase activity was similar in the two groups, but the resulting decline in HDL-C levels was significantly greater in normolipidemic men. These data suggest that the effect of a polymorphism on gene expression can vary among individuals, and that the resulting phenotype may be further modified by interactions with other factors. Three novel LIPC polymorphisms were identified in the study (-1596insC, -2740A>G, and -2880G>C).

Adult↗

PubMed2002

Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.

Mutations in either ATP-binding cassette (ABC) G5 or ABCG8 cause sitosterolemia, an autosomal recessive disorder of sterol trafficking. To determine the site of action of ABCG5 and ABCG8, we expressed recombinant, epitope-tagged mouse ABCG5 and ABCG8 in cultured cells. Both ABCG5 and ABCG8 underwent N-linked glycosylation. When either protein was expressed individually in cells, the N-linked sugars remained sensitive to Endoglycosidase H (Endo H). When ABCG5 and ABCG8 were coexpressed, the attached sugars were Endo H-resistant and neuraminidase-sensitive, indicating that the proteins were transported to the trans-Golgi complex. The mature, glycosylated forms of ABCG5 and ABCG8 coimmunoprecipitated, consistent with heterodimerization of these two proteins. The Endo H-sensitive forms of ABCG5 and ABCG8 were confined to the endoplasmic reticulum (ER), whereas the mature forms were present in non-ER fractions in cultured hepatocytes. Immunoelectron microscopy revealed ABCG5 and ABCG8 on the plasma membrane of these cells. In polarized WIF-B cells, recombinant ABCG5 localized to the apical (canalicular) membrane when coexpressed with ABCG8, but not when expressed alone. To our knowledge this is the first direct demonstration that trafficking of an ABC half-transporter to the cell surface requires the presence of its dimerization partner.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2002

Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.

Two ATP-binding cassette (ABC) transporters, ABCG5 and ABCG8, have been proposed to limit sterol absorption and to promote biliary sterol excretion in humans. To test this hypothesis, a P1 clone containing the human ABCG5 and ABCG8 genes was used to generate transgenic mice. The transgenes were expressed primarily in the liver and small intestine, mirroring the expression pattern of the endogenous genes. Transgene expression only modestly affected plasma and liver cholesterol levels but profoundly altered cholesterol transport. The fractional absorption of dietary cholesterol was reduced by about 50%, and biliary cholesterol levels were increased more than fivefold. Fecal neutral sterol excretion was increased three- to sixfold and hepatic cholesterol synthesis increased two- to fourfold in the transgenic mice. No significant changes in the pool size, composition, and fecal excretion of bile acids were observed in the transgenic mice. Transgene expression attenuated the increase in hepatic cholesterol content induced by consumption of a high cholesterol diet. These results demonstrate that increased expression of ABCG5 and ABCG8 selectively drives biliary neutral sterol secretion and reduces intestinal cholesterol absorption, leading to a selective increase in neutral sterol excretion and a compensatory increase in cholesterol synthesis.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2002

Heritability of plasma noncholesterol sterols and relationship to DNA sequence polymorphism in ABCG5 and ABCG8.

The plasma concentrations of cholesterol precursor sterols and plant sterols vary over a 5- to 10-fold range among normolipidemic individuals, and provide indices of the relative rates of cholesterol synthesis and fractional absorption. In the present study, we examined the relative contributions of genetic and environmental factors to variation in the plasma concentrations and sterol-cholesterol ratios of five noncholesterol sterols, including the 5alpha-saturated derivative of cholesterol (cholestanol), two precursors in the cholesterol biosynthesis pathway (desmosterol and lathosterol), and two phytosterols (campesterol and sitosterol). Plasma sterol concentrations were highly stable in 30 individuals measured over a 48 week period. Regression of offspring sterol levels on the parental values indicated that plasma levels of all five noncholesterol sterols were highly heritable. Analysis of monozygotic and dizygotic twin pairs also indicated strong heritability of all five sterols. Two common sequence variations (D19H and T400K) in ABCG8, an ABC half-transporter defective in sitosterolemia, were associated with lower concentrations of plant sterols in parents, and in their offspring.Taken together, these findings indicate that variation in the plasma concentrations of noncholesterol sterols is highly heritable, and that polymorphism in ABCG8 contributes to genetic variation in the plasma concentrations of plant sterols.

ATP Binding Cassette Transporter, Subfamily G, Mem↗