leupeptins and Hyperinsulinism

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.

Topics: Alleles; Amino Acid Substitution; Animals; ATP-Binding Cassette Transporters; Carbohydrate Metabolism, Inborn Errors; Cysteine Proteinase Inhibitors; Disulfides; Endoplasmic Reticulum; Glycosylation; HEK293 Cells; Humans; Hyperinsulinism; Hypoglycemia; K562 Cells; Leupeptins; Mice; Mitochondrial Proteins; Mutation, Missense; NIH 3T3 Cells; Potassium Channels, Inwardly Rectifying; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Structure, Tertiary; Protein Transport; Receptors, Drug; Sulfonylurea Receptors

In the current study, we examined the mechanisms that regulate hepatic apolipoprotein B (apoB)-containing lipoprotein secretion in Psammomys obesus, a good animal model for the investigation of insulin resistance and diabetes.. When fed chow ad libitum, 22% maintained normoglycemia and normoinsulinemia (group A), 33% exhibited normoglycemia and appreciable hyperinsulinemia (group B), and 45% developed overt diabetes (group C). Body weight gain, plasma free fatty acid elevation, hypertriglyceridemia, and hypercholesterolemia characterized groups B and C. Triton WR-1339 injection, at fasting, resulted in higher plasma VLDL-triglyceride and VLDL-apoB accumulation in groups B and C, suggesting increased VLDL production by the liver. Pulse-chase labeling experiments in cultured hepatocytes with [35S]methionine revealed reduced intracellular degradation and enhanced secretion of newly synthesized apoB in groups B and C. Concomitant with the raised triglyceride and cholesterol contents in the livers of groups B and C, there was an increase in lipogenesis and in the activity of microsomal triglyceride transfer protein, monoacylglycerol acyltransferase, and diacylglycerol transferase. Pretreatment of hepatocytes with proteasomal inhibitors eliminated the differences in apoB secretion among groups A, B, and C.. Our data indicate that both insulin resistance and diabetes triggered the intracellular machinery involved in VLDL assembly and secretion.

Topics: Acetylcysteine; Animals; Apolipoproteins B; Cells, Cultured; Cysteine Endopeptidases; Diabetes Mellitus; Disease Models, Animal; Gerbillinae; Hepatocytes; Hypercholesterolemia; Hyperinsulinism; Hypertriglyceridemia; Insulin; Insulin Resistance; Leupeptins; Lipoproteins, VLDL; Liver; Multienzyme Complexes; Protease Inhibitors; Proteasome Endopeptidase Complex