Sitosterolemia is a lipid disorder characterized by the accumulation of dietary xenosterols in plasma and tissues caused by mutations in either ABCG5 or ABCG8. ABCG5 ABCG8 encodes a pair of ABC half transporters that form a heterodimer (G5G8), which then traffics to the surface of hepatocytes and enterocytes and promotes the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. We review the literature from the initial description of the disease, the discovery of its genetic basis, current therapy, and what has been learned from animal, cellular, and molecular investigations of the transporter in the twenty years since its discovery. The genomic era has revealed that there are far more carriers of loss of function mutations and likely pathogenic variants of ABCG5 ABCG8 than previously thought. The impact of these variants on G5G8 structure and activity are largely unknown. We propose a classification system for ABCG5 ABCG8 mutants based on previously published systems for diseases caused by defects in ABC transporters. This system establishes a framework for the comprehensive analysis of disease-associated variants and their impact on G5G8 structure–function.
Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in development. Interestingly, several vertebrate transmembrane Sema classes are capable of producing functional soluble ectodomains. However, little is known of soluble Sema6 ectodomains in the nervous system. Herein, we show that the soluble Sema6A ectodomain, sSema6A, exhibits natural and protein kinase C (PKC)‐induced release. We show that PKC mediates Sema6A phosphorylation at specific sites and while this phosphorylation is not the primary mechanism regulating sSema6A production, we found that the intracellular domain confers resistance to ectodomain release. Finally, sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This suggests that in addition to its canonical contact‐mediated functions, Sema6A may have regulated, long‐range, forward‐signaling capacity.
Background: Nonalcoholic fatty liver disease (NAFLD) affects almost 1 billion people worldwide and is associated with risk factors such as obesity and dyslipidemia. Studies show linked variants and methylation status of carnitine palmitoytransferase 1a (CPT1a) to disrupt very low-density lipoprotein (VLDL) cholesterol and triglyceride (TG) levels. We exhibit liver-specific deletion of CPT1a in mice lowers plasma cholesterol and TG while exacerbating NAFLD and inflammation. Methods: Eight-week old Cpt1a floxed mice with the human apoB100 transgene (Cpt1a fl/fl /B100 Tg ) were administered control adenoassociated virus (AAV) or AAV encoding Cre-recombinase under control of a liver specific promoter (TBG-Cre). Control and Cpt1a liver-specific knock out (LKO) mice were placed on low-fat control or western-type diet (WTD; 42% kcal fat, 0.2% cholesterol) for 16 weeks. Body weights were recorded weekly and body composition by MRI was performed at the study midpoint and end. Tissues and plasma were collected and analyzed for lipid composition and gene and protein expression by QPCR and immunoblotting, respectively. Results: CPT1a LKO mice had lower plasma cholesterol and TG irrespective of diet. The reduction in plasma cholesterol was limited to the LDL pool in FPLC-fractionated plasma. Hepatic TG was elevated in mice fed WTD and LKO Cpt1a mice. Loss of hepatic Cpt1a had no effect on hepatic cholesterol in male mice, but increased total and free cholesterol by 2- and 2.5-fold, respectively, in females. The rise in hepatic free cholesterol in female Cpt1a LKO mice associated with increases in Kupffer cell ( Clec4f ) and collagen ( Col1a1 ) gene expression. Free cholesterol levels were not related to differences in transcripts for enzymes involved in cholesterol synthesis or secretion ( Srebp2, Hmgcr, Hmgcs, Soat2, Mttp ), hepatic clearance ( Ldlr ), metabolism ( Cpy7a1, Cyp8b1 ), or biliary secretion ( Abcg5, Abcg8 ). Conclusions: Liver-specific deletion of CPT1a reduces plasma LDL-cholesterol and increases cholesterol levels in female, but not male mice. The increase in hepatic free cholesterol did not alter expression of cholesterol-responsive genes, suggesting cholesterol accumulation outside of the regulatory pool.
Background: The proteostasis network is essential for the maintenance of cellular integrity by ensuring the correct folding of newly synthesized proteins. However, the high fidelity of this system contributes to diseases such as Cystic Fibrosis (CF, ABCC7), Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3, ABCB4), and Sitosterolemia (ABCG5/ABCG8). Interference with proteostasis and allowing sub-optimally folded ATP-Binding Cassette (ABC) transporters to transit to the cell surface partially restores function, with clinical benefit for CF and PFIC3. We sought to delineate the molecular underpinnings of Sitosterolemia, an autosomal recessive form of Familial Hypercholesterolemia (FH), characterized by accumulation of phytosterols in the plasma and tissues. ABCG5 ABCG8 form an obligate heterodimer at the surface of the liver and small intestine, and mediates sterol transport into bile and the intestinal lumen. Case studies reveal 57 loss of function and over 40 missense mutations of ABCG5 associated with Sitosterolemia. Recent pharmaceutics show promise with rescue of mutations in CF and PFIC3 with small molecule chaperones and potentiators. Through our analysis of ABCG5 missense mutations causing Sitosterolemia, we anticipate rescue by these compounds as well. Methods: We established a classification system for missense mutations of ABCG5, similar to CF and PFIC3. Mutations were generated by site-directed mutagenesis, and confirmed through Sanger sequencing. Native and mutant ABCG5 were co-transfected with native ABCG8 into human hepatocytes and evaluated for protein abundance and trafficking beyond the endoplasmic reticulum (ER), by SDS-PAGE and immunoblot analysis. Results: Nine mutants of ABCG5 have been generated. Co-expression of ABCG5 with ABCG8 i n vitro demonstrated I68N, A98G, E146Q, and R419P successfully traffic while R284S, T305R, R389H, R419H, and N437K were arrested within the proteostasis network. Conclusions: Similar to CF and PFIC3, Sitosterolemia-associated mutations result in compromised heterodimer formation and trafficking to the apical surface. Small molecule chaperones and potentiators may partially rescue the mutants and provide clinical benefit for Sitosterolemia.
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