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.
Background: Sitosterolemia is a rare form of Familial Hypercholesterolemia (FH) which is unique from other forms of FH due to the accumulation of phytosterols in the plasma and tissues. Sitosterolemia is autosomal recessive and caused by mutations in either the ABCG5 or ABCG8 gene. ABCG5 and ABCG8 form a heterodimer (ABCG5/ABCG8) that functions at the apical surface of hepatocytes and enterocytes to promote cholesterol and phytosterol excretion. Small molecule modulators classified as either correctors or potentiators have therapeutic benefit when used to treat mutants of ABCB4 (PFIC3) or ABCC7 (Cystic Fibrous) that have impaired folding, stability, or activity. There are over 40 missense mutations in ABCG5/ABCG8 which have been clinically confirmed in patients with Sitosterolemia. Methods: We developed a classification system for ABCG5 and ABCG8 mutations based on the underlying molecular defect for ABCG5/ABCG8 dysfunction (maturation, trafficking, activity, etc.). We used site directed mutagenesis to introduce ABCG8 missense mutations located in the N-terminal, cytosolic domain of human ABCG8. Normal and mutant ABCG5/ABCG8 constructs were transiently transfected into a human hepatocyte cell line, Huh-7, and analyzed for heterodimerization, trafficking beyond the ER, and localization at the cell surface. Results: Of the ABCG8 cytosolic mutants studied, R184H, L196Q, L228P, and R263Q failed to traffic beyond the ER to form a stable heterodimer (class II) while P231T, T400K, N409D, N409I, and P415H were trafficking competent. Conclusions: Forty four percent (4 of 9) of cytosolic, ABCG8 mutations that cause Sitosterolemia are due to failures in complex formation and trafficking beyond the ER. Fifty six (5 of 9) percent were trafficking competent, indicating a stable ABCG5/G8 complex, but loss of activity due to impaired ATP hydrolysis or substrate binding. ABCG8 mutants may be potentially restored by proteostatic regulators and/or potentiators & correctors that have shown to be effective in disease-causing mutations in other ABC transporters.
Background: ABCG5 and ABCG8 form a heterodimer that promotes biliary secretion and opposes intestinal absorption of cholesterol and phytosterols. Whole-genome and exome sequencing has revealed over 2000 variants of ABCG5 and ABCG8, many of which are predicted to be pathogenic or likely pathogenic. Missense mutations in clinically confirmed cases of Sitosterolemia are of particular interest for their potential to reveal structure-function relationships in ABCG5 ABCG8 and other ABC transporter family members. Mutations in ABCG5 ABCG8 with clinically confirmed Sitosterolemia generally cluster within the ATP binding cassette and transmembrane spanning segments that form the substrate-binding domain. However, four disease-causing mutations occur in the short extracellular loop in ABCG8, suggesting that these mutations may reveal novel elements of the ABCG5 ABCG8 function. Methods: Mutants of ABCG8 were generated by site-directed mutagenesis. Plasmids encoding normal ABCG5 and normal or mutant ABCG8s were co-transfected into human Huh7 hepatocytes, lysates prepared, and analyzed by SDS-PAGE and immunoblot analysis. Maturation and trafficking of the ABCG5 ABCG8 heterodimer beyond the endoplasmic reticulum was assessed by the appearance of the higher molecular weight, mature form of each glycoprotein. Results: Mutants G574R, G575D, and L596R failed to support trafficking of ABCG5 ABCG8 and were designated as Class II mutants causative in Sitosterolemia. Only G574E supported maturation of the ABCG5 ABCG8 transporter in cultured human hepatocytes and remains unclassified. Conclusion: Formation and trafficking of the ABCG5 ABCG8 heterodimer is dependent on key residues within the third extracellular loop in ABCG8. Maturation of ABCG5 ABCG8 is tolerant of substitutions in glycine 574, including the addition of a larger and negatively charged residue. However, neither substitution forms a functional ABCG5 ABCG8 transporter.
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