RESUMEN
The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Adenosina Trifosfatasas/metabolismo , Ésteres del Colesterol/metabolismo , Colesterol/metabolismo , Ácido Cólico/metabolismo , Lipoproteínas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Sitios de Unión , Transporte Biológico , Colesterol/química , Ésteres del Colesterol/química , Ácido Cólico/química , Expresión Génica , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Enfermedades Intestinales/genética , Enfermedades Intestinales/metabolismo , Enfermedades Intestinales/patología , Cinética , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/metabolismo , Errores Innatos del Metabolismo Lipídico/patología , Lipoproteínas/química , Lipoproteínas/genética , Modelos Moleculares , Mutación , Fitosteroles/efectos adversos , Fitosteroles/genética , Fitosteroles/metabolismo , Pichia/química , Pichia/genética , Pichia/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
The ATP-binding cassette (ABC) proteins play critical roles in maintaining lipid and sterol homeostasis in higher eukaryotes. In humans, several subfamily-A and -G members function as cholesterol transporters across the cellular membranes. Deficiencies of these ABC proteins can cause dyslipidemia that is associated with health conditions such as atherosclerosis, diabetes, fatty liver disease, and neurodegeneration. The physiological roles of ABC cholesterol transporters have been implicated in mediating cholesterol efflux for reverse cholesterol transport and in maintaining membrane integrity for cell survival. The precise role of these ABC transporters in cells remains elusive, and little is known about the sterol-transport mechanism. The membrane constituents of ABC transporters have been postulated to play a key role in determining the transport substrates and the translocation mechanisms via the transmembrane domains. Recent breakthroughs in determining high-resolution structures of the human sterol transporter ABCG5/G8 and its functional homologs have shed light on new structural features of ABC transporters, providing a more relevant framework for mechanistic analysis of cholesterol-transport ABC proteins. This minireview outlines what is known about ABCG cholesterol transporters, addresses key structural features in the putative sterol translocation pathway on the transmembrane domains, and concludes by proposing a mechanistic model of ABC cholesterol transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Colesterol/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Colesterol/metabolismo , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
The ABCG5/G8 heterodimer is the primary neutral sterol transporter in hepatobiliary and transintestinal cholesterol excretion. Inactivating mutations on either the ABCG5 or ABCG8 subunit cause Sitosterolemia, a rare genetic disorder. In 2016, a crystal structure of human ABCG5/G8 in an apo state showed the first structural information on ATP-binding cassette (ABC) sterol transporters and revealed several structural features that were observed for the first time. Over the past decade, several missense variants of ABCG5/G8 have been associated with non-Sitosterolemia lipid phenotypes. In this review, we summarize recent pathophysiological and structural findings of ABCG5/G8, interpret the structure-function relationship in disease-causing variants and describe the available evidence that allows us to build a mechanistic view of ABCG5/G8-mediated sterol transport.