RESUMEN
Cholesterol homeostasis and trafficking are critical to the maintenance of the asymmetric plasma membrane of eukaryotic cells. Disruption or dysfunction of cholesterol trafficking leads to numerous human diseases. ATP-binding cassette (ABC) transporters play several critical roles in this process, and mutations in these sterol transporters lead to disorders such as Tangier disease and sitosterolemia. Biochemical and structural information on ABC sterol transporters is beginning to emerge, with published structures of ABCA1 and ABCG5/G8; these two proteins function in the reverse cholesterol transport pathway and mediate the efflux of cholesterol and xenosterols to high-density lipoprotein and bile salt micelles, respectively. Although both of these transporters belong to the ABC family and mediate the efflux of a sterol substrate, they have many distinct differences. Here, we summarize the current understanding of sterol transport driven by ABC transporters, with an emphasis on these two extensively characterized transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico/fisiología , Esteroles/metabolismo , Animales , Colesterol/metabolismo , HumanosRESUMEN
Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate xyloglucan xylosyltransferase 1 (XXT1), a GT involved in the synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from uridine diphosphate (UDP)-xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-xylose and UDP-glucose, while reactions with UDP-arabinose and UDP-galactose are over 10-fold slower. Using kcat/KM as a measure of efficiency, UDP-xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting that there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems.
Asunto(s)
Glucanos/biosíntesis , Pentosiltransferasa/genética , Proteínas de Plantas/genética , Plantas/enzimología , Glucanos/genética , Cinética , Pentosiltransferasa/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Especificidad por SustratoRESUMEN
The plant cell wall is primarily a polysaccharide mesh of the most abundant biopolymers on earth. Although one of the richest sources of biorenewable materials, the biosynthesis of the plant polysaccharides is poorly understood. Structures of many essential plant glycosyltransferases are unknown and suitable substrates are often unavailable for in vitro analysis. The dearth of such information impedes the development of plants better suited for industrial applications. Presented here are structures of Arabidopsis xyloglucan xylosyltransferase 1 (XXT1) without ligands and in complexes with UDP and cellohexaose. XXT1 initiates side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. Structures here and the properties of mutant XXT1s are consistent with a SNi-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. The XXT1 dimer alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed by the acceptor binding cleft. Homology modeling of XXT2 and XXT5, the other two xylosyltransferases involved in xyloglucan biosynthesis, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. An assembly of the three XXTs can produce the xylosylation patterns of native xyloglucans, suggesting the involvement of an organized multienzyme complex in the xyloglucan biosynthesis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/ultraestructura , Pentosiltransferasa/metabolismo , Pentosiltransferasa/ultraestructura , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Pared Celular/metabolismo , Cristalografía por Rayos X/métodos , Glucanos/genética , Glucanos/metabolismo , Modelos Biológicos , Pentosiltransferasa/genética , Xilanos/genética , Xilanos/metabolismo , UDP Xilosa Proteína XilosiltransferasaRESUMEN
Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a ß-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Pentosiltransferasa/metabolismo , Secuencias de Aminoácidos/genética , Proteínas de Arabidopsis/genética , Escherichia coli/genética , Mutagénesis , Oligosacáridos/metabolismo , Pentosiltransferasa/genética , Plantas Modificadas Genéticamente , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Uridina Difosfato Xilosa/metabolismoRESUMEN
In dicotyledonous plants, xyloglucan (XyG) is the most abundant hemicellulose of the primary cell wall. The enzymes involved in XyG biosynthesis have been identified through reverse-genetics and activity was characterized by heterologous expression. Currently, there is no information on the atomic structures or amino acids involved in activity or substrate binding of any of the Golgi-localized XyG biosynthetic enzymes. A homology model of the xyloglucan xylosyltransferase 2 (XXT2) catalytic domain was built on the basis of the crystal structure of A64Rp. Molecular dynamics simulations revealed that the homology model retains the glycosyltransferase (GT)-A fold of the template structure used to build the homology model indicating that XXT2 likely has a GT-A fold. According to the XXT2 homology model, six amino acids (Phe204, Lys207, Asp228, Ser229, Asp230, His378) were selected and their contribution in catalytic activity was investigated. Site-directed mutagenesis studies show that Asp228, Asp230 and His378 are critical for XXT2 activity and are predicted to be involved in coordination of manganese ion. Lys207 was also found to be critical for protein activity and the homology model indicates a critical role in substrate binding. Additionally, Phe204 mutants have less of an impact on XXT2 activity with the largest effect when replaced with a polar residue. This is the first study that investigates the amino acids involved in substrate binding of the XyG-synthesizing xylosyltransferases and contributes to the understanding of the mechanisms of polysaccharide-synthesizing GTs and XyG biosynthesis.
Asunto(s)
Aminoácidos/química , Pared Celular/química , Pentosiltransferasa/química , Conformación Proteica , Secuencia de Aminoácidos/genética , Aminoácidos/genética , Arabidopsis/química , Arabidopsis/genética , Sitios de Unión , Dominio Catalítico , Pared Celular/enzimología , Glucanos/biosíntesis , Glucanos/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pentosiltransferasa/genética , Unión Proteica , Homología Estructural de Proteína , Especificidad por Sustrato , Xilanos/biosíntesis , Xilanos/químicaRESUMEN
High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.
Asunto(s)
Apolipoproteínas , Lipocalinas , Humanos , Ratones , Animales , Apolipoproteínas/metabolismo , Apolipoproteínas/farmacología , Lipocalinas/metabolismo , Lipocalinas/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Apolipoproteínas M , Inflamación , Lipoproteínas HDL/farmacología , Lipoproteínas HDL/metabolismo , Lisofosfolípidos/farmacología , Lisofosfolípidos/metabolismo , EsfingosinaRESUMEN
The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer's disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-Å resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.
Asunto(s)
Transportador 1 de Casete de Unión a ATP , Membrana Celular , Lípidos de la Membrana , Imagen Individual de Molécula , Esteroles , Humanos , Apolipoproteína A-I/metabolismo , Transportador 1 de Casete de Unión a ATP/química , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Membrana Celular/química , Fosfolípidos/química , Esteroles/química , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Mutación , Membrana Dobles de Lípidos/química , Imagen Individual de Molécula/métodosRESUMEN
Xyloglucan is the most abundant hemicellulose in the primary cell wall of dicotyledonous plants. In Arabidopsis, three xyloglucan xylosyltransferases, XXT1, XXT2, and XXT5, participate in xylosylation of the xyloglucan backbone. Despite the importance of these enzymes, there is a lack of information on their structure and the critical residues required for substrate binding and transferase activity. In this study, the roles of different domains of XX2 in protein expression and catalytic activity were investigated by constructing a series of N- and C-terminal truncations. XXT2 with an N-terminal truncation of 31 amino acids after the predicted transmembrane domain showed the highest protein expression, but truncations of more than 31 residues decreased protein expression and catalytic activity. XXT2 constructs with C-terminal truncations showed increased protein expression but decreased activity, particularly for truncations of 44 or more amino acids. Site-directed mutagenesis was also used to investigate six positively charged residues near the C-terminus and found that four of the mutants showed decreased enzymatic activity. We conclude that the N- and C-termini of XXT2 have important roles in protein folding and enzymatic activity: the stem region (particularly the N-terminus of the catalytic domain) is critical for protein folding and the C-terminus is essential for enzymatic activity but not for protein folding.