RESUMEN
The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Microscopía por Crioelectrón , Animales , Humanos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Ratones , Sitio Alostérico , Células HEK293 , Sitios de Unión , Conformación ProteicaRESUMEN
Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Microscopía por Crioelectrón , Prolina , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Prolina/metabolismo , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , COVID-19/virología , COVID-19/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Modelos MolecularesRESUMEN
The neutral amino acid transporter subfamily that consists of six members, consecutively SLC6A15-SLC620, also called orphan transporters, represents membrane, sodium-dependent symporter proteins that belong to the family of solute carrier 6 (SLC6). Primarily, they mediate the transport of neutral amino acids from the extracellular milieu toward cell or storage vesicles utilizing an electric membrane potential as the driving force. Orphan transporters are widely distributed throughout the body, covering many systems; for instance, the central nervous, renal, or intestinal system, supplying cells into molecules used in biochemical, signaling, and building pathways afterward. They are responsible for intestinal absorption and renal reabsorption of amino acids. In the central nervous system, orphan transporters constitute a significant medium for the provision of neurotransmitter precursors. Diseases related with aforementioned transporters highlight their significance; SLC6A19 mutations are associated with metabolic Hartnup disorder, whereas altered expression of SLC6A15 has been associated with a depression/stress-related disorders. Mutations of SLC6A18-SLCA20 cause iminoglycinuria and/or hyperglycinuria. SLC6A18-SLC6A20 to reach the cellular membrane require an ancillary unit ACE2 that is a molecular target for the spike protein of the SARS-CoV-2 virus. SLC6A19 has been proposed as a molecular target for the treatment of metabolic disorders resembling gastric surgery bypass. Inhibition of SLC6A15 appears to have a promising outcome in the treatment of psychiatric disorders. SLC6A19 and SLC6A20 have been suggested as potential targets in the treatment of COVID-19. In this review, we gathered recent advances on orphan transporters, their structure, functions, related disorders, and diseases, and in particular their relevance as therapeutic targets. SIGNIFICANCE STATEMENT: The following review systematizes current knowledge about the SLC6A15-SLCA20 neutral amino acid transporter subfamily and their therapeutic relevance in the treatment of different diseases.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Aminoácidos Neutros , COVID-19 , Humanos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Riñón/metabolismo , Aminoácidos/metabolismo , Aminoácidos Neutros/metabolismo , COVID-19/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Mammalian target of rapamycin complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Some amino acids signal to mTORC1 through the Rag GTPase, whereas glutamine and asparagine activate mTORC1 through a Rag GTPase-independent pathway. Here, we show that the lysosomal glutamine and asparagine transporter SNAT7 activates mTORC1 after extracellular protein, such as albumin, is macropinocytosed. The N terminus of SNAT7 forms nutrient-sensitive interaction with mTORC1 and regulates mTORC1 activation independently of the Rag GTPases. Depletion of SNAT7 inhibits albumin-induced mTORC1 lysosomal localization and subsequent activation. Moreover, SNAT7 is essential to sustain KRAS-driven pancreatic cancer cell growth through mTORC1. Thus, SNAT7 links glutamine and asparagine signaling from extracellular protein to mTORC1 independently of the Rag GTPases and is required for macropinocytosis-mediated mTORC1 activation and pancreatic cancer cell growth.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Lisosomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Pinocitosis , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Asparagina/metabolismo , Glutamina/metabolismo , Humanos , Lisosomas/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de SeñalRESUMEN
It is known that 4F2hc and rBAT are the heavy subunits of the heteromeric amino acid transporters (HATs). These heavy subunits are N-glycosylated proteins, with an N-terminal domain, one transmembrane domain and a bulky extracellular domain (ectodomain) that belongs to the α-amylase family. The heavy subunits are covalently linked to a light subunit from the SLC7 family, which is responsible for the amino acid transport activity, forming a heterodimer. The functions of 4F2hc and rBAT are related mainly to the stability and trafficking of the HATs in the plasma membrane of vertebrates, where they exert the transport activity. Moreover, 4F2hc is a modulator of integrin signaling, has a role in cell fusion and it is overexpressed in some types of cancers. On the other hand, some mutations in rBAT are found to cause the malfunctioning of the b0,+ transport system, leading to cystinuria. The ectodomains of 4F2hc and rBAT share both sequence and structure homology with α-amylase family members. Very recently, cryo-EM has revealed the structure of several HATs, including the ectodomains of rBAT and 4F2hc. Here, we analyze available data on the ectodomains of rBAT and 4Fhc and their relationship with the α-amylase family. The physiological relevance of this relationship remains largely unknown.
Asunto(s)
Sistemas de Transporte de Aminoácidos/química , alfa-Glucosidasas/química , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Dominio Catalítico , Microscopía por Crioelectrón , Cadena Pesada de la Proteína-1 Reguladora de Fusión/química , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Humanos , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína , alfa-Glucosidasas/genéticaRESUMEN
We modeled 3D structures of all SARS-CoV-2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that Ë6% of the proteome mimicked human proteins, while Ë7% was implicated in hijacking mechanisms that reverse post-translational modifications, block host translation, and disable host defenses; a further Ë29% self-assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is-and is not-known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria-COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Interacciones Huésped-Patógeno/genética , Procesamiento Proteico-Postraduccional , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Sitios de Unión , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Biología Computacional/métodos , Proteínas de la Envoltura de Coronavirus/química , Proteínas de la Envoltura de Coronavirus/genética , Proteínas de la Envoltura de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Humanos , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Modelos Moleculares , Imitación Molecular , Neuropilina-1/química , Neuropilina-1/genética , Neuropilina-1/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas/métodos , Multimerización de Proteína , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Viroporinas/química , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismo , Replicación ViralRESUMEN
SLC38A6 (SNAT6) is the only known member of the SLC38 family that is expressed exclusively in the excitatory neurons of the brain. It has been described as an orphan transporter with an unknown substrate profile, therefore very little is known about SNAT6. In this study, we addressed the substrate specificity, mechanisms for internalization of SNAT6, and the regulatory role of SNAT6 with specific insights into the glutamate-glutamine cycle. We used tritium-labeled amino acids in order to demonstrate that SNAT6 is functioning as a glutamine and glutamate transporter. SNAT6 revealed seven predicted transmembrane segments in a homology model and was localized to caveolin rich sites at the plasma membrane. SNAT6 has high degree of specificity for glutamine and glutamate. Presence of these substrates enables formation of SNAT6-caveolin complexes that aids in sodium dependent trafficking of SNAT6 off the plasma membrane. To further understand its mode of action, several potential interacting partners of SNAT6 were identified using bioinformatics. Among them where CTP synthase 2 (CTPs2), phosphate activated glutaminase (Pag), and glutamate metabotropic receptor 2 (Grm2). Co-expression analysis, immunolabeling with co-localization analysis and proximity ligation assays of these three proteins with SNAT6 were performed to investigate possible interactions. SNAT6 can cycle between cytoplasm and plasma membrane depending on availability of substrates and interact with Pag, synaptophysin, CTPs2, and Grm2. Our data suggest a potential role of SNAT6 in glutamine uptake at the pre-synaptic terminal of excitatory neurons. We propose here a mechanistic model of SNAT6 trafficking that once internalized influences the glutamate-glutamine cycle in presence of its potential interacting partners.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Caveolinas/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Caveolinas/química , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Ratones , Modelos Biológicos , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , ARN Interferente Pequeño/genética , Transducción de Señal , Sodio/metabolismo , Relación Estructura-ActividadRESUMEN
Biallelic pathogenic variants in solute carrier family 38 member 8, SLC38A8, cause a pan-ocular autosomal recessive condition known as foveal hypoplasia 2, FVH2, characterised by foveal hypoplasia, nystagmus and optic nerve chiasmal misrouting. Patients are often clinically diagnosed with ocular albinism, but foveal hypoplasia can occur in several other ocular disorders. Here we describe nine patients from seven families who had molecularly confirmed biallelic recessive variants in SLC38A8 identified through whole genome sequencing or targeted gene panel testing. We identified four novel sequence variants (p.(Tyr88*), p.(Trp145*), p.(Glu233Gly) and c.632+1G>A). All patients presented with foveal hypoplasia, nystagmus and reduced visual acuity; however, one patient did not exhibit any signs of chiasmal misrouting, and three patients had features of anterior segment dysgenesis. We highlight these findings in the context of 30 other families reported to date. This study reinforces the importance of obtaining a molecular diagnosis in patients whose phenotype overlap with other inherited ocular conditions, in order to support genetic counselling, clinical prognosis and family planning. We expand the spectrum of SLC38A8 mutations which will be relevant for treatment through future genetic-based therapies.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/genética , Enfermedades Hereditarias del Ojo/genética , Fóvea Central/patología , Mutación , Enfermedades de la Retina/genética , Alelos , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Anomalías del Ojo , Enfermedades Hereditarias del Ojo/patología , Femenino , Humanos , Masculino , Linaje , Dominios Proteicos , Enfermedades de la Retina/patología , Agudeza Visual , Población Blanca/genética , Secuenciación Completa del GenomaRESUMEN
Electrochemical sensing performance is often compromised by electrode biofouling (e.g., proteins nonspecific binding) in complex biological fluids; however, the design and construction of a robust biointerface remains a great challenge. Herein, inspired by nature, we demonstrate a robust polydopamine-engineered biointerfacing, to tailing zwitterionic molecules (i.e., sulfobetaine methacrylate, SBMA) through Michael Addition. The SBMA-PDA biointerface can resist proteins nonspecific binding in complex biological fluids while enhancing interfacial electron transfer and electrochemical stability of the electrode. In addition, this sensing interface can be integrated with tissue-implantable electrode for in vivo analysis with improved sensing performance, preserving ca. 92.0% of the initial sensitivity after 2 h of implantation in brain tissue, showing low acute neuroinflammatory responses and good stability both in normal and in Parkinson's disease (PD) rat brain tissue.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/química , Técnicas Electroquímicas/métodos , Indoles/química , Polímeros/química , Animales , Ratas , Propiedades de SuperficieRESUMEN
Protein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/ultraestructura , Proteínas Portadoras/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Unión Proteica/genética , Conformación Proteica , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sitios de Unión/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutamina/genética , Cinética , Ligandos , Cadenas de Markov , Simulación de Dinámica MolecularRESUMEN
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has challenged the speed at which laboratories can discover the viral composition and study health outcomes. The small â¼30-kb ssRNA genome of coronaviruses makes them adept at cross-species spread while enabling a robust understanding of all of the proteins the viral genome encodes. We have employed protein modeling, molecular dynamics simulations, evolutionary mapping, and 3D printing to gain a full proteome- and dynamicome-level understanding of SARS-CoV-2. We established the Viral Integrated Structural Evolution Dynamic Database (VIStEDD at RRID:SCR_018793) to facilitate future discoveries and educational use. Here, we highlight the use of VIStEDD for nsp6, nucleocapsid (N), and spike (S) surface glycoprotein. For both nsp6 and N, we found highly conserved surface amino acids that likely drive protein-protein interactions. In characterizing viral S protein, we developed a quantitative dynamics cross-correlation matrix to gain insights into its interactions with the angiotensin I-converting enzyme 2 (ACE2)-solute carrier family 6 member 19 (SLC6A19) dimer. Using this quantitative matrix, we elucidated 47 potential functional missense variants from genomic databases within ACE2/SLC6A19/transmembrane serine protease 2 (TMPRSS2), warranting genomic enrichment analyses in SARS-CoV-2 patients. These variants had ultralow frequency but existed in males hemizygous for ACE2. Two ACE2 noncoding variants (rs4646118 and rs143185769) present in â¼9% of individuals of African descent may regulate ACE2 expression and may be associated with increased susceptibility of African Americans to SARS-CoV-2. We propose that this SARS-CoV-2 database may aid research into the ongoing pandemic.
Asunto(s)
Betacoronavirus/química , Betacoronavirus/genética , Infecciones por Coronavirus/metabolismo , Bases de Datos de Proteínas , Simulación de Dinámica Molecular , Neumonía Viral/metabolismo , Proteoma , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Enzima Convertidora de Angiotensina 2 , Población Negra/genética , COVID-19 , Infecciones por Coronavirus/virología , Proteínas de la Nucleocápside de Coronavirus , Predisposición Genética a la Enfermedad , Variación Genética , Interacciones Huésped-Patógeno , Humanos , Masculino , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Pandemias , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Fosfoproteínas , Neumonía Viral/virología , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , SARS-CoV-2 , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
The global pandemic caused by the SARS-CoV-2 infection is a health emergency that needs to be addressed immediately. The international scientific community, following World Health Organization (WHO) indications, launched different trials for testing drugs putatively able to block the SARS-CoV-2 infection or treat the COVID-19 disease symptoms. In parallel, studies devoted to a better understanding of SARS-CoV-2 biology are in the course for designing an effective vaccine. One of the human membrane proteins known to be docked by the virus is angiotensin-converting enzyme 2 (ACE2), proposed to be responsible for viral entry in target cells. Recently, the 3D structure of ACE2 has been obtained, showing its physical interaction with B0AT1 (SLC6A19), a plasma membrane transporter involved in the trafficking of amino acids in cells. The receptor targeted by SARS-CoV-2 is a supercomplex formed by a dimer of ACE2-B0AT1, in which ACE2 binds the viral protein and B0AT1 stabilizes the heterodimer. As a serendipity occurrence, nimesulide was shown to abolish the transport function of B0AT1. Here we suggest including nimesulide in the list of drugs to be tested for the identification of co-adjuvants in the treatment of COVID-19.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antivirales/farmacología , SARS-CoV-2/patogenicidad , Sulfonamidas/farmacología , Sistemas de Transporte de Aminoácidos Neutros/química , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Reposicionamiento de Medicamentos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Serina Endopeptidasas/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
Carnitine (CAR), an amino acid derivative, has great potential as a facial exfoliating agent owing to its calcium chelating property under weakly acidic or neutral conditions. However, its application is limited by its poor transdermal penetration. To optimise its exfoliation efficacy with minimal concentration, we propose the ion-pair method. The ionic interaction between CAR and a zwitterionic substance was successfully monitored by measuring conductivity. The alterations of penetration and exfoliation efficacy for CAR addition to different types of counter ions were investigated in vitro and in vivo. We found that hydrogenated soya phosphatidylcholine (HSC), an amphiphilic counter ion, significantly increases the stratum corneum penetration and exfoliation efficacy of CAR. The changes of the CAR-HSC ionic interaction in the presence of calcium ions were also investigated by 1H nuclear magnetic resonance (NMR) spectroscopy. The NMR spectra for amino groups of CAR first decreased with HSC and then gradually recovered and shifted as calcium ions were added. From the results, a noble exfoliating complex of CAR with high exfoliation efficacy could be proposed. Moreover, the results demonstrate that NMR spectroscopy is useful to obtain direct experimental evidence of the molecular dynamics simulations of the alteration of an exfoliating complex as it penetrates.
Asunto(s)
Administración Cutánea , Carnitina/farmacología , Sistemas de Liberación de Medicamentos/métodos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos , Antiinflamatorios no Esteroideos/química , Carnitina/metabolismo , Iones , Espectroscopía de Resonancia Magnética , Piel/metabolismo , Absorción Cutánea/fisiologíaRESUMEN
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Modelos Moleculares , Conformación Proteica , Simportadores/química , Simportadores/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Aminoácidos , Sitios de Unión , Mutación , Unión Proteica , Proteínas Recombinantes , Relación Estructura-Actividad , Especificidad por Sustrato , Simportadores/genéticaRESUMEN
Cystinuria is an autosomal recessive defect in reabsorptive transport of cystine and the dibasic amino acids ornithine, arginine, and lysine from renal tubule and small intestine. Mutations in two genes: SLC3A1, encoding the heavy chain rbAT of the renal cystine transport system and SLC7A9, the gene of its light chain b0, + AT have a crucial role in the diseases. In our previous studies from Iranian populations with Cystinuria totally six and eleven novel mutations respectively identified in SLC3A1 and SLC7A9 genes. In this study, we conducted an in silico functional analysis to explore the possible association between these genetic mutations and Cystinuria. MutationTaster, PolyPhen-2, PANTHER, FATHMM. PhDSNP and MutPred was applied to predict the degree of pathogenicity for the missense mutations. Furthermore, Residue Interaction Network (RIN) and Intron variant analyses was performed using Cytoscape and Human Slicing Finder softwares. These genetic variants can provide a better understanding of genotype-phenotype relationships in patients with Cystinuria. In the future, the findings may also facilitate the development of new molecular diagnostic markers for the diseases.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Biología Computacional/métodos , Cistinuria/genética , Mutación Missense , Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Neutros/química , Sitios de Unión , Simulación por Computador , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Irán , Modelos Moleculares , Conformación Proteica , Programas InformáticosAsunto(s)
Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Treonina/fisiología , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Transporte Biológico/genética , Femenino , Proteínas de Insectos/genética , Activación del Canal Iónico/genética , Manduca , Potenciales de la Membrana/genética , Oocitos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Treonina/química , Treonina/genética , Xenopus laevisRESUMEN
Solubilization of parenteral drugs is a high unmet need in both preclinical and clinical drug development. Recently, co-amorphous drug formulation has emerged as a new strategy to solubilize orally dosed drugs. The aim of the present study is to explore the feasibility of using the co-amorphous strategy to enable the dosing of parenteral zwitterionic drugs at a high concentration. A new screening procedure was established with solubility as the indicator for co-amorphous co-former selection, and lyophilization was established as the method for co-amorphous formulation preparation. Various amino acids were screened, and tryptophan was found to be the most powerful in improving the solubility of ofloxacin when lyophilized with ofloxacin at a 1:1 weight ratio, with more than 10 times solubility increase. X-ray powder diffraction showed complete amorphization of both components, and an elevated Tg compared with the theoretical value was observed in differential scanning calorimetry. Fourier transform infrared spectroscopy revealed that hydrogen bonding and π-π stacking were possibly involved in the formation of a co-amorphous system in the solid state. Further solution-state characterization revealed the involvement of ionic interactions and π-π stacking in maintaining a high concentration of ofloxacin in solution. Furthermore, co-amorphous ofloxacin/tryptophan at 1:1 weight ratio was both physically and chemically stable for at least 2 months at 40 °C/75% RH. Lastly, the same screening procedure was validated with two more zwitterionic compounds, showing its promise as a routine screening methodology to solubilize and enable the parenteral delivery of zwitterionic compounds.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/química , Aminoácidos/química , Liofilización , Enlace de Hidrógeno , Ofloxacino/química , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Triptófano/químicaAsunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Autofagia , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/química , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/química , Plantas/genética , Plantas/metabolismo , Estructura Secundaria de Proteína , Transporte de ProteínasRESUMEN
BACKGROUND: Cystinuria is an inherited disease that results in the formation of cystine stones in the kidney, which can have serious health complications. Two genes (SLC7A9 and SLC3A1) that form an amino acid transporter are known to be responsible for the disease. Variants that cause the disease disrupt amino acid transport across the cell membrane, leading to the build-up of relatively insoluble cystine, resulting in formation of stones. Assessing the effects of each mutation is critical in order to provide tailored treatment options for patients. We used various computational methods to assess the effects of cystinuria associated mutations, utilising information on protein function, evolutionary conservation and natural population variation of the two genes. We also analysed the ability of some methods to predict the phenotypes of individuals with cystinuria, based on their genotypes, and compared this to clinical data. RESULTS: Using a literature search, we collated a set of 94 SLC3A1 and 58 SLC7A9 point mutations known to be associated with cystinuria. There are differences in sequence location, evolutionary conservation, allele frequency, and predicted effect on protein function between these mutations and other genetic variants of the same genes that occur in a large population. Structural analysis considered how these mutations might lead to cystinuria. For SLC7A9, many mutations swap hydrophobic amino acids for charged amino acids or vice versa, while others affect known functional sites. For SLC3A1, functional information is currently insufficient to make confident predictions but mutations often result in the loss of hydrogen bonds and largely appear to affect protein stability. Finally, we showed that computational predictions of mutation severity were significantly correlated with the disease phenotypes of patients from a clinical study, despite different methods disagreeing for some of their predictions. CONCLUSIONS: The results of this study are promising and highlight the areas of research which must now be pursued to better understand how mutations in SLC3A1 and SLC7A9 cause cystinuria. The application of our approach to a larger data set is essential, but we have shown that computational methods could play an important role in designing more effective personalised treatment options for patients with cystinuria.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Neutros/química , Cistinuria/genética , Modelos Moleculares , Mutación Puntual , Índice de Severidad de la Enfermedad , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Biología Computacional , Cistinuria/metabolismo , Estudios de Asociación Genética , Humanos , Medicina de Precisión , Conformación ProteicaRESUMEN
Glutamine-Binding Protein (GlnBP) of Escherichia coli, an important member of the periplasmic binding protein family, is responsible for the first step in the active transport of glutamine across the cytoplasmic membrane. In this work, the functionally key regulation sites of GlnBP were identified by utilizing a perturbation method proposed by our group, in which the residues whose perturbations markedly change the binding free energy between GlnBP and glutamine are considered to be functionally key residues. The results show that besides the substrate binding sites, some other residues distant from the binding pocket, including the ones in the hinge regions between the two domains, the front- and back- door channels and the exposed region, are important for the function of glutamine binding and transport. The predicted results are well consistent with the theoretical and experimental data, which indicates that our method is an effective approach to identify the key residues important for both ligand binding and long-range allosteric signal transmission. This work can provide some insights into the function performance of GlnBP and the physical mechanism of its allosteric regulation.