RESUMO
Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. We previously described abnormalities in the branched-chain amino acid (BCAA) catabolic pathway as a cause of ASD. Here, we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation, and severe neurological abnormalities. Furthermore, we identified several patients with autistic traits and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. Finally, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for the BCAA in human brain function.
Assuntos
Transtorno do Espectro Autista/genética , Barreira Hematoencefálica/fisiopatologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Mutação , Aminoácidos/administração & dosagem , Aminoácidos/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Transtorno do Espectro Autista/fisiopatologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Feminino , Humanos , Lactente , Recém-Nascido , Transportador 1 de Aminoácidos Neutros Grandes/genética , Masculino , Camundongos , Camundongos Knockout , Linhagem , Biossíntese de Proteínas , Receptor TIE-2/genéticaRESUMO
Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.
Assuntos
Antiporters , Prótons , Antiporters/genéticaRESUMO
BACKGROUND: SLC38A2 is a ubiquitously expressed Na+-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II diabetes and cancer. Despite its relevance in human physio-pathology, structure/function relationship studies and identification of ligands with regulatory roles are still in infancy. METHODS AND RESULTS: The cDNA coding for SLC38A2 was cloned in the pET-28-Mistic vector, and the BL21 codon plus RIL strain was transformed with the recombinant construct. 0.5% glucose and oxygen availability were crucial for protein expression. The over-expressed hSNAT2-Mistic chimera was cleaved on column and purified by nickel-chelating affinity chromatography, with a yield of about 60 mg/Liter cell culture. The purified hSNAT2 was reconstituted in proteoliposomes in an active form with a right-side-out orientation with respect to the native membrane. CONCLUSIONS: The addition of a Mistic tag at the N-terminus of the SNAT2 protein was crucial for its over-expression and purification. The purified protein was functionally active, representing a powerful tool for performing structure/function studies and testing ligands as inhibitors and/or activators.
Assuntos
Sistema A de Transporte de Aminoácidos , Humanos , Sistema A de Transporte de Aminoácidos/biossíntese , Proteínas de Membrana TransportadorasRESUMO
Riboflavin is an essential water-soluble vitamin that needs to be provided through the diet because of the conversion into flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), important cofactors in hundreds of flavoenzymes. The adsorption and distribution of riboflavin is mediated by transmembrane transporters of the SLC52 family, namely RFVT1-3, whose mutations are mainly associated with two diseases, MADD and the Brown-Vialetto-Van Laere syndrome. Interest in RFVTs as pharmacological targets has increased in the last few years due to their overexpression in several cancer cells, which can be exploited both by blocking the uptake of riboflavin into the cancerous cells, and by performing cancer targeted delivery of drugs with a high affinity for RFVTs. In this work, we propose three-dimensional structural models for all three human riboflavin transporters obtained by state-of-the-art artificial intelligence-based methods, which were then further refined with molecular dynamics simulations. Furthermore, two of the most notable mutations concerning RFVT2 and RFVT3 (W31S and N21S, respectively) were investigated studying the interactions between the wild-type and mutated transporters with riboflavin.
Assuntos
Inteligência Artificial , Perda Auditiva Neurossensorial , Humanos , Riboflavina/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Perda Auditiva Neurossensorial/genética , Relação Estrutura-Atividade , Mononucleotídeo de Flavina , Flavina-Adenina Dinucleotídeo/metabolismoRESUMO
The large Amino Acid Transporter 1 (LAT1) is an interesting target in drug discovery since this transporter is overexpressed in several human cancers. Furthermore, due to its location in the blood-brain barrier (BBB), LAT1 is interesting for delivering pro-drugs to the brain. In this work, we focused on defining the transport cycle of LAT1 using an in silico approach. So far, studies of the interaction of LAT1 with substrates and inhibitors have not considered that the transporter must undergo at least four different conformations to complete the transport cycle. We built outward-open and inward-occluded conformations of LAT1 using an optimized homology modelling procedure. We used these 3D models and the cryo-EM structures in outward-occluded and inward-open conformations to define the substrate/protein interaction during the transport cycle. We found that the binding scores for the substrate depend on the conformation, with the occluded states as the crucial steps affecting the substrate affinity. Finally, we analyzed the interaction of JPH203, a high-affinity inhibitor of LAT1. The results indicate that conformational states must be considered for in silico analyses and early-stage drug discovery. The two built models, together with the available cryo-EM 3D structures, provide important information on the LAT1 transport cycle, which could be used to speed up the identification of potential inhibitors through in silico screening.
Assuntos
Benzoxazóis , Transportador 1 de Aminoácidos Neutros Grandes , Tirosina , Humanos , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Neoplasias/metabolismo , Tirosina/química , Tirosina/farmacologia , Benzoxazóis/química , Benzoxazóis/farmacologiaRESUMO
The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in E. coli. Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with C12E8 and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na+-independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.
Assuntos
Escherichia coli , Proteolipídeos , Serina , Humanos , Escherichia coli/genética , Transporte Biológico , Transporte de ÍonsRESUMO
The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.
Assuntos
Patulina , Humanos , Animais , Ratos , Escherichia coli/metabolismo , Cisteína/metabolismo , Reagentes de Sulfidrila/farmacologia , Carnitina/farmacologia , Carnitina/metabolismo , Glutationa/metabolismo , Proteínas de Membrana TransportadorasRESUMO
The carnitine/organic cation transporter novel 2 (OCTN2) is responsible for the cellular uptake of carnitine in most tissues. Being a transmembrane protein OCTN2 must interact with the surrounding lipid microenvironment to function. Among the main lipid species that constitute eukaryotic cells, cholesterol has highly dynamic levels under a number of physiopathological conditions. This work describes how plasma membrane cholesterol modulates OCTN2 transport of L-carnitine in human embryonic kidney 293 cells overexpressing OCTN2 (OCTN2-HEK293) and in proteoliposomes harboring human OCTN2. We manipulated the cholesterol content of intact cells, assessed by thin layer chromatography, through short exposures to empty and/or cholesterol-saturated methyl-ß-cyclodextrin (mßcd), whereas free cholesterol was used to enrich reconstituted proteoliposomes. We measured OCTN2 transport using [3H]L-carnitine, and expression levels and localization by surface biotinylation and Western blotting. A 20-min preincubation with mßcd reduced the cellular cholesterol content and inhibited L-carnitine influx by 50% in comparison with controls. Analogously, the insertion of cholesterol in OCTN2-proteoliposomes stimulated L-carnitine uptake in a dose-dependent manner. Carnitine uptake in cells incubated with empty mßcd and cholesterol-saturated mßcd to preserve the cholesterol content was comparable with controls, suggesting that the mßcd effect on OCTN2 was cholesterol dependent. Cholesterol stimulated L-carnitine influx in cells by markedly increasing the affinity for L-carnitine and in proteoliposomes by significantly enhancing the affinity for Na+ and, in turn, the L-carnitine maximal transport capacity. Because of the antilipogenic and antioxidant features of L-carnitine, the stimulatory effect of cholesterol on L-carnitine uptake might represent a novel protective effect against lipid-induced toxicity and oxidative stress.
Assuntos
Carnitina/metabolismo , Colesterol/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Transporte Biológico , Células HEK293 , Humanos , Proteolipídeos/metabolismoRESUMO
Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proliferação de Células , Queratinócitos/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/etiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Humanos , Queratinócitos/imunologia , Queratinócitos/virologia , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Riboflavin transporter deficiency 2 (RTD2) is a rare neurological disorder caused by mutations in the Solute carrier family 52 member 2 (Slc52a2) gene encoding human riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed and mediates tissue distribution of riboflavin, a water-soluble vitamin that, after conversion into FMN and FAD, plays pivotal roles in carbohydrate, protein, and lipid metabolism. The 3D structure of RFVT2 has been constructed by homology modeling using three different templates that are equilibrative nucleoside transporter 1 (ENT1), Fucose: proton symporter, and glucose transporter type 5 (GLUT5). The structure has been validated by several approaches. All known point mutations of RFVT2, associated with RTD2, have been localized in the protein 3D model. Six of these mutations have been introduced in the recombinant protein for functional characterization. The mutants W31S, S52F, S128L, L312P, C325G, and M423V have been expressed in E. coli, purified, and reconstituted into proteoliposomes for transport assay. All the mutants showed impairment of function. The Km for riboflavin of the mutants increased from about 3 to 9 times with respect to that of WT, whereas Vmax was only marginally affected. This agrees with the improved outcome of most RTD2 patients after administration of high doses of riboflavin.
Assuntos
Doenças Neurodegenerativas , Receptores Acoplados a Proteínas G , Perda Auditiva Neurossensorial/genética , Humanos , Mutação , Doenças Neurodegenerativas/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Nucleotidiltransferases , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Doenças Neuromusculares/genética , Nucleotidiltransferases/genética , Riboflavina/metabolismo , Vitaminas/metabolismoRESUMO
BACKGROUND: Escherichia coli is a widely used tool for the over-expression of human proteins for studying structure and function. The toxicity of human proteins for E. coli often hampers the expression. This study aims to find conditions for the expression of a membrane transporter known as the carnitine transporter CT2. The knowledge on this transporter is scarce, thus obtaining the recombinant protein is very important for further studies. METHODS AND RESULTS: The cDNAs coding for human CT2 (hCT2) was cloned in the pH6EX3 vector and different transformed E. coli strains were cultured in absence or in presence of glucose. hCT2 expression was obtained. The protein was purified and reconstituted into proteoliposomes in a functionally active state. CONCLUSIONS: Using the appropriate IPTG concentration, together with the addition of glucose, hCT2 has been expressed in E. coli. The protein is active and shows capacity to transport carnitine in proteoliposomes. The results have a great interest in basic biochemistry of membrane transporters and applications to human health since hCT2 is involved in human pathology.
Assuntos
Carnitina , Escherichia coli , Carnitina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas Recombinantes/metabolismoRESUMO
The Novel Organic Cation Transporter, OCTN1, is the first member of the OCTN subfamily; it belongs to the wider Solute Carrier family SLC22, which counts many members including cation and anion organic transporters. The tertiary structure has not been resolved for any cation organic transporter. The functional role of OCNT1 is still not well assessed despite the many functional studies so far conducted. The lack of a definitive identification of OCTN1 function can be attributed to the different experimental systems and methodologies adopted for studying each of the proposed ligands. Apart from the contradictory data, the international scientific community agrees on a role of OCTN1 in protecting cells and tissues from oxidative and/or inflammatory damage. Moreover, the involvement of this transporter in drug interactions and delivery has been well clarified, even though the exact profile of the transported/interacting molecules is still somehow confusing. Therefore, OCTN1 continues to be a hot topic in terms of its functional role and structure. This review focuses on the most recent advances on OCTN1 in terms of functional aspects, physiological roles, substrate specificity, drug interactions, tissue expression, and relationships with pathology.
Assuntos
Biomarcadores , Suscetibilidade a Doenças , Interações Medicamentosas , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Acetilação , Animais , Sítios de Ligação , Transporte Biológico , Ergotioneína/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Modelos Moleculares , Conformação Molecular , Especificidade de Órgãos , Proteínas de Transporte de Cátions Orgânicos/química , Ligação Proteica , Relação Estrutura-Atividade , Simportadores/químicaRESUMO
Ten percent of human genes encode for membrane transport systems, which are key components in maintaining cell homeostasis. They are involved in the transport of nutrients, catabolites, vitamins, and ions, allowing the absorption and distribution of these compounds to the various body regions. In addition, roughly 60% of FDA-approved drugs interact with membrane proteins, among which are transporters, often responsible for pharmacokinetics and side effects. Defects of membrane transport systems can cause diseases; however, knowledge of the structure/function relationships of transporters is still limited. Among the expression of hosts that produce human membrane transport systems, E. coli is one of the most favorable for its low cultivation costs, fast growth, handiness, and extensive knowledge of its genetics and molecular mechanisms. However, the expression in E. coli of human membrane proteins is often toxic due to the hydrophobicity of these proteins and the diversity in structure with respect to their bacterial counterparts. Moreover, differences in codon usage between humans and bacteria hamper translation. This review summarizes the many strategies exploited to achieve the expression of human transport systems in bacteria, providing a guide to help people who want to deal with this topic.
Assuntos
Escherichia coli , Proteínas de Membrana Transportadoras , Bactérias/metabolismo , Transporte Biológico , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismoRESUMO
The plasma membrane transporter ASCT2 is a well-known Na+-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [3H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na+-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [3H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLTPh.
Assuntos
Substituição de Aminoácidos , Sistema ASC de Transporte de Aminoácidos/química , Sistema ASC de Transporte de Aminoácidos/metabolismo , Cisteína/metabolismo , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Sítios de Ligação , Clonagem Molecular , Glutamina/metabolismo , Humanos , Transporte de Íons , Antígenos de Histocompatibilidade Menor/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimentoRESUMO
Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos/metabolismoRESUMO
The localization of membrane transporters at the forefront of natural barriers makes these proteins very interesting due to their involvement in the absorption and distribution of nutrients and xenobiotics, including drugs. Over the years, structure/function relationship studies have been performed employing several strategies, including chemical modification of exposed amino acid residues. These approaches are very meaningful when applied to membrane transporters, given that these proteins are characterized by both hydrophobic and hydrophilic domains with a different degree of accessibility to employed chemicals. Besides basic features, the chemical targeting approaches can disclose information useful for pharmacological applications as well. An eminent example of this picture is the histidine/large amino acid transporter SLC7A5, known as LAT1 (Large Amino Acid Transporter 1). This protein is crucial in cell life because it is responsible for mediating the absorption and distribution of essential amino acids in peculiar body districts, such as the blood brain barrier and placenta. Furthermore, LAT1 can recognize a large variety of molecules of pharmacological interest and is also considered a hot target for drugs due to its over-expression in virtually all human cancers. Therefore, it is not surprising that the chemical targeting approach, coupled with bioinformatics, site-directed mutagenesis and transport assays, proved fundamental in describing features of LAT1 such as the substrate binding site, regulatory domains and interactions with drugs that will be discussed in this review. The results on LAT1 can be considered to have general applicability to other transporters linked with human diseases.
Assuntos
Histidina/antagonistas & inibidores , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Preparações Farmacêuticas/química , Biomarcadores/análise , Biomarcadores/metabolismo , Biologia Computacional , Histidina/metabolismo , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/genéticaRESUMO
It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5-1%), at low temperature (15 °C), and low inducer concentrations (10-100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.
Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Pneumonia Viral/virologia , Sistema ASC de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Enzima de Conversão de Angiotensina 2 , COVID-19 , Temperatura Baixa , DNA Complementar/genética , Descoberta de Drogas , Escherichia coli/genética , Expressão Gênica , Humanos , Antígenos de Histocompatibilidade Menor/genética , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2RESUMO
This editorial aims to summarize the 19 scientific papers that contributed to this Special Issue.
Assuntos
Aminoácidos/metabolismo , Transporte Biológico/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Humanos , Transportador 1 de Aminoácidos Neutros Grandes , ProteínasRESUMO
The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MßCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MßCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.