RESUMO
Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Cistinose , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Transporte Biológico , Cistina/metabolismo , Cistinose/genética , Humanos , Lisossomos/metabolismo , PrótonsRESUMO
IDO1 oxidizes tryptophan (TRP) to generate kynurenine (KYN), the substrate for 1-carbon and NAD metabolism, and is implicated in pro-cancer pathophysiology and infection biology. However, the mechanistic relationships between IDO1 in amino acid depletion versus product generation have remained a longstanding mystery. We found an unrecognized link between IDO1 and cell survival mediated by KYN that serves as the source for molecules that inhibit ferroptotic cell death. We show that this effect requires KYN export from IDO1-expressing cells, which is then available for non-IDO1-expressing cells via SLC7A11, the central transporter involved in ferroptosis suppression. Whether inside the "producer" IDO1+ cell or the "receiver" cell, KYN is converted into downstream metabolites, suppressing ferroptosis by ROS scavenging and activating an NRF2-dependent, AHR-independent cell-protective pathway, including SLC7A11, propagating anti-ferroptotic signaling. IDO1, therefore, controls a multi-pronged protection pathway from ferroptotic cell death, underscoring the need to re-evaluate the use of IDO1 inhibitors in cancer treatment.
Assuntos
Sistema y+ de Transporte de Aminoácidos , Ferroptose , Cinurenina , Neoplasias , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Cinurenina/farmacologia , Neoplasias/metabolismo , Transdução de Sinais , Triptofano/metabolismoRESUMO
Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.
Assuntos
Antiporters/química , Antiporters/metabolismo , Cistina/metabolismo , Ácido Glutâmico/metabolismo , Glutationa/biossíntese , Sistema y+ de Transporte de Aminoácidos/química , Sistema y+ de Transporte de Aminoácidos/metabolismo , Antiporters/genética , Bioquímica , Microscopia Crioeletrônica , Cisteína/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/química , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Células HEK293 , Humanos , Neoplasias , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
The SLC15 family of proton-coupled solute carriers PepT1 and PepT2 play a central role in human physiology as the principal route for acquiring and retaining dietary nitrogen. A remarkable feature of the SLC15 family is their extreme substrate promiscuity, which has enabled the targeting of these transporters for the improvement of oral bioavailability for several prodrug molecules. Although recent structural and biochemical studies on bacterial homologs have identified conserved sites of proton and peptide binding, the mechanism of peptide capture and ligand promiscuity remains unclear for mammalian family members. Here, we present the cryo-electron microscopy structure of the outward open conformation of the rat peptide transporter PepT2 in complex with an inhibitory nanobody. Our structure, combined with molecular dynamics simulations and biochemical and cell-based assays, establishes a framework for understanding peptide and prodrug recognition within this pharmaceutically important transporter family.
Assuntos
Pró-Fármacos , Simportadores , Animais , Microscopia Crioeletrônica , Mamíferos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transportador 1 de Peptídeos/química , Peptídeos/metabolismo , Prótons , RatosRESUMO
Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.
Assuntos
Microscopia Crioeletrônica , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/metabolismo , Pemetrexede/química , Pemetrexede/metabolismo , Transportador de Folato Acoplado a Próton/química , Transportador de Folato Acoplado a Próton/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Transporte Biológico , Humanos , Modelos Moleculares , Transportador de Folato Acoplado a Próton/ultraestrutura , PrótonsRESUMO
POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Peptídeos/metabolismo , Prótons , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Xanthomonas/química , Xanthomonas/metabolismoRESUMO
Post-translational modification by the ubiquitin-like protein SUMO is often regulated by cellular signals that restrict the modification to appropriate situations. Nevertheless, many SUMO-specific ligases do not exhibit much target specificity, and--compared with the diversity of sumoylation substrates--their number is limited. This raises the question of how SUMO conjugation is controlled in vivo. We report here an unexpected mechanism by which sumoylation of the replication clamp protein, PCNA, from budding yeast is effectively coupled to S phase. We find that loading of PCNA onto DNA is a prerequisite for sumoylation in vivo and greatly stimulates modification in vitro. To our surprise, however, DNA binding by the ligase Siz1, responsible for PCNA sumoylation, is not strictly required. Instead, the stimulatory effect of DNA on conjugation is mainly attributable to DNA binding of PCNA itself. These findings imply a change in the properties of PCNA upon loading that enhances its capacity to be sumoylated.
Assuntos
Regulação Fúngica da Expressão Gênica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína SUMO-1/metabolismo , Alelos , Cromatina/metabolismo , Cisteína Endopeptidases/química , DNA/química , Modelos Biológicos , Modelos Genéticos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina-Proteína Ligases/químicaRESUMO
REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.