RESUMO
Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.
Assuntos
Imunidade Adaptativa/fisiologia , Imunidade Inata/fisiologia , Canais Iônicos/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Síndromes de Imunodeficiência/tratamento farmacológico , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Imunoterapia/métodos , Canais Iônicos/genética , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Terapia de Alvo Molecular , Mutação , Transdução de SinaisRESUMO
Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.
Assuntos
Proteínas de Transporte de Cátions/química , Complexos Multiproteicos/química , Nexinas de Classificação/química , Proteínas de Transporte Vesicular/química , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Espalhamento a Baixo Ângulo , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Calcium (Ca2+) is an essential nutrient for plants and a cellular signal, but excessive levels can be toxic and inhibit growth1,2. To thrive in dynamic environments, plants must monitor and maintain cytosolic Ca2+ homeostasis by regulating numerous Ca2+ transporters3. Here we report two signalling pathways in Arabidopsis thaliana that converge on the activation of vacuolar Ca2+/H+ exchangers (CAXs) to scavenge excess cytosolic Ca2+ in plants. One mechanism, activated in response to an elevated external Ca2+ level, entails calcineurin B-like (CBL) Ca2+ sensors and CBL-interacting protein kinases (CIPKs), which activate CAXs by phosphorylating a serine (S) cluster in the auto-inhibitory domain. The second pathway, triggered by molecular patterns associated with microorganisms, engages the immune receptor complex FLS2-BAK1 and the associated cytoplasmic kinases BIK1 and PBL1, which phosphorylate the same S-cluster in CAXs to modulate Ca2+ signals in immunity. These Ca2+-dependent (CBL-CIPK) and Ca2+-independent (FLS2-BAK1-BIK1/PBL1) mechanisms combine to balance plant growth and immunity by regulating cytosolic Ca2+ homeostasis.
Assuntos
Arabidopsis , Cálcio , Homeostase , Imunidade Vegetal , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citosol/metabolismo , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Antiporters/metabolismoRESUMO
Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Trifosfato de Adenosina , Animais , Cálcio/metabolismo , Canais de Cálcio , Proteínas de Ligação ao Cálcio/genética , Dissulfetos/metabolismo , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Osteoarthritis (OA), primarily characterized by cartilage degeneration, is caused by an imbalance between anabolic and catabolic factors. Here, we investigated the role of zinc (Zn2+) homeostasis, Zn2+ transporters, and Zn(2+)-dependent transcription factors in OA pathogenesis. Among Zn2+ transporters, the Zn2+ importer ZIP8 was specifically upregulated in OA cartilage of humans and mice, resulting in increased levels of intracellular Zn2+ in chondrocytes. ZIP8-mediated Zn2+ influx upregulated the expression of matrix-degrading enzymes (MMP3, MMP9, MMP12, MMP13, and ADAMTS5) in chondrocytes. Ectopic expression of ZIP8 in mouse cartilage tissue caused OA cartilage destruction, whereas Zip8 knockout suppressed surgically induced OA pathogenesis, with concomitant modulation of Zn2+ influx and matrix-degrading enzymes. Furthermore, MTF1 was identified as an essential transcription factor in mediating Zn2+/ZIP8-induced catabolic factor expression, and genetic modulation of Mtf1 in mice altered OA pathogenesis. We propose that the zinc-ZIP8-MTF1 axis is an essential catabolic regulator of OA pathogenesis.
Assuntos
Osteoartrite/metabolismo , Osteoartrite/patologia , Transdução de Sinais , Proteínas ADAM/metabolismo , Idoso , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Regulação para Cima , Zinco/metabolismoRESUMO
Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S. cerevisiae HOL1 mRNA is under translational control by polyamines, and we reveal that the encoded membrane transporter Hol1 is a high-affinity polyamine transporter and is required for yeast growth under limiting polyamine conditions. Moreover, we show that polyamine inhibition of the translation factor eIF5A impairs translation termination at a Pro-Ser-stop motif in a conserved upstream open reading frame on the HOL1 mRNA to repress Hol1 synthesis under conditions of elevated polyamines. Our findings reveal that polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Fases de Leitura Aberta , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Several intracellular pathogens, including Salmonella enterica and Mycobacterium tuberculosis, require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm, whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Infecções por Salmonella/microbiologia , Salmonella typhimurium/citologia , Salmonella typhimurium/patogenicidade , Fatores de Virulência/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C3H , Subunidades Proteicas/antagonistas & inibidores , Salmonella typhimurium/enzimologia , VirulênciaRESUMO
Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sobrevivência Celular , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Apoptose , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/metabolismoRESUMO
Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Mitofagia , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Dinaminas , Metabolismo Energético , GTP Fosfo-Hidrolases/genética , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Mutação , Neurônios/metabolismo , Neurônios/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de Tempo , Zinco/metabolismoRESUMO
Transcription regulation underlies stem cell function and development. Here, we elucidate an unexpected role of an essential ribogenesis factor, WDR43, as a chromatin-associated RNA-binding protein (RBP) and release factor in modulating the polymerase (Pol) II activity for pluripotency regulation. WDR43 binds prominently to promoter-associated noncoding/nascent RNAs, occupies thousands of gene promoters and enhancers, and interacts with the Pol II machinery in embryonic stem cells (ESCs). Nascent transcripts and transcription recruit WDR43 to active promoters, where WDR43 facilitates releases of the elongation factor P-TEFb and paused Pol II. Knockdown of WDR43 causes genome-wide defects in Pol II release and pluripotency-associated gene expression. Importantly, auxin-mediated rapid degradation of WDR43 drastically reduces Pol II activity, precluding indirect consequences. These results reveal an RNA-mediated recruitment and feedforward regulation on transcription and demonstrate an unforeseen role of an RBP in promoting Pol II elongation and coordinating high-level transcription and translation in ESC pluripotency.
Assuntos
Proteínas de Transporte de Cátions/genética , Cromatina/química , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , RNA Polimerase II/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Proteínas de Peixe-Zebra/genética , Animais , Sítios de Ligação , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular , Linhagem Celular , Cromatina/metabolismo , Embrião de Mamíferos , Elementos Facilitadores Genéticos , Deleção de Genes , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas , Proteólise , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismoRESUMO
Intracellular sensors detect changes in levels of essential metals to initiate homeostatic responses. But, a mammalian manganese (Mn) sensor is unknown, representing a major gap in understanding of Mn homeostasis. Using human-relevant models, we recently reported that: 1) the primary homeostatic response to elevated Mn is upregulation of hypoxia-inducible factors (HIFs), which increases expression of the Mn efflux transporter SLC30A10; and 2) elevated Mn blocks the prolyl hydroxylation of HIFs by prolyl hydroxylase domain (PHD) enzymes, which otherwise targets HIFs for degradation. Thus, the mammalian mechanism for sensing elevated Mn likely relates to PHD inhibition. Moreover, 1) Mn substitutes for a catalytic iron (Fe) in PHD structures; and 2) exchangeable cellular levels of Fe and Mn are comparable. Therefore, we hypothesized that elevated Mn directly inhibits PHD by replacing its catalytic Fe. In vitro assays using catalytically active PHD2, the primary PHD isoform, revealed that Mn inhibited, and Fe supplementation rescued, PHD2 activity. However, a mutation in PHD2 (D315E) that selectively reduced Mn binding without substantially impacting Fe binding or enzymatic activity resulted in complete insensitivity of PHD2 to Mn in vitro. Additionally, hepatic cells expressing full-length PHD2D315E were less sensitive to Mn-induced HIF activation and SLC30A10 upregulation than PHD2wild-type. These results: 1) define a fundamental Mn sensing mechanism for controlling Mn homeostasis-elevated Mn inhibits PHD2, which functions as a Mn sensor, by outcompeting its catalytic Fe, and PHD2 inhibition activates HIF signaling to up-regulate SLC30A10; and 2) identify a unique mode of metal sensing that may have wide applicability.
Assuntos
Homeostase , Prolina Dioxigenases do Fator Induzível por Hipóxia , Manganês , Humanos , Manganês/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células HEK293 , Ferro/metabolismoRESUMO
ABSTRACT: Iron-mediated induction of bone morphogenetic protein (BMP)6 expression by liver endothelial cells is essential for iron homeostasis regulation. We used multiple dietary and genetic mouse cohorts to demonstrate a minor functional role for the metal-ion transporter ZIP8 in regulating BMP6 expression under high-iron conditions.
Assuntos
Proteína Morfogenética Óssea 6 , Proteínas de Transporte de Cátions , Ferro , Animais , Proteína Morfogenética Óssea 6/metabolismo , Proteína Morfogenética Óssea 6/genética , Camundongos , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Ferro/metabolismo , Células Endoteliais/metabolismo , Camundongos Knockout , Regulação da Expressão Gênica , Fígado/metabolismo , Camundongos Endogâmicos C57BL , HomeostaseRESUMO
Ferroportin (Fpn) is the only known iron exporter in humans and is essential for maintaining iron homeostasis. Fpn activity is suppressed by hepcidin, an endogenous peptide hormone, which inhibits iron export and promotes endocytosis of Fpn. Hepcidin deficiency leads to hemochromatosis and iron-loading anemia. Previous studies have shown that small peptides that mimic the first few residues of hepcidin, i.e., minihepcidins, are more potent than hepcidin. However, the mechanism of enhanced inhibition by minihepcidins remains unclear. Here, we report the structure of human ferroportin in complex with a minihepcidin, PR73 that mimics the first 9 residues of hepcidin, at 2.7 Å overall resolution. The structure reveals novel interactions that were not present between Fpn and hepcidin. We validate PR73-Fpn interactions through binding and transport assays. These results provide insights into how minihepcidins increase inhibition potency and will guide future development of Fpn inhibitors.
Assuntos
Proteínas de Transporte de Cátions , Hemocromatose , Humanos , Hepcidinas/metabolismo , Hepcidinas/farmacologia , Ferro/metabolismo , Proteínas de Transporte de Cátions/metabolismoRESUMO
BACKGROUND: Calcium (Ca2+) uptake by mitochondria occurs via the mitochondrial Ca2+ uniporter. Mitochondrial Ca2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca2+]m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca2+-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS: MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.
Assuntos
Proteínas de Ligação ao Cálcio , Cálcio , Camundongos Knockout , Mitocôndrias Cardíacas , Proteínas de Transporte da Membrana Mitocondrial , Miócitos Cardíacos , Animais , Feminino , Humanos , Masculino , Camundongos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cardiomegalia/metabolismo , Cardiomegalia/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/genética , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Miócitos Cardíacos/metabolismoRESUMO
The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.
Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Microscopia Crioeletrônica , Hepcidinas/metabolismo , Homeostase , Ferro/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Sítios de Ligação , Proteínas de Transporte de Cátions/ultraestrutura , Cobalto/química , Cobalto/metabolismo , Hepcidinas/química , Humanos , Ferro/química , Simulação de Dinâmica Molecular , Domínios Proteicos , ProteóliseRESUMO
Plant roots forage the soil for iron, the concentration of which can be dramatically lower than those needed for growth. Soil iron uptake uses the broad metal spectrum IRT1 transporter that also transports zinc, manganese, cobalt, and cadmium. Sophisticated iron-dependent transcriptional regulatory mechanisms allow plants to tightly control the abundance of IRT1, ensuring optimal absorption of iron. Here, we uncover that IRT1 acts as a transporter and receptor (transceptor), directly sensing excess of its non-iron metal substrates in the cytoplasm, to regulate its own degradation. Direct metal binding to a histidine-rich stretch in IRT1 triggers its phosphorylation by the CIPK23 kinase and facilitates the subsequent recruitment of the IDF1 E3 ligase. CIPK23-driven phosphorylation and IDF1-mediated lysine-63 polyubiquitination are jointly required for efficient endosomal sorting and vacuolar degradation of IRT1. Thus, IRT1 directly senses elevated non-iron metal concentrations and integrates multiple substrate-dependent regulations to optimize iron uptake and protect plants from highly reactive metals.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Metais/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Endocitose , Regulação da Expressão Gênica de Plantas , Peptídeos e Proteínas de Sinalização Intracelular , Metais/toxicidade , Plantas Geneticamente Modificadas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Transporte Proteico , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ubiquitinação , Vacúolos/metabolismoRESUMO
Proper control of the mitochondrial Ca2+ uniporter's pore (MCU) is required to allow Ca2+-dependent activation of oxidative metabolism and to avoid mitochondrial Ca2+ overload and cell death. The MCU's gatekeeping and cooperative activation is mediated by the Ca2+-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU's DIME motif, the selectivity filter. This led us to recognize in MICU1's sequence a putative DIME interacting domain (DID), which is required for both gatekeeping and cooperative activation of MCU and for cell survival. Thus, we propose that MICU1 has to interact with the D-ring formed by the DIME domains in MCU to control the uniporter.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Compostos de Rutênio/farmacologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genéticaRESUMO
The current state of knowledge on how copper metallochaperones support the maturation of cuproproteins is reviewed. Copper is needed within mitochondria to supply the Cu(A) and intramembrane Cu(B) sites of cytochrome oxidase, within the trans-Golgi network to supply secreted cuproproteins and within the cytosol to supply superoxide dismutase 1 (Sod1). Subpopulations of copper-zinc superoxide dismutase also localize to mitochondria, the secretory system, the nucleus and, in plants, the chloroplast, which also requires copper for plastocyanin. Prokaryotic cuproproteins are found in the cell membrane and in the periplasm of gram-negative bacteria. Cu(I) and Cu(II) form tight complexes with organic molecules and drive redox chemistry, which unrestrained would be destructive. Copper metallochaperones assist copper in reaching vital destinations without inflicting damage or becoming trapped in adventitious binding sites. Copper ions are specifically released from copper metallochaperones upon contact with their cognate cuproproteins and metal transfer is thought to proceed by ligand substitution.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Metalochaperonas/metabolismo , Bactérias/metabolismo , Proteínas de Transporte de Cátions/química , Citosol/metabolismo , Células Eucarióticas/metabolismo , Metalochaperonas/química , Mitocôndrias/metabolismo , Proteoma/análiseRESUMO
Shank3 is a synaptic scaffolding protein that assists in tethering and organizing structural proteins and glutamatergic receptors in the postsynaptic density of excitatory synapses. The localization of Shank3 at excitatory synapses and the formation of stable Shank3 complexes is regulated by the binding of zinc to the C-terminal sterile-alpha-motif (SAM) domain of Shank3. Mutations in the SAM domain of Shank3 result in altered synaptic function and morphology, and disruption of zinc in synapses that express Shank3 leads to a reduction of postsynaptic proteins important for synaptic structure and function. This suggests that zinc supports the localization of postsynaptic proteins via Shank3. Many regions of the brain are highly enriched with free zinc inside glutamatergic vesicles at presynaptic terminals. At these synapses, zinc transporter 3 (ZnT3) moves zinc into vesicles where it is co-released with glutamate. Alterations in ZnT3 are implicated in multiple neurodevelopmental disorders, and ZnT3 knock-out (KO) mice-which lack synaptic zinc-show behavioral deficits associated with autism spectrum disorder and schizophrenia. Here we show that male and female ZnT3 KO mice have smaller dendritic spines and miniature excitatory postsynaptic current amplitudes than wildtype (WT) mice in the auditory cortex. Additionally, spine size deficits in ZnT3 KO mice are restricted to synapses that express Shank3. In WT mice, synapses that express both Shank3 and ZnT3 have larger spines compared to synapses that express Shank3 but not ZnT3. Together these findings suggest a mechanism whereby presynaptic ZnT3-dependent zinc supports postsynaptic structure and function via Shank3 in a synapse-specific manner.
Assuntos
Córtex Auditivo , Proteínas de Transporte de Cátions , Espinhas Dendríticas , Proteínas do Tecido Nervoso , Sinapses , Animais , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Sinapses/metabolismo , Espinhas Dendríticas/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Córtex Auditivo/metabolismo , Feminino , Masculino , Camundongos Knockout , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Potenciais Pós-Sinápticos Excitadores/fisiologiaRESUMO
The stepwise addition of monosaccharides to N-glycans attached to client proteins to generate a repertoire of mature proteins involves a concerted action of many glycosidases and glycosyltransferases. Here, we report that Golgi α-mannosidase II (GMII), a pivotal enzyme catalyzing the first step in the conversion of hybrid- to complex-type N-glycans, is activated by Zn2+ supplied by the early secretory compartment-resident ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in marked accumulation of hybrid-type and complex/hybrid glycans with concomitant reduction of complex- and high-mannose-type glycans. In cells lacking the ZNT5-6 and ZNT7 functions, the GMII activity is substantially decreased. In contrast, the activity of its homolog, lysosomal mannosidase (LAMAN), is not decreased. Moreover, we show that the growth of pancreatic cancer MIA PaCa-2 cells lacking ZNT5-6 and ZNT7 is significantly decreased in a nude mouse xenograft model. Our results indicate the integral roles of ZNT5-6 and ZNT7 in N-glycosylation and highlight their potential as novel target proteins for cancer therapy.