RESUMO
Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.
Assuntos
Anticorpos Monoclonais , Membrana Celular , Inflamação , Fígado , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Animais , Camundongos , Alanina Transaminase , Alarminas , Anticorpos Monoclonais/imunologia , Aspartato Aminotransferases , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/imunologia , Moléculas de Adesão Celular Neuronais/ultraestrutura , Morte Celular , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Concanavalina A , Galactosamina , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Inflamação/patologia , Lactato Desidrogenases , Fígado/patologia , Microscopia Eletrônica , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/ultraestrutura , Infiltração de Neutrófilos , Traumatismo por Reperfusão/patologiaRESUMO
Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB-TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB-TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H(+) antiport.
Assuntos
Antiporters/metabolismo , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/toxicidade , Tolerância a Medicamentos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismoRESUMO
SUMO proteases can regulate the amounts of SUMO-conjugated proteins in the cell by cleaving off the isopeptidic bond between SUMO and the target protein. Of the six members that constitute the human SENP/ULP protease family, SENP6 and SENP7 are the most divergent members in their conserved catalytic domain. The SENP6 and SENP7 subclass displays a clear proteolytic cleavage preference for SUMO2/3 isoforms. To investigate the structural determinants for such isoform specificity, we have identified a unique sequence insertion in the SENP6 and SENP7 subclass that is essential for their proteolytic activity and that forms a more extensive interface with SUMO during the proteolytic reaction. Furthermore, we have identified a region in the SUMO surface determinant for the SUMO2/3 isoform specificity of SENP6 and SENP7. Double point amino acid mutagenesis on the SUMO surface allows us to swap the specificity of SENP6 and SENP7 between the two SUMO isoforms. Structure-based comparisons combined with biochemical and mutagenesis analysis have revealed Loop 1 insertion in SENP6 and SENP7 as a platform to discriminate between SUMO1 and SUMO2/3 isoforms in this subclass of the SUMO protease family.
Assuntos
Cisteína Endopeptidases/química , Endopeptidases/química , Proteína SUMO-1/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Ubiquitinas/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Humanos , Mutagênese , Isoformas de Proteínas , Estrutura Secundária de Proteína , Proteólise , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologia , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
The ß1-adrenergic receptor (ß1-AR) can activate two families of G proteins. When coupled to Gs, ß1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey ß1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with ß1-AR, the overall interacting modes between ß1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by ß1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Estrutura Terciária de Proteína/fisiologia , Receptores Adrenérgicos beta 1/metabolismo , Animais , Débito Cardíaco/genética , Débito Cardíaco/fisiologia , Linhagem Celular , Microscopia Crioeletrônica , AMP Cíclico/metabolismo , Ativação Enzimática/fisiologia , Células HEK293 , Cardiopatias/patologia , Humanos , Hipertensão/patologia , Isoproterenol/química , Estrutura Secundária de Proteína/fisiologia , Células Sf9 , Transdução de Sinais/fisiologiaRESUMO
Multidrug resistance is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. In the following review, we present a synthesis of current understanding of the Escherichia coli multidrug resistance transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily (MFS).
Assuntos
Antiporters/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antiporters/química , Antiporters/genética , Transporte Biológico , Farmacorresistência Bacteriana Múltipla , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Família MultigênicaRESUMO
Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins.
Assuntos
Antiporters/metabolismo , Farmacorresistência Bacteriana Múltipla , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Aminoácidos/química , Antibacterianos/química , Antiporters/química , Antiporters/genética , Transporte Biológico , Cloranfenicol/química , Simulação por Computador , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Oniocompostos/química , Compostos Organofosforados/química , Ligação Proteica , Conformação ProteicaRESUMO
Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Animais , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Multimerização ProteicaRESUMO
Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters.
RESUMO
The SENP proteases regulate the SUMO conjugates in the cell by cleaving SUMO from target proteins. SENP6 and SENP7 are the most divergent members of the SENP/ULP protease family in humans by the presence of insertions in their catalytic domains. Loop1 insertion is determinant for the SUMO2/3 activity and specificity on SENP6 and SENP7. To gain structural insights into the role of Loop1, we have designed a chimeric SENP2 with the insertion of Loop1 into its sequence. The structure of SENP2-Loop1 in complex with SUMO2 was solved at 2.15 Å resolution, and reveals the details of an interface exclusive to SENP6/7 and the formation of unique contacts between both proteins. Interestingly, functional data with SUMO substrates showed an increase of the proteolytic activity in the SENP2-Loop1 chimera for diSUMO2 and polySUMO2 substrates.