RESUMO
AIMS: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. RESULTS: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1K72A and catalytic site mutant Trx-1C32/35S rescued ADAM17 activity, although the interaction with Trx-1C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1K72A mutant showed similar oxidant levels to Trx-1C32/35S, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. INNOVATION: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. CONCLUSION: This unexpected Trx-1K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.
Assuntos
Proteína ADAM17/metabolismo , Tiorredoxinas/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Modelos Moleculares , Oxirredução , Tiorredoxinas/análise , Tiorredoxinas/genética , Células Tumorais CultivadasRESUMO
Leishmania parasites have evolved a number of strategies to cope with the harsh environmental changes during mammalian infection. One of these mechanisms involves the functional gain that allows mitochondrial 2-Cys peroxiredoxins to act as molecular chaperones when forming decamers. This function is critical for parasite infectivity in mammals, and its activation has been considered to be controlled exclusively by the enzyme redox state under physiological conditions. Herein, we have revealed that magnesium and calcium ions play a major role in modulating the ability of these enzymes to act as molecular chaperones, surpassing the redox effect. These ions are directly involved in mitochondrial metabolism and participate in a novel mechanism to stabilize the decameric form of 2-Cys peroxiredoxins in Leishmania mitochondria. Moreover, we have demonstrated that a constitutively dimeric Prx1m mutant impairs the survival of Leishmania under heat stress, supporting the central role of the chaperone function of Prx1m for Leishmania parasites during the transition from insect to mammalian hosts.
Assuntos
Cálcio/metabolismo , Leishmania/metabolismo , Magnésio/metabolismo , Proteínas Mitocondriais/metabolismo , Peroxirredoxinas/metabolismo , Proteínas de Protozoários/metabolismo , Anisotropia , Cromatografia , Dissulfetos/química , Fluorometria , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Luz , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/química , Multimerização Proteica , Espalhamento de Radiação , TemperaturaRESUMO
Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.
Assuntos
Venenos de Crotalídeos/enzimologia , Calicreínas/metabolismo , Metaloproteases/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Bothrops , Hidrólise , Cinética , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Prolil Oligopeptidases , Radioimunoensaio , Especificidade por SubstratoRESUMO
Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.
Assuntos
Miosina Tipo V/química , Sítios de Ligação , Transporte Biológico Ativo/fisiologia , Humanos , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Peroxissomos/química , Peroxissomos/genética , Peroxissomos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
The flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/ß)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein flexibility. Moreover, the binding mode of ß-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.
Assuntos
NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Trypanosoma cruzi/enzimologia , Cristalografia por Raios X , Modelos Moleculares , NADPH Desidrogenase/genética , Conformação Proteica , Especificidade por Substrato , TermodinâmicaRESUMO
The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Xanthomonas , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Permeabilidade da Membrana Celular , Dissulfetos/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Molibdênio/metabolismo , Mutação , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , TemperaturaRESUMO
ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.
Assuntos
Proteínas ADAM/metabolismo , Tiorredoxinas/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Reagentes de Ligações Cruzadas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Células HeLa , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Acetato de Tetradecanoilforbol/farmacologia , Tiorredoxinas/químicaRESUMO
Pathogenic bacteria constitute an important cause of hospital-acquired infections. However, the misuse of available bactericidal agents has led to the appearance of antibiotic-resistant strains. Thus, efforts to seek new antimicrobials with different action mechanisms would have an enormous impact. Here, a novel antimicrobial protein (SiAMP2) belonging to the 2S albumin family was isolated from Sesamum indicum kernels and evaluated against several bacteria and fungi. Furthermore, in silico analysis was conducted in order to identify conserved residues through other 2S albumin antimicrobial proteins (2S-AMPs). SiAMP2 specifically inhibited Klebsiella sp. Specific regions in the molecule surface where cationic (RR/RRRK) and hydrophobic (MEYWPR) residues are exposed and conserved were proposed as being involved in antimicrobial activity. This study reinforces the hypothesis that plant storage proteins might also play as pathogen protection providing an insight into the mechanism of action for this novel 2S-AMP and evolutionary relations between antimicrobial activity and 2S albumins.