RESUMO
Objective- PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results- Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47phox and PDI and an intramolecular bond between Cys 196 and 378 in p47phox. PDI catalytic Cys 400 and p47phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47phox, whereas PDI mutant did not promote this effect. Mutation of p47phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47phox occurred only in PDI family members with the a' redox active site. Conclusions- PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.
Assuntos
Dissulfetos/química , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidase 1/metabolismo , NADPH Oxidases/química , Isomerases de Dissulfetos de Proteínas/química , Animais , Movimento Celular , Células Cultivadas , Ativação Enzimática , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Oxirredução , Superóxidos/metabolismoRESUMO
BACKGROUND AND AIM: Oxidative stress arising from inflammatory processes is a serious cause of cell and tissue damage. Tempol is an efficient antioxidant with superoxide dismutase-like activity. The purpose of this paper is to address the inhibition of protein disulfide isomerase (PDI), an essential redox chaperone whose active sites contain the Cys-Gly-His-Cys (CXXC) motif, by the nitroxide Tempol. RESULTS: In the presence of Tempol (5-120 µM), the reductase activity of PDI was reversibly affected both in vitro and in activated mice neutrophils, with an IC50 of 22.9 ± 10.8 µM. Inhibitory activity was confirmed by using both the insulin method and fluorescent formation of eosin-glutathione (E-GSH). The capacity of Tempol to bind the enzyme was determined by EPR and mass spectrometry. EPR Tempol signal decreased in the presence of PDI while remained unaffected when PDI thiols were previously blocked with NEM. When total protein was analyzed, 1 and 4 molecules of Tempol were bound to the protein. However, only one was found to be covalently bound to PDI at the a'active site. More specifically, Cys400 was modified by Tempol. CONCLUSION: We have shown that the nitroxide Tempol acts as an inhibitor of PDI through covalent binding to the Cys400 of the protein structure. Since PDI is coupled with the assembly of the NADPH oxidase complex of phagocytes, these findings reveal a novel action of Tempol that presents potential clinical applications for therapeutic intervention to target PDI knockdown in pathological processes in which this protein is engaged.
Assuntos
Óxidos N-Cíclicos/metabolismo , Cisteína/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Óxidos N-Cíclicos/química , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Dissulfeto de Glutationa/química , Dissulfeto de Glutationa/metabolismo , Masculino , Camundongos , Neutrófilos/enzimologia , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Marcadores de Spin , Espectrometria de Massas em TandemRESUMO
Essentials Inhibitors of protein disulfide isomerase (PDI) have been considered a new antithrombotic class. CxxC is a PDI-targeted peptide that has been previously shown to inhibit its reductase activity. CxxC binds to surface PDI and inhibits ADP- and thrombin-evoked platelet activation and aggregation. CxxC binds to Cys400 on CGHC redox motif of PDI a' domain, a site for PDI prothrombotic activity. SUMMARY: Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.
Assuntos
Inibidores da Agregação Plaquetária/química , Pró-Colágeno-Prolina Dioxigenase/química , Isomerases de Dissulfetos de Proteínas/química , Alanina/química , Motivos de Aminoácidos , Plaquetas/metabolismo , Domínio Catalítico , Cisteína/química , Dissulfetos , Humanos , Oxirredução , Peptídeos/química , Ativação Plaquetária , Agregação Plaquetária , Ligação Proteica , Domínios Proteicos , Dobramento de ProteínaRESUMO
The Pseudomonas aeruginosa plasmid pUM505 contains in a pathogenicity island the dsbA2 gene, which encodes a product with similarity to DsbA protein disulfide isomerases, enzymes that catalyze formation and isomerization of disulfide bonds in protein cysteine residues. Using transcriptional fusions, it was found that dsbA2 gene promoter is activated during the stationary phase, suggesting that DsbA2 protein may be required for adaptive changes that occur during this stage of bacterial growth. Transfer of the pUM505 dsbA2 gene to a cadmium-sensitive P. aeruginosa PAO1-derivative affected in the chromosomal dsbA gene, restored cadmium resistance, suggesting a role of DsbA2 in protecting protein disulfide bonds. PAO1 dsbA2 transformants displayed increased sensitivity to intercalating agent mitomycin C, indicating that DsbA2 functions as a thioredoxin enzyme able to modify and activate toxicity of this compound. These results highlight the adaptive role of the pUM505 plasmid in its P. aeruginosa hosts.
Assuntos
Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , Isomerases de Dissulfetos de Proteínas/genética , Tiorredoxinas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cádmio/farmacologia , Cádmio/toxicidade , Clonagem Molecular , Farmacorresistência Bacteriana , Ordem dos Genes , Mitomicina/farmacologia , Isomerases de Dissulfetos de Proteínas/química , Pseudomonas aeruginosa/genética , Tiorredoxinas/químicaRESUMO
Toxoplasma protein disulfide isomerase (PDI) is a 52 KDa thioredoxin of interest because have a great immunogenicity for humans. We cloned and produced a recombinant protein (recTgPDI) used to test its effect during infection to different human cell lines (epithelial and retinal). We also determine if there were differences in gen expression during in vitro infection. Expression of the gen was lower after entry into the host cells. PDI's inhibitors bacitracin and nitroblue tetrazolium reduced the percent of infected cells and small amounts of recTgPDI proteins interfered with the invasion step. All these results support a role of Toxoplasma PDI during the first steps of infection (adhesion and invasion). Toxoplasma PDI is a protein linked to early steps of invasion, it would be of importance to identify the host proteins substrates during invasion steps.
Assuntos
Isomerases de Dissulfetos de Proteínas/metabolismo , Tiorredoxinas/metabolismo , Toxoplasma/enzimologia , Toxoplasma/fisiologia , Linhagem Celular , Clonagem Molecular , Células Ependimogliais/parasitologia , Fibroblastos/parasitologia , Regulação Enzimológica da Expressão Gênica , Células HeLa/parasitologia , Humanos , Modelos Estruturais , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Toxoplasma/genéticaRESUMO
Disturbance of endoplasmic reticulum (ER) proteostasis is observed in Prion-related disorders (PrDs). The protein disulfide isomerase ERp57 is a stress-responsive ER chaperone up-regulated in the brain of Creutzfeldt-Jakob disease patients. However, the actual role of ERp57 in prion protein (PrP) biogenesis and the ER stress response remained poorly defined. We have recently addressed this question using gain- and loss-of-function approaches in vitro and animal models, observing that ERp57 regulates steady-state levels of PrP. Our results revealed that ERp57 modulates the biosynthesis and maturation of PrP but, surprisingly, does not contribute to the global cellular reaction against ER stress in neurons. Here we discuss the relevance of ERp57 as a possible therapeutic target in PrDs and other protein misfolding disorders.
Assuntos
Doenças Priônicas/metabolismo , Proteínas Priônicas/biossíntese , Proteínas Priônicas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Modelos Biológicos , Proteínas Priônicas/análise , Proteínas Priônicas/química , Isomerases de Dissulfetos de Proteínas/química , Dobramento de ProteínaRESUMO
Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution.
Assuntos
Proteínas de Bactérias/química , Isomerases de Dissulfetos de Proteínas/química , Multimerização Proteica , Xylella/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cobre/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oxirredução , Doenças das Plantas/microbiologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios X , Xylella/genética , Xylella/fisiologiaRESUMO
Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.
Assuntos
Entamoeba histolytica/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Antibacterianos/farmacologia , Bacitracina/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Insulina/metabolismo , Chaperonas Moleculares/metabolismo , Muramidase/química , Muramidase/metabolismo , Oxirredutases/metabolismo , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Dobramento de Proteína , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismoRESUMO
Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.
Assuntos
Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Xylella/enzimologia , Substituição de Aminoácidos , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Histidina/química , Família Multigênica , Oxirredução , Isomerases de Dissulfetos de Proteínas/genética , Especificidade por Substrato , Xylella/genéticaRESUMO
Vascular cell NADPH oxidase complexes are key sources of signaling reactive oxygen species (ROS) and contribute to disease pathophysiology. However, mechanisms that fine-tune oxidase-mediated ROS generation are incompletely understood. Besides known regulatory subunits, upstream mediators and scaffold platforms reportedly control and localize ROS generation. Some evidence suggest that thiol redox processes may coordinate oxidase regulation. We hypothesized that thiol oxidoreductases are involved in this process. We focused on protein disulfide isomerase (PDI), a ubiquitous dithiol disulfide oxidoreductase chaperone from the endoplasmic reticulum, given PDI's unique versatile role as oxidase/isomerase. PDI is also involved in protein traffic and can translocate to the cell surface, where it participates in cell adhesion and nitric oxide internalization. We recently provided evidence that PDI exerts functionally relevant regulation of NADPH oxidase activity in vascular smooth muscle and endothelial cells, in a thiol redox-dependent manner. Loss-of-function experiments indicate that PDI supports angiotensin II-mediated ROS generation and Akt phosphorylation. In addition, PDI displays confocal co-localization and co-immunoprecipitates with oxidase subunits, indicating close association. The mechanisms of such interaction are yet obscure, but may involve subunit assembling stabilization, assistance with traffic, and subunit disposal. These data may clarify an integrative view of oxidase activation in disease conditions, including stress responses.