scholarly journals Analysis of the Isomerase and Chaperone-Like Activities of an Amebic PDI (EhPDI)

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Rosa E. Mares ◽  
Alexis Z. Minchaca ◽  
Salvador Villagrana ◽  
Samuel G. Meléndez-López ◽  
Marco A. Ramos
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Molecular Mechanisms ◽  
Thermal Inactivation ◽  
Initial Step ◽  
E Coli ◽  
Isomerase Activity ◽  
Disulfide Isomerases ◽  
Protein Disulfide

Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. TheEhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation inE. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant ofE. coli, to test whetherEhPDI exhibits isomerase activityin vivo. Our preliminary results showed thatEhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed thatEhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization ofEhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding inE. histolytica.

scholarly journals Protein Disulfide Isomerase Superfamily in Disease and the Regulation of Apoptosis

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
C. Grek ◽  
D.M. Townsend
Keyword(s):  
Protein Folding ◽  
Er Stress ◽  
Cell Fate ◽  
Molecular Chaperones ◽  
Molecular Mechanisms ◽  
Disulfide Isomerases ◽  
Apoptotic Pathways ◽  
Er Homeostasis ◽  
Protein Disulfide

AbstractCellular homeostasis requires the balance of a multitude of signaling cascades that are contingent upon the essential proteins being properly synthesized, folded and delivered to appropriate subcellular locations. In eukaryotic cells the endoplasmic reticulum (ER) is a specialized organelle that is the central site of synthesis and folding of secretory, membrane and a number of organelletargeted proteins. The integrity of protein folding is enabled by the presence of ATP, Ca++, molecular chaperones, as well as an oxidizing redox environment. The imbalance between the load and capacity of protein folding results in a cellular condition known as ER stress. Failure of these pathways to restore ER homeostasis results in the activation of apoptotic pathways. Protein disulfide isomerases (PDI) compose a superfamily of oxidoreductases that have diverse sequences and are localized in the ER, nucleus, cytosol, mitochondria and cell membrane. The PDI superfamily has multiple functions including, acting as molecular chaperones, protein-binding partners, and hormone reservoirs. Recently , PDI family members have been implicated in the regulation of apoptotic signaling events. The complexities underlying the molecular mechanisms that define the switch from pro-survival to pro-death response are evidenced by recent studies that reveal the roles of specific chaperone proteins as integration points in signaling pathways that determine cell fate. The following review discusses the dual role of PDI in cell death and survival during ER stress.

β2-integrin activity: the role of thiols

Blood ◽  
2013 ◽  
Vol 121 (19) ◽  
pp. 3779-3780 ◽  
Author(s):  
Alexander Zarbock
Keyword(s):  
Disulfide Bonds ◽  
Protein Disulfide Isomerase ◽  
Neutrophil Recruitment ◽  
Regulatory Effect ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Β2 Integrin ◽  
Protein Disulfide

In this issue of Blood, Hahm and colleagues identify the extracellular protein disulfide isomerase (PDI) as an essential regulator of the adhesiveness of the β2-integrin macrophage-1 antigen (Mac-1) on neutrophils.1 In the absence of PDI, Mac-1–dependent neutrophil adhesion and crawling is reduced in vivo. Rescue experiments with exogenous PDI showed that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. This intriguing finding suggests that disulfide bonds in Mac-1 regulate integrin activity and neutrophil recruitment.

scholarly journals Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4891-4896 ◽  
Author(s):  
Ji Qiu ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Disulfide Bonds ◽  
Specific Activity ◽  
Active Form ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
E Coli ◽  
Disulfide Isomerases

ABSTRACT The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins inE. coli without the need for in vitro refolding.

scholarly journals Protein disulfide isomerase activity is released by activated platelets

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2226-2228 ◽  
Author(s):  
K Chen ◽  
Y Lin ◽  
TC Detwiler
Keyword(s):  
Disulfide Bonds ◽  
Protein Disulfide Isomerase ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Activated Platelets ◽  
Protein Disulfide ◽  
Supernatant Solution ◽  
Disulfide Isomerase Activity

Abstract The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin- thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.

scholarly journals Full Capacity of Recombinant Escherichia coliHeat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

2000 ◽  
Vol 68 (7) ◽  
pp. 4064-4074 ◽  
Author(s):  
Isabelle Batisson ◽  
Maurice Der Vartanian ◽  
Brigitte Gaillard-Martinie ◽  
Michel Contrepois
Keyword(s):  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Biological Properties ◽  
Leader Peptide ◽  
Dependent Manner ◽  
Reporter Protein ◽  
E Coli ◽  
Heat Stable

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

scholarly journals Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

2006 ◽  
Vol 282 (7) ◽  
pp. 4437-4446 ◽  
Author(s):  
Markus Liebscher ◽  
Günther Jahreis ◽  
Christian Lücke ◽  
Susanne Grabley ◽  
Satish Raina ◽  
...  
Keyword(s):  
Protein Folding ◽  
Minimal Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Secondary Amide ◽  
Fatty Acyl Chain ◽  
Isomerase Activity

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. Nα-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 ± 20 μg/ml. The compounds were shown to compete with fluorophore-labeled σ32-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.

scholarly journals The protein disulfide isomerase AGR2 is essential for production of intestinal mucus

2009 ◽  
Vol 106 (17) ◽  
pp. 6950-6955 ◽  
Author(s):  
Sung-Woo Park ◽  
Guohua Zhen ◽  
Catherine Verhaeghe ◽  
Yasuhiro Nakagami ◽  
Louis T. Nguyenvu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Disulfide Bonds ◽  
Critical Role ◽  
Cysteine Residue ◽  
Direct Role ◽  
Mucus Production ◽  
Intestinal Mucus ◽  
Mucus Gel ◽  
Protein Disulfide

Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production.

Protective effects of Cathelicidin against glomerulonephritis through promotion of host defense

2014 ◽  
Vol 2 (3) ◽  
pp. 189-198
Author(s):  
Ajay H. Bahl ◽  
Wanda Lee
Keyword(s):  
Host Defense ◽  
Disulfide Bonds ◽  
Helical Conformation ◽  
Antimicrobial Protein ◽  
Protective Effects ◽  
In Vivo Models ◽  
E Coli ◽  
Risk Of Death

Cathelicidin-related antimicrobial peptides are a family of polypeptides found in lysosomes of macrophages and polymorphonuclear leukocytes (PMNs). Some of these peptides can assume an alpha-helical conformation, others contain one or two disulfide bonds, still others are Pro- and Arg-rich, or Trp-rich. Higher levels of human cathelicidin antimicrobial protein (hCAP18), which are up-regulated by vitamin D, appear to significantly reduce the risk of death from infection in dialysis patients. Using in vitro and in vivo models of kidney infection, we demonstrate key antimicrobial and host immunomodulatory properties of cathelicidins. To directly assess the role of endogenous cathelicidin in the development of glomerulonephritis, WT and mCRAMP KO mice were provided with 5% DSS to induce glomerulonephritis. Some mice groups were administered with E. coli DNA I.P. Our findings showed that mCRAMP KO mice develop more severe glomerulonephritis. These data demonstrate key roles for cathelicidins in host defense against glomerulonephritis and the potential to inform the development of synthetic analogues to modulate specific host-pathogen interactions as novel antimicrobial therapeutics.

scholarly journals Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

2014 ◽  
Vol 82 (5) ◽  
pp. 1801-1812 ◽  
Author(s):  
Sylvia Kleta ◽  
Marcel Nordhoff ◽  
Karsten Tedin ◽  
Lothar H. Wieler ◽  
Rafal Kolenda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Single Infection ◽  
Content Type ◽  
E Coli ◽  
Initial Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probioticE. colistrain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While bothin vitroandin vivostudies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenicE. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenicE. coli(aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

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