The Chaperone Activity of Protein Disulfide Isomerase Is Affected Cyclophilin B and Cyclosporin A In Vitro

2002 ◽  
Vol 132 (3) ◽  
pp. 401-407 ◽  
Author(s):  
T. Horibe ◽  
C. Yosho ◽  
S. Okada ◽  
M. Tsukamoto ◽  
H. Nagai ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Protein Disulfide Isomerase ◽  
Chaperone Activity ◽  
Cyclophilin B ◽  
Disulfide Isomerase ◽  
Protein Disulfide

scholarly journals Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Audrey Beaussart ◽  
Tristan Brandhorst ◽  
Yves F. Dufrêne ◽  
Bruce S. Klein
Keyword(s):  
Protein Disulfide Isomerase ◽  
Tandem Repeats ◽  
Pathogenic Fungi ◽  
Heparin Binding ◽  
Binding Studies ◽  
Disulfide Linkage ◽  
Disulfide Isomerase ◽  
Atomic Force ◽  
Protein Disulfide

ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. IMPORTANCE Pathogenic fungi and other microbes must adhere to host tissue to initiate infection. Surface adhesins promote this event and may be required for disease pathogenesis. We studied a fungal adhesin essential for virulence (BAD-1; Blastomyces adhesin-1) and found that host products induce its structural reconfiguration and foster its optimal binding to tissue structures.

Protein Disulfide Isomerase Regulates Sickle Erythrocyte Volume Via ET-1 Dependent Casein Kinase II Mechanism

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2114-2114
Author(s):  
Shirley D Valentin-Berrios ◽  
Jose R Romero ◽  
Alicia Rivera
Keyword(s):  
Plasma Membrane ◽  
Sickle Cell ◽  
Protein Disulfide Isomerase ◽  
Disulfide Isomerase ◽  
Sickle Erythrocyte ◽  
Cellular Dehydration ◽  
Sickle Erythrocytes ◽  
Protein Disulfide

Abstract Abstract 2114 Disordered K+ efflux and osmotically induced water loss leads to red blood cell (RBC) dehydration and plays a role in the pathophysiology of Sickle Cell Disease. We previously reported that activation of endothelin-1 (ET-1) receptors in sickle erythrocyte was partially responsible for dense sickle cell formation. However, the mechanism by which ET-1 regulates RBC volume remains unclear. Serine/threonine kinases have been shown to regulate K+ transport in RBC. Casein Kinase II (CK2), a serine/threonine kinase, phosphorylates acidic proteins, regulates calmodulin activity and cytoskeletal proteins and is present in RBC. CK2 activity is blocked by apigenin, emodin, heparin, and ornithine decarboxylase. Previous reports have shown a role for flavonoids such as apigenin as substrates for erythrocyte plasma membrane oxidoreductases. We recently observed a role for Protein Disulfide Isomerase (PDI) in regulating cellular hydration and K+ efflux in human RBC. PDI catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. However the relationship between CK2 and PDI in the setting of cellular hydration status is un-explored. Our results indicate that erythrocyte membrane CK2 activity increases when sickle cells are incubated with 500 nM ET-1 for 30 min (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell, n=3, p<0.04) and 20 μM apigenin (2.7 ± 0.4 nmol/min/mL * 106 cell, n=3, p<0.04). We examined the role of CK2 activation on cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), a specific CK2 inhibitor, and measured the changes in erythrocyte density by phthalate oil density analysis. We observed that inhibition of CK2 led to reduced deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). We then studied the role of CK2 inhibitors on PDI activity by Insulin Turbidity Assay and observed that apigenin and TBB led to significant reductions in PDI activity in vitro (64% and 42% respectively). We also studied the effects of the flavonoids: naringenin, naringin, apigenin and rutin on PDI activity and observed reductions in PDI activity that were greater with apigenin>rutin>TBB>naringin>naringenin (n=2, P<0.05). Furthermore, we observed that K+ flux via Gardos channel activation is correlated with PDI activity in vitro in sickle erythrocytes. Taken together our results implicate CK2 and PDI as intermediate regulators of ET-1 stimulated cellular volume systems in red blood cells. Supported by NIH R01-HL09632 to AR. Disclosures: No relevant conflicts of interest to declare.

Functional analysis of tunicamycin-inducible gene A polypeptide from Aspergillus niger

2005 ◽  
Vol 83 (5) ◽  
pp. 654-658 ◽  
Author(s):  
Yurong Liang ◽  
Wei Li ◽  
Qing Ma ◽  
Yuying Zhang
Keyword(s):  
Functional Analysis ◽  
Aspergillus Niger ◽  
Substrate Specificity ◽  
Protein Disulfide Isomerase ◽  
Ribonuclease A ◽  
Chaperone Activity ◽  
Disulfide Isomerase ◽  
Chaperone Protein ◽  
Protein Disulfide ◽  
Inducible Gene

Tunicamycin-inducible gene A polypeptide (TIGA) is a member of the protein disulfide isomerase (PDI) family and is suggested to facilitate the folding of nascent polypeptides. The functional properties of TIGA were investigated here. TIGA acted as an isomerase, catalyzing the refolding of denatured and reduced ribonuclease A. TIGA also exhibited chaperone activity in the refolding of denatured prochymosin but not in the refolding of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), indicating that it had substrate specificity with respect to chaperone activity. Detailed study with a series of thioredoxin-motif (trx-motif) mutants revealed that the 2 trx-motifs of TIGA were not equal in activity. The N-terminal trx-motif was more active than the C-terminal trx-motif, and the first cysteine in each trx-motif was necessary for isomerase activity.Key words: tunicamycin-inducible gene A polypeptide (TIGA), protein disulfide isomerase, chaperone, protein refolding.

scholarly journals Murine Factor H Co-Produced in Yeast With Protein Disulfide Isomerase Ameliorated C3 Dysregulation in Factor H-Deficient Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Heather Kerr ◽  
Andrew P. Herbert ◽  
Elisavet Makou ◽  
Dariusz Abramczyk ◽  
Talat H. Malik ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Mouse Serum ◽  
Dna Encoding ◽  
Disulfide Isomerase ◽  
Age Related ◽  
Modified Dna ◽  
Protein Disulfide

Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen.

Abstract 17269: Extracelllular Protein Disulfide Isomerase Reshapes Vascular Architecture Against Constrictive Remodeling

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Haniel A Araujo ◽  
Leonardo Y Tanaka ◽  
Gustavo K Hironaka ◽  
Thais L Araujo ◽  
Celso K Takimura ◽  
...  
Keyword(s):  
Vascular Remodeling ◽  
Protein Disulfide Isomerase ◽  
Fiber Type ◽  
Vascular Diseases ◽  
Collagen Type I ◽  
Type I ◽  
Disulfide Isomerase ◽  
Protein Disulfide

INTRODUCTION: Vascular remodeling orchestrates a complex network of signaling pathways responsible for pathological changes in many vascular diseases such as atherosclerosis. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase (PDI) and the extracellular PDI (ecPDI) pool in vascular caliber and architecture during vascular repair and remodeling after injury (AI). METHODS AND RESULTS: After rabbit iliac artery balloon injury, PDI is markedly increased at mRNA and protein levels (25-fold vs. basal 14 days AI), with increase in both intracellular and ecPDI. Silencing PDI by siRNA in vitro induced ER stress markers upregulation and apoptosis (assessed by TUNEL assay). PDI knockdown also upregulated proliferation marker PCNA and decreased differentiation marker calponin-C. Furthermore, ecPDI inhibition prevents injury-increased hydrogen peroxide generation and decreases arterial nitrate (NO3-) level. EcPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted 25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. EcPDI neutralization promoted striking changes in collagen, with switch from circumferential to radial fiber orientation towards a more rigid fiber type. Collagen type I and III were decreased after ecPDI inhibition in arteries 14 days AI. Cytoskeleton architecture was also disrupted, with loss of stress fiber coherent organization and switch from thin to medium-thickness actin fibers. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. There was decreased PDI expression in media and intima from plaques exhibiting constrictive remodeling and, conversely, enhanced PDI expression in media of plaques depicting outward remodeling. CONCLUSIONS: Thus, PDI is highly upregulated after injury and reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect. Such findings suggest an important role for PDI in lumen maintenance during vascular remodeling.

scholarly journals Htm1p–Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

2016 ◽  
Vol 113 (28) ◽  
pp. E4015-E4024 ◽  
Author(s):  
Yi-Chang Liu ◽  
Danica Galonić Fujimori ◽  
Jonathan S. Weissman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Protein Disulfide Isomerase ◽  
Misfolded Proteins ◽  
Disulfide Isomerase ◽  
Licensing Factor ◽  
Folded Proteins ◽  
Protein Disulfide

Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p–Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae. We reconstitute this step in vitro with Htm1p–Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p–Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p–Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

scholarly journals Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins

2018 ◽  
Vol 19 (11) ◽  
pp. 3418 ◽  
Author(s):  
Henrik O’Brien ◽  
Shingo Kanemura ◽  
Masaki Okumura ◽  
Robert Baskin ◽  
Pradip Bandyopadhyay ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Venom Gland ◽  
Cone Snail ◽  
Disulfide Isomerase ◽  
Folding Rates ◽  
Cone Snails ◽  
And Function ◽  
Protein Disulfide ◽  
Insight Into

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.

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