scholarly journals Protein disulfide-isomerase interacts with soluble guanylyl cyclase via a redox-based mechanism and modulates its activity

2013 ◽  
Vol 452 (1) ◽  
pp. 161-169 ◽  
Author(s):  
Erin J. Heckler ◽  
Pierre-Antoine Crassous ◽  
Padmamalini Baskaran ◽  
Annie Beuve
Keyword(s):  
Guanylyl Cyclase ◽  
Protein Disulfide Isomerase ◽  
Cellular Localization ◽  
Redox Regulation ◽  
Soluble Guanylyl Cyclase ◽  
Proximity Ligation Assay ◽  
Western Blots ◽  
Disulfide Isomerase ◽  
Protein Disulfide

NO binds to the receptor sGC (soluble guanylyl cyclase), stimulating cGMP production. The NO–sGC–cGMP pathway is a key component in the cardiovascular system. Discrepancies in sGC activation and deactivation in vitro compared with in vivo have led to a search for endogenous factors that regulate sGC or assist in cellular localization. In our previous work, which identified Hsp (heat-shock protein) 70 as a modulator of sGC, we determined that PDI (protein disulfide-isomerase) bound to an sGC-affinity matrix. In the present study, we establish and characterize this interaction. Incubation of purified PDI with semi-purified sGC, both reduced and oxidized, resulted in different migration patterns on non-reducing Western blots indicating a redox component to the interaction. In sGC-infected COS-7 cells, transfected FLAG-tagged PDI and PDI CXXS (redox active site ‘trap mutant’) pulled down sGC. This PDI–sGC complex was resolved by reductant, confirming a redox interaction. PDI inhibited NO-stimulated sGC activity in COS-7 lysates, however, a PDI redox-inactive mutant PDI SXXS did not. Together, these data unveil a novel mechanism of sGC redox modulation via thiol-disulfide exchange. Finally, in SMCs (smooth muscle cells), endogenous PDI and sGC co-localize by in situ proximity ligation assay, which suggests biological relevance. PDI-dependent redox regulation of sGC NO sensitivity may provide a secondary control over vascular homoeostasis.

scholarly journals Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0143523 ◽  
Author(s):  
Erin J. Heckler ◽  
Vladyslav Kholodovych ◽  
Mohit Jain ◽  
Tong Liu ◽  
Hong Li ◽  
...  
Keyword(s):  
Guanylyl Cyclase ◽  
Protein Disulfide Isomerase ◽  
Soluble Guanylyl Cyclase ◽  
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.

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.

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