scholarly journals Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway ofAspergillus niger

2000 ◽  
Vol 66 (2) ◽  
pp. 775-782 ◽  
Author(s):  
Celina Ngiam ◽  
David J. Jeenes ◽  
Peter J. Punt ◽  
Cees A. M. J. J. Van Den Hondel ◽  
David B. Archer

ABSTRACT Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene,pdiA, encoding PDIA was previously isolated fromAspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A.pdiA also complemented PDI function in aSaccharomyces cerevisiae Δpdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

2020 ◽  
Vol 21 (24) ◽  
pp. 9351
Author(s):  
Shingo Kanemura ◽  
Motonori Matsusaki ◽  
Kenji Inaba ◽  
Masaki Okumura

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.


2010 ◽  
Vol 21 (18) ◽  
pp. 3093-3105 ◽  
Author(s):  
Lori A. Rutkevich ◽  
Myrna F. Cohen-Doyle ◽  
Ulf Brockmeier ◽  
David B. Williams

To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1351-1351
Author(s):  
Alexandra Lozano ◽  
Christopher Vega ◽  
Yaritza Inostroza-Nieves ◽  
Lorena Rivera González ◽  
Pablo J. López ◽  
...  

Abstract Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units (RFU); P<0.005, n=6 and 9, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. Disclosures No relevant conflicts of interest to declare.


2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Joao Wosniak ◽  
Renata C Gonçalves ◽  
Leonardo Y Tanaka ◽  
Daniela B Zanatta ◽  
Bryan E Strauss ◽  
...  

Objective: Vascular smooth muscle cell (VSMC) phenotype switch depends on extrinsic/intrinsic cues including NOX NADPH oxidase-linked redox signaling. Growth factor-triggered NOX1 expression/activity requires the chaperone oxidoreductase protein disulfide isomerase-A1 (PDI). Acute PDI overexpression induces agonist-independent NOX1 expression. PDI is required for VSMC migration and cytoskeleton organization, and extracellular PDI supports expansive vascular remodeling via cytoskeleton reshaping. Such PDI effects led us to hypothesize that PDI may orchestrate VSMC phenotypic alterations. Approach and Results: In primary VSMC, PDI silencing spontaneously decreased differentiation marker expression. Transfection with a doxycycline-inducible lentiviral vector encoding PDI showed that sustained PDI overexpression (72h) enhanced actin branching pattern vs. baseline (anisotropy index, 0.103±0.019 vs. 0.220±0.027, 72h vs. 0h, mean±SEM, N=5, P<0.05), increased cell length and induced expression of differentiation marker calponin (2-fold, 72h vs 0h, N=5, P<0.05), alpha-actin and smoothelin, which were abrogated upon catalase incubation. Intracellular superoxide enhanced upon 48h of PDI overexpression (2-hydroxyethidium levels, 0.998±0.102 vs. 2.887±0.227 AU, N=4, P<0.05) and was NOX1-dependent, based on inhibition with GKT136901 (by 48%) or NOXA1ds peptide (by 54%) (N=6, P<0.05). Increased NOX1 mRNA occurred early after PDI overexpression (74%, 24h vs. 0h, N=4, P<0.05), while NOX4 mRNA was upregulated only after long-term PDI induction (100%, 72h vs. 0h, N=4, P<0.05). In rabbit restenosis model exhibiting strong PDI upregulation (12-fold at day 14 after injury), ex vivo PDI silencing 7 or 14 days after injury reversed PCNA expression, while promoting increased NOX1 and decreased NOX4 mRNA levels (+54% and -45%, respectively, siPDI vs. siSCR, N=3, P<0.05 for both). Conclusions: While short-term PDI overexpression supports NOX1 activation/expression, sustained PDI overexpression drives NOX4 expression and VSMC differentiation. Effects on cytoskeleton, NOX1/4 activation and temporal control of NOX1/4 expression suggest a central role for PDI as a hub for redox-mediated VSMC phenotype regulation.


2013 ◽  
Vol 452 (1) ◽  
pp. 161-169 ◽  
Author(s):  
Erin J. Heckler ◽  
Pierre-Antoine Crassous ◽  
Padmamalini Baskaran ◽  
Annie Beuve

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.


Author(s):  
Thomas Chetot ◽  
Etienne Benoit ◽  
Véronique Lambert ◽  
Virginie Lattard

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is the target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and have been suspected to confer resistance to VKA and/or affect its velocity. Nevertheless, the results between studies have been conflicting, the functional characterization of these mutations in a cell system being complex due to the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to globally evaluate the vitamin K cycle in the HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when the VKORC1/VKORC1L1 were knocked out, the K/KO balance decreased significantly due to an accumulation of vitamin KO. On the contrary, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating a limitation of the VKOR activity. This limitation was shown to be due to an insufficient expression of the activation partner of VKORC1, as overexpressing the protein disulfide isomerase (PDI) overcomes the limitation. This study is the first to demonstrate a functional interaction between VKORC1 and the PDI enzyme.


1989 ◽  
Vol 37 (12) ◽  
pp. 1835-1844 ◽  
Author(s):  
S Akagi ◽  
A Yamamoto ◽  
T Yoshimori ◽  
R Masaki ◽  
R Ogawa ◽  
...  

We investigated the intracellular distribution of protein disulfide isomerase (PDI) in rat epiphyseal chondrocytes by immunocytochemistry, using a post-embedding protein A-gold technique. Gold particles were localized primarily in the cisternal space of the rough endoplasmic reticulum (ER) and nuclear envelopes. The ER cisternae of the chondrocytes in all the differentiating epiphyseal zones--resting, proliferative, pre-hypertrophic, and hypertrophic--were equally and highly labeled. The labeling density of the cisternal space of the dilated ER, probably reflecting marked accumulation of secretory proteins such as procollagen, was always higher than that of the non-dilated ER. In the dilated cisternal space, gold particles were freely and evenly distributed, without preferential binding to the luminal surface of the ER membranes. We suggest that PDI catalyzes the formation of disulfide bonds of various secretory proteins, perhaps type II procollagen, in the cisternal space of the ER in epiphyseal chondrocytes. The exclusive localization of gold particles in the cisternal space of the ER and nuclear envelopes and the lack of gold particles in the Golgi apparatus, including cis-Golgi cisternae, indicate that PDI is an ER-soluble protein in the chondrocytes and is presumably sorted out in some pre-Golgi compartment and not transported to the Golgi apparatus.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2271-2271
Author(s):  
Arelys Ramos-Rivera ◽  
Alicia Rivera ◽  
Enrique D. Machado-Fiallo ◽  
Josue A. Benabe-Carlo ◽  
Gregory N. Prado ◽  
...  

Abstract Aldosterone (ALDO) has been shown to play an important role in inflammatory responses in addition to its well described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). However, its effects on polymorphonuclear leukocytes (PMNC) are not well described. We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for the myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses. We incubated PMNC with ALDO (10–9–10–7M) for 30 min and observed a dose-dependent rise in β–glucuronidase release with an EC50 of 6.11 nM (P<0.001, n=3), an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). In addition, our results show that incubation of human PMNC with 10-8M ALDO likewise led to increases in myeloperoxidase ([MPO], P<0.05, n=3) and protein disulfide isomerase ([PDI], P<0.01, n=4), a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions (Prado, 2013 FASEB J). We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. Our results likewise show an increase in MPO responses upon 10–8M ALDO stimulation as compared to vehicle (AUC: 1090±147 to 505±48, P<0.02, n=3). We have recently reported that aldosterone stimulates increases of striatin, a scaffolding protein that interacts with caveolin-1, and co-precipitates with striatin and as such may facilitate cross talk of signaling complexes. As there are no pharmacological inhibitors of striatin we used a molecular approach to reduce striatin levels. In differentiated HL-60 cells, siRNA against striatin led to reduced MPO responses (AUC: 590±14 to 528±13, P<0.05, n=3) that were associated with significantly reduced striatin mRNA levels but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and β-microglobulin used as an endogenous control (P<0.01, n=3). These results suggest that striatin plays an important role in ALDO-stimulated degranulation responses. Of importance we also observed that incubation with ALDO (10–9–10–7M) in differentiated HL60 cells led to increases in the oxidative-respiratory burst [superoxide production] in a dose- and time-dependent manner (P<0.01, n=4). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). As there is evidence that activated neutrophils, MPO and PDI are elevated in Sickle Cell Disease, we studied the in vivo effects of MR blockade in BERK sickle transgenic mice, a model of increased oxidative stress. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and blood collected. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units [RFU]; P<0.005, n=6 and 9) and lower MPO levels (AUC: 214±11 to 73±20, P<0.03, n=3); events that were associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). Thus, our results suggest that MR activation by ALDO is a novel mechanism for neutrophil stimulation and as such represents a novel therapeutic target aimed at ameliorating the vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 (AR) and R01HL096518 (JRR). Disclosures: No relevant conflicts of interest to declare.


2004 ◽  
Vol 9 (7) ◽  
pp. 614-620 ◽  
Author(s):  
Anthony M. Smith ◽  
John Chan ◽  
Donna Oksenberg ◽  
Roman Urfer ◽  
David S. Wexler ◽  
...  

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.


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