Thioredoxin and protein-disulfide isomerase selectivity for redox regulation of proteins in <i>Corynebacterium glutamicum</i>

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.

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S-Glutathionylation of Protein Disulfide Isomerase Regulates Estrogen ReceptorαStability and Function

International Journal of Cell Biology ◽

10.1155/2012/273549 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 21

Author(s):

Ying Xiong ◽

Yefim Manevich ◽

Kenneth D. Tew ◽

Danyelle M. Townsend

Keyword(s):

Protein Disulfide Isomerase ◽

Estrogen Receptor Alpha ◽

Redox Regulation ◽

Gene Products ◽

Disulfide Isomerase ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

And Function ◽

Protein Response ◽

Protein Disulfide

S-Glutathionylation of cysteine residues within target proteins is a posttranslational modification that alters structure and function. We have shown that S-glutathionylation of protein disulfide isomerase (PDI) disrupts protein folding and leads to the activation of the unfolded protein response (UPR). PDI is a molecular chaperone for estrogen receptor alpha(ERα). Our present data show in breast cancer cells that S-glutathionylation of PDI interferes with its chaperone activity and abolishes its capacity to form a complex withERα. Such drug treatment also reverses estradiol-induced upregulation of c-Myc, cyclinD1, andP21Cip, gene products involved in cell proliferation. Expression of an S-glutathionylation refractory PDI mutant diminishes the toxic effects of PABA/NO. Thus, redox regulation of PDI causes its S-glutathionylation, thereby mediating cell death through activation of the UPR and abrogation ofERαstability and signaling.

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

Biochemical Journal ◽

10.1042/bj20130298 ◽

2013 ◽

Vol 452 (1) ◽

pp. 161-169 ◽

Cited By ~ 15

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.

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