Protein disulfide isomerase redox-dependent association with p47phox: evidence for an organizer role in leukocyte NADPH oxidase activation
Abstract
Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 μg/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by ∼40%), whereas PDIred diminished (by ∼60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by ∼70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47phox in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47phox. PDI-p47phox association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47phox in cytosol. Incubation of purified PDI (>80% reduced) and p47phox in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re)cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer.
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Use of p47phox−/− as a surrogate for NOX2 deletion is partly limited by the involvement of p47phox in other protein complexes, including noncanonical activation of NOX1 and interaction with protein disulfide isomerase.
“It should be noted that the use of p47phox−/− as a surrogate for the NOX2 deletion is partly limited by the involvement of p47phox in other protein complexes, including not only the noncanonical activation of NOX1, as already mentioned above but also its interaction with protein disulfide isomerase (214, 559).”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [214]
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Self-citation
- Authors
- de A. Paes, A. M., Veríssimo-Filho, S., Guimarães, L. L., Silva, A. C. B., Takiuti, J. T., Santos, C. X. C., Janiszewski, M., Laurindo, F. R. M., & Lopes, L. R.
- Affiliations
- Redox Signaling Laboratory, Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo , São Paulo, Brazil
- Universidade de São Paulo (BR)
- Vascular Biology Laboratory, Heart Institute (InCor), School of Medicine, University of São Paulo , São Paulo, Brazil
- Corresponding author
- Lopes, Lucia R
- Venue
- Journal of Leukocyte Biology
- Publisher
- Oxford University Press (OUP)
- Published
- 2011-07-26
- Volume
- 90
- Issue
- 4
- Pages
- 799-810
- ISSN
- 0741-5400 (ISSN-L)
- 1938-3673
- OpenAlex
- W2144320777
- Cited by
- 95
- References
- 56
- Citations by year
- 2026: 1 · 2025: 5 · 2024: 2 · 2023: 3 · 2022: 5 · 2021: 4 · 2020: 8 · 2019: 6 · 2018: 9 · 2017: 11 · 2016: 6 · 2015: 11 · 2014: 10 · 2013: 6 · 2012: 7
- FWCI
- 3.38
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Leukocyte Biology (publishedVersion)
- PubMed (publishedVersion)
- Scientific Electronic Library Online (São Paulo Research Foundation, Latin American and Caribbean Center on Health Sciences Information, Conselho Nacional de Desenvolvimento Científico e Tecnológico) (publishedVersion)
- Funders
- Fundação de Amparo à Pesquisa do Estado de São Paulo
- Conselho Nacional de Desenvolvimento Científico e Tecnológico
- Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Keywords
- protein disulfide isomerase, p47phox, NADPH oxidase, neutrophils, superoxide production, redox regulation, oxidoreductase activity, azurophilic granules, PMA stimulation, arachidonic acid
- MeSH terms
- Anti-Bacterial Agents, Bacitracin, Cell Membrane, Cytosol, Enzyme Activation, Enzyme Inhibitors, Humans, Neutrophils, Oxidation-Reduction, Superoxides, NADPH Oxidases, Protein Disulfide-Isomerases, Amino Acid Substitution, Mutation, Missense, Protein Transport
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