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Cited by 63Redox biology and oxidative stressNitric Oxide and Endothelin EffectsNeutrophil, Myeloperoxidase and Oxidative Mechanisms

Identification of the Binding Site for the Regulatory Calcium-Binding Domain in the Catalytic Domain of NOX5

Tirone, Fabiana, Radu, Laura, Craescu, Constantin T., Cox, Jos A.·2010·Biochemistry
DOI 10.1021/bi901846y
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Abstract

NADPH oxidases (NOX) are important superoxide producing enzymes that regulate a variety of physiological and pathological processes such as bacteria killing, angiogenesis, sperm-oocyte fusion, and oxygen sensing. NOX5 is a member of the NOX family but distinct from the others by the fact that it contains a long N-terminus with four EF-hand Ca(2+)-binding sites (NOX5-EF). NOX5 generates superoxide in response to intracellular Ca(2+) elevation in vivo and in a cell-free system. Previously, we have shown that the regulatory N-terminal EF-hand domain interacts directly and in a Ca(2+)-dependent manner with the catalytic C-terminal catalytic dehydrogenase domain (CDHD) of the enzyme, leading to its activation. Here we have characterized the interaction site for the regulatory NOX5-EF in the catalytic CDHD of NOX5 using cloned fragments and synthetic peptides of the CDHD. The interaction was monitored with pull-down techniques, cross-linking experiments, tryptophan fluorescence, hydrophobic exposure, isothermal titration calorimetry, and cell-free system enzymatic assays. This site is composed of two short segments: the 637-660 segment, referred to as the regulatory EF-hand-binding domain (REFBD), and the 489-505 segment, previously identified as the phosphorylation region (PhosR). NOX5-EF binds to these two segments in a Ca(2+)-dependent way, and the superoxide generation by NOX5 depends on this interaction. Controlled proteolysis suggests that the REFBD is autoinhibitory and inhibition is relieved by NOX5-EF.

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  • In the cardiovascular system, peroxynitrite can oxidize heme proteins, such as Hb and Mb, resulting in the oxidation of their heme oxygen-carrying site from ferrous iron to ferric heme.

    “In the cardiovascular system, peroxynitrite can oxidize heme proteins, such as Hb and Mb, resulting in the oxidation of their heme oxygen–carrying site from ferrous iron to ferric heme (101, 942).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [942]

  • In the mitochondrion, peroxynitrite oxidizes the electron transport protein cytochrome c.

    “In the mitochondrion, a similar reaction oxidizes the electron transport protein cytochrome c (942).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [942]

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Authors
Tirone, F., Radu, L., Craescu, C. T., & Cox, J. A.
Affiliations
  • University of Geneva (CH)
  • Inserm (FR)
Corresponding author
  • Tirone, Fabiana
  • Cox, Jos A.
ORCID
Radu, Laura
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2010-01-05
Volume
49
Issue
4
Pages
761-771
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi901846y
OpenAlex
W1982280480
Cited by
63
References
40
Citations by year
2026: 3 · 2025: 1 · 2024: 4 · 2023: 6 · 2022: 1 · 2021: 3 · 2020: 8 · 2019: 7 · 2018: 1 · 2017: 6 · 2016: 2 · 2015: 5 · 2014: 2 · 2013: 2 · 2012: 6
FWCI
1.61
Type
journal article
Language
en
Open access
closed
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Keywords
NADPH oxidase 5, NADPH oxidase, EF-hand calcium-binding domain, calcium-dependent protein-protein interaction, superoxide production, autoinhibition, enzyme activation, intracellular calcium signaling
MeSH terms
NADPH Oxidase 5, Amino Acid Sequence, Binding Sites, Calcium, Cell Line, Humans, Membrane Proteins, Molecular Sequence Data, Protein Conformation, Transfection, NADPH Oxidases, Catalytic Domain, EF Hand Motifs

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