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A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity.

Publication ,  Journal Article
Mizrahi, A; Berdichevsky, Y; Casey, PJ; Pick, E
Published in: J Biol Chem
August 13, 2010

The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b(559), a membrane-associated heterodimer composed of two subunits (Nox2 and p22(phox)), and four cytosolic proteins (p47(phox), p67(phox), Rac, and p40(phox)). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b(559) and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47(phox), p67(phox), and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122-22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47(phox)-p67(phox)-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47(phox) phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp(193) in p47(phox) persists. Prenylated GFP-p47(phox)-p67(phox)-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors.

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Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

August 13, 2010

Volume

285

Issue

33

Start / End Page

25485 / 25499

Location

United States

Related Subject Headings

  • rac1 GTP-Binding Protein
  • Spodoptera
  • Protein Prenylation
  • Phosphoproteins
  • Phospholipids
  • NADPH Oxidases
  • Macrophages
  • Liposomes
  • Humans
  • Guinea Pigs
 

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Mizrahi, A., Berdichevsky, Y., Casey, P. J., & Pick, E. (2010). A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity. J Biol Chem, 285(33), 25485–25499. https://doi.org/10.1074/jbc.M110.113779
Mizrahi, Ariel, Yevgeny Berdichevsky, Patrick J. Casey, and Edgar Pick. “A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity.J Biol Chem 285, no. 33 (August 13, 2010): 25485–99. https://doi.org/10.1074/jbc.M110.113779.
Mizrahi A, Berdichevsky Y, Casey PJ, Pick E. A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity. J Biol Chem. 2010 Aug 13;285(33):25485–99.
Mizrahi, Ariel, et al. “A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity.J Biol Chem, vol. 285, no. 33, Aug. 2010, pp. 25485–99. Pubmed, doi:10.1074/jbc.M110.113779.
Mizrahi A, Berdichevsky Y, Casey PJ, Pick E. A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity. J Biol Chem. 2010 Aug 13;285(33):25485–25499.

Published In

J Biol Chem

DOI

EISSN

1083-351X

Publication Date

August 13, 2010

Volume

285

Issue

33

Start / End Page

25485 / 25499

Location

United States

Related Subject Headings

  • rac1 GTP-Binding Protein
  • Spodoptera
  • Protein Prenylation
  • Phosphoproteins
  • Phospholipids
  • NADPH Oxidases
  • Macrophages
  • Liposomes
  • Humans
  • Guinea Pigs