Inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of small GTP-binding proteins. Lack of amino acid sequence specificity and importance of polybasic motif.
The small GTP-binding protein (G protein) Rac1 is an obligatory participant in the assembly of the superoxide (O2-.)-generating NADPH oxidase complex of macrophages. We investigated the effect of synthetic peptides, mapping within the near carboxyl-terminal domains of Rac1 and of related G proteins, on the activity of NADPH oxidase in a cell-free system consisting of solubilized guinea pig macrophage membrane, a cytosolic fraction enriched in p47phox and p67phox (or total cytosol), highly purified Rac1-GDP dissociation inhibitor for Rho (Rho GDI) complex, and the activating amphiphile, lithium dodecyl sulfate. Peptides Rac1-(178-188) and Rac1-(178-191), but not Rac2-(178-188), inhibited NADPH oxidase activity in a Rac1-dependent system when added prior to or simultaneously with the initiation of activation. However, undecapeptides corresponding to the near carboxyl-terminal domains of RhoA and RhoC and, most notably, a peptide containing the same amino acids as Rac1-(178-188), but in reversed orientation, were also inhibitory. Surprisingly, O2-. production in a Rac2-dependent cell-free system was inhibited by Rac1-(178-188) but not by Rac2-(178-188). Finally, basic polyamino acids containing lysine, histidine, or arginine, also inhibited NADPH oxidase activation. We conclude that inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of certain small G proteins is not amino acid sequence-specific but related to the presence of a polybasic motif. It has been proposed that such a motif serves as a plasma membrane targeting signal for a number of small G proteins (Hancock, J.F., Paterson, H., and Marshall, C.J. (1990) Cell 63, 133-139).
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
G
G
Joseph
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Y
Gorzalczany
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V
Koshkin
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E
Pick
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002474:Cell-Free System; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019255:NADPH Oxidases; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D020744:rac GTP-Binding Proteins
D010455:Peptides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins