A recently developed proteomics strategy, designated tagging-viasubstrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and͞or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.
NADPH oxidase activation involves the assembly of membrane-localized cytochrome b 559 with the cytosolic components p47 phox , p67 phox , and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67 phox and fulllength Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47 phox . We subjected chimera p67 phox -(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67 phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67 phox and Rac1, in the p67 phox -(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67 phox . This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67 phox to the membrane; the other is to induce an "activating" conformational change in p67 phox . . production, is probably the consequence of a conformational change in gp91 phox , caused by the interaction of cytochrome b 559 with one or several of the cytosolic oxidase components (8). Establishing contact with cytochrome b 559 requires the translocation to the plasma membrane of p47 phox , p67 phox , and Rac, a process known as oxidase assembly (reviewed in Refs. 9 and 10). Stimulus-elicited activation of the oxidase in intact cells can be mimicked in a cell-free system, in which phagocyte membranes or purified and relipidated cytochrome b 559 are mixed with the cytosolic components p47 phox , p67 phox , and Rac, in the presence of an anionic amphiphile, serving as an activator (11-15). The amphiphile acts by causing a conformational change in p47 phox (16) and, possibly, also by the induction of structural changes in cytochrome b 559 (17).The impairment of O 2 . production in the p47 phox -deficient form of chronic granulomatous disease shows that p47 phox is * This work was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grant 428/01, the Roberts-Guthman Chair in Immunopharmacology (to E. P.), and National Institutes of Health Grant GM46372 (to C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefor...
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