A simple and highly sensitive method to quantify the rates of production of OH in biological systems is described. This method employs the reaction between OH and dimethyl sulfoxide to generate quantitatively a methyl radical, which then reacts with a fluorescamine-derivatized nitroxide to produce the stable O-methylhydroxylamine. This O-methylhydroxylamine is separated by reversed-phase high-performance liquid chromatography and quantified fluorometrically. The estimated detection limit of the O-methylhydroxylamine is 3.5 nM for a 50 microL injection at a signal to noise ratio of 2. The method is applied to the determination of the rates of OH production in a biologically relevant model system and in a mouse epidermal cell line treated with a quinone anticancer compound.

The Rho-GTPase Cdc42 is important for the establishment and maintenance of epithelial polarity. Signaling from Cdc42 is propagated via its effector molecules that specifically bind to Cdc42 in the GTP-bound form. The cell-cell contact regulator and actin-binding protein IQGAP1 is described as effector of Cdc42 and Rac. Unexpectedly, we show in this study that IQGAP1 bound also directly nucleotide-depleted Cdc42 (Cdc42-ND). This interaction was enhanced in the presence of phosphatase inhibitors and in epithelial cells without cellcell contacts. Tandem mass spectrometry analysis and immunoprecipitation experiments revealed that IQGAP1 was Ser 1443 -phosphorylated in vivo, potentially by protein kinase C⑀ and upon loss of cell-cell contacts. In addition, we identified two independent domains of the IQGAP1 C terminus that bound exclusively Cdc42-ND. These domains interacted with each other, favoring the binding to Cdc42-GTP. Moreover, phosphorylation on Ser 1443 strongly inhibited this intramolecular interaction. Thus, we unraveled a molecular mechanism that reveals a novel type of Rho-GTPase regulator. We propose that, depending on its phosphorylation state, IQGAP1 might serve as an effector or sequester nucleotide-free Cdc42 to prevent signaling.Epithelial polarity is regulated by the Rho-GTPase Cdc42, which is implicated in signal transduction, gene regulation, cell cycle progression, and cytoskeletal regulation (1). In epithelial cells, Cdc42 regulates establishment and maintenance of basolateral polarity by controlling endocytic and secretory trafficking (2-4). The basolateral membrane of epithelial cells is formed upon establishment of cell-cell contacts. Trans-activation of nectins and formation of E-cadherin mediated cell-cell contacts lead to the activation of Cdc42 (5, 6). However, how cell-cell contact formation favors the activation of Cdc42 is not known.The most direct activators of GTPases are guanine nucleotide exchange factors (GEFs), 1 which are characterized by four properties. GEFs bind to a GDP-bound GTPase, they catalyze the displacement of GDP and stabilize the resulting nucleotidefree GTPase. Due to the high ratio of GTP to GDP in cells, the nucleotide-depleted GTPase-GEF complex dissociates into GEF and GTP-bound (active) GTPase. The active GTPase can then bind selectively to effectors and elicit downstream effects.Activation of GEFs for Cdc42 at sites of cell-cell contacts might involve several modes. Some GEFs are activated by phosphorylation, which abolishes intramolecular inhibition (7-9). Other GEFs need additional factors to exhibit GEF activity. For example, interaction of intersectin with the Cdc42 effector protein N-WASP causes the activation of intersectin GDP displacement activity (10). Also, interaction of DOCK180 with the protein ELMO might be necessary for its activation as a GEF for Rac (11,12).Mammalian IQGAPs are scaffolding proteins with conserved homologues in budding yeast, Dictyostelium, and Caenorhabditis elegans and have been shown to regulate actomyosin ring formation ...

The o-quinone forms of 2,3- and 3,4-catechol estrogens have been implicated in the carcinogenicity of these hormones. The concomitant production of reactive oxygen species during reduction of the o-quinone estrogens has been inferred to play a mechanistic role in their mutagenic potential. Conclusive evidence documenting the production of hydrogen peroxide, the hydroxyl radical, and the estrone 3,4-semiquinone in estrone 3,4-quinone (3,4-EQ)-treated human breast cancer subcellular fractions was demonstrated in the absence of exogenously added catalysts. Subcellular fractions of MCF-7 cells treated with 3,4-EQ and NADPH, including nuclei, mitochondria, and microsomes, were shown to support significant amounts of hydrogen peroxide production. Hydrogen peroxide production in 3,4-EQ-treated cellular fractions and the chromosomal DNA damage induced in 3,4-EQ-treated MCF-7 cells were abolished by the addition of catalase. A significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH resulting in hydrogen peroxide production was demonstrated. The results unequivocally demonstrate that free radicals are produced during the metabolism of estrone 3,4-quinone in human cells.

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-␤-alltrans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are: in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. Based on the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.

No abstract

Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartic acid residues. This chemical cleavage has been applied to ovalbumin and several model peptides to test the effect on some of the more common post-translational modifications. No oxidation of methionine or cysteine was observed; however, hydrolysis of phosphate groups proceeds at a detectable rate. Acid cleavage was also extended to the yeast ribosome model proteome, where it provided information on 74% of that proteome. Aspartic acid occurs across the proteome with approximately half the frequency of the combined occurrence of the trypsin residues lysine and arginine, and implications of this are considered.

No abstract