Critical cysteines in Akt1 regulate its activity and proteasomal degradation: implications for neurodegenerative diseases.

Impaired Akt1 signaling is observed in neurodegenerative diseases, including Parkinson׳s disease (PD). In PD models oxidative modification of Akt1 leads to its dephosphorylation and consequent loss of its kinase activity. To explore the underlying mechanism we exposed Neuro2A cells to cadmium, a pan inhibitor of protein thiol disulfide oxidoreductases, including glutaredoxin 1 (Grx1), or downregulated Grx1, which led to dephosphorylation of Akt1, loss of its kinase activity, and also decreased Akt1 protein levels. Mutation of cysteines to serines at 296 and 310 in Akt1 did not affect its basal kinase activity but abolished cadmium- and Grx1 downregulation-induced reduction in Akt1 kinase activity, indicating their critical role in redox modulation of Akt1 function and turnover. Cadmium-induced decrease in phosphorylated Akt1 correlated with increased association of wild-type (WT) Akt1 with PP2A, which was absent in the C296-310S Akt1 mutant and was also abolished by N-acetylcysteine treatment. Further, increased proteasomal degradation of Akt1 by cadmium was not seen in the C296-310S Akt1 mutant, indicating that oxidation of cysteine residues facilitates degradation of WT Akt1. Moreover, preventing oxidative modification of Akt1 cysteines 296 and 310 by mutating them to serines increased the cell survival effects of Akt1. Thus, in neurodegenerative states such as PD, maintaining the thiol status of cysteines 296 and 310 in Akt1 would be critical for Akt1 kinase activity and for preventing its degradation by proteasomes. Preventing downregulation of Akt signaling not only has long-range consequences for cell survival but could also affect the multiple roles that Akt plays, including in the Akt-mTOR signaling cascade.

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder.

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

Indolyl-quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation-mediated control of tumor vasculature.

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.

Characteristics of thiol:protein disulfide oxidoreductase from wheat (Triticum aestivum L.) grain.

Biochemical properties of a homogenous preparation of thiol:protein disulfide oxidoreductase (TPDO, EC 1.8.4.2) isolated for the first time from mature wheat (Triticum aestivum L.) grain were studied. According to polyacrylamide gel electrophoresis data, the molecular weight of TPDO is around 167 kD, the enzyme consisting of two subunits of 77 and 73 kD, which differentiates TPDO from known enzymes of SH/SS-metabolism of wheat caryopses. In substrate specificity and enzymatic characteristics (pH and temperature optima) TPDO is similar to analogous enzymes of animal tissues. Inhibition of disulfide reductase activity by alkylating agents and heavy metal ions suggests the participation of active center SH-groups in the catalytic act and classes the enzyme as a member of the thioredoxin superfamily. The SS-reductase reduces aggregating capacity of acetic acid-soluble fraction of wheat storage proteins. The proposed physiological role of TPDO is participation in creation and regulation of SH/SS-status of wheat endosperm proteins and formation of the rheological properties of gluten.

Recent advances in understanding the molecular mechanisms of HIV-1 entry and fusion: revisiting current targets and considering new options for therapeutic intervention.

Recent advances in our understanding of the cellular and molecular mechanisms of HIV-1 entry provide the basis for novel therapeutic strategies that prevent viral penetration of the target cell-membrane, while reducing detrimental virus and treatment effects on cells and prolonging virion exposure to immune defenses. A number of potential sites for therapeutic intervention become accessible during the narrow window between virus attachment and the subsequent fusion of viral envelope with the cell membrane. Initial approaches considered for prevention of HIV-1 entry included the use of natural ligands, small-molecule inhibitors and/or monoclonal antibodies, which could interfere with gp120-CD4 and/or gp120-CXCR4/CCR5 interaction. Others avenues pursued were the use of agents that interfere with the conformational changes of gp120 or gp41 leading to subsequent fusion of viral and cellular membranes. More recently, strategies have emerged that involve inhibition of thiol/disulfide oxidoreductases (factors which facilitate Env transition from an inactive to a fusion-competent conformation) to block redox exchange, thereby impeding the entry process. This review provides a summary of the cellular and viral factors mediating the HIV-1 entry process, with an emphasis on novel therapeutics targeting Env-receptor/coreceptor interaction, Env conformational change and the membrane fusion process.

Zinc fingers and thiol-disulfide oxidoreductase activities of chaperone DnaJ.

Chaperone DnaJ is a homodimer with each subunit containing 10 cysteine residues and two Zn(II) ions, which have been identified to form two zinc fingers, C(144)DVC(147)Zn(II)C(197)NKC(200) (Zn1) and C(161)PTC(164)Zn(II)C(183)PHC(186) (Zn2), with C(265) and C(323) in reduced form. Guanidine hydrochloride at 6.4 M destroys only Zn1, which does not reform after refolding. p-Hydroxymercuriphenylsulfonate acid, but not ethylenediaminetetraacetic acid (EDTA) even at high concentrations, can remove two Zn(II) ions from DnaJ, but only Zn2 can be reconstituted. After removal of Zn(II) ions, only C(144) and C(147) in Zn1 are oxidation-resistant, and the other six cysteines are easily oxidizable. DnaJ shows reductase activity and oxidase activity but little, if any, isomerase activity. The reductase activity is reversibly inhibited by EDTA. Zn2 is important for the enzymatic activity, and only -C(183)PHC(186)- among the four motifs of -CXXC- functions as the active site of the enzyme. A C-terminal (Q(181)-R(376)) fragment shows a zinc finger of C(183)PHC(186)Zn(II)C(197)NKC(200) and full enzymatic activity of DnaJ. The N-terminal half sequence (M(1)-Q(180)) and Zn1 are not required for the enzymatic activity but are important for the chaperone activity of DnaJ.

Inhibition of glutathione-insulin transhydrogenase by metal ions and activation by histidine and other chelating agents.

The catalytic activity of purified glutathione-insulin transhydrogenase (thiol:protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2) from bovine pancreas is markedly stimulated by histidine and other chelating agents. The activation produced was highest with EDTA, followed by EGTA, 8-hydroxyquinoline and 1,10-phenanthroline. Of the many amino acids tested, histidine was the only one that activated the enzyme; the structurally related compounds, 3-methylhistidine and imidazole also stimulated the enzyme, but 1-methylhistidine and histamine were without effect. The activation of EDTA was negated by metal ions, most effectively by Se2+, Hg2+, Cu2+ and Zn2+, and less effectively by Ca2+ and Ni2+. Likewise, activation by histidine was negated by Zn2+ but not by Ca2+ or Mg2+. Thus, activation of glutathione-insulin transhydrogenase is apparently achieved in part by the chelation of inhibitory metal ion(s). These findings are consistent with a regulatory scheme for glutathione-insulin transhydrogenase in which (a) the enzyme is inhibited by selenium and heavy metal ions normally present in tissues and (b) this inhibition can be relieved by the addition of histidine or chelating agents.