[show abstract][hide abstract] ABSTRACT: Reversible protein cysteine nitrosylation (S-nitrosylation) is a common mechanism utilized in signal transduction and other diverse cellular processes. Protein denitrosylation is largely mediated by cysteine denitrosylases, but the functional scope and significance of these enzymes are incompletely defined, in part due to limited information on their cognate substrates. Here, using Jurkat cells, we employed stable isotope labeling by amino acids in cell culture (SILAC), coupled to the biotin switch technique and mass spectrometry, to identify 46 new substrates of one denitrosylase, thioredoxin 1. These substrates are involved in a wide range of cellular functions including cytoskeletal organization, cellular metabolism, signal transduction, and redox homeostasis. We also identified multiple S-nitrosylated proteins that are not substrates of thioredoxin 1. A verification of our principal findings was made in a second cell type (RAW264.7 cells). Our results point to thioredoxin 1 as a major protein denitrosylase in mammalian cells and demonstrate the utility of quantitative proteomics for large-scale identification of denitrosylase substrates.
[show abstract][hide abstract] ABSTRACT: Nitric oxide exerts a plethora of biological effects via protein S-nitrosylation, a redox-based reaction that converts a protein Cys thiol to a S-nitrosothiol. However, although the regulation of protein S-nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus-coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin-interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin-mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S-nitrosylation.
Journal of Biological Chemistry 10/2009; 284(52):36160-6. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: S-Nitrosylation, the redox-based modification of Cys thiol side chains by nitric oxide, is a common mechanism in signal transduction. Dysregulated S-nitrosylation contributes to a range of human pathologies. New roles for protein denitrosylation in regulating S-nitrosylation are being revealed. Recently, several denitrosylases - the enzymes that mediate Cys denitrosylation - have been discovered, of which two enzyme systems in particular, the S-nitrosoglutathione reductase and thioredoxin systems, have been shown to be physiologically relevant. These highly conserved enzymes regulate signalling through multiple classes of receptors and influence diverse cellular responses. In addition, they protect from nitrosative stress in microorganisms, mammals and plants, thereby exerting profound effects on host-microbe interactions and innate immunity.
[show abstract][hide abstract] ABSTRACT: Protein S-nitrosylation, the posttranslational modification of cysteine thiols to form S-nitrosothiols, is a principle mechanism of nitric oxide-based signaling. Studies have demonstrated myriad roles for S-nitrosylation in organisms from bacteria to humans, and recent efforts have greatly advanced our scientific understanding of how this redox-based modification is dynamically regulated during physiological and pathophysiological conditions. The focus of this review is the biotin-switch technique (BST), which has become a mainstay assay for detecting S-nitrosylated proteins in complex biological systems. Potential pitfalls and modern adaptations of the BST are discussed, as are future directions for this assay in the burgeoning field of protein S-nitrosylation.
[show abstract][hide abstract] ABSTRACT: Nitric oxide acts substantially in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. The mechanisms that might subserve protein denitrosylation in cellular signaling remain uncharacterized. Our search for denitrosylase activities focused on caspase-3, an exemplar of stimulus-dependent denitrosylation, and identified thioredoxin and thioredoxin reductase in a biochemical screen. In resting human lymphocytes, thioredoxin-1 actively denitrosylated cytosolic caspase-3 and thereby maintained a low steady-state amount of S-nitrosylation. Upon stimulation of Fas, thioredoxin-2 mediated denitrosylation of mitochondria-associated caspase-3, a process required for caspase-3 activation, and promoted apoptosis. Inhibition of thioredoxin-thioredoxin reductases enabled identification of additional substrates subject to endogenous S-nitrosylation. Thus, specific enzymatic mechanisms may regulate basal and stimulus-induced denitrosylation in mammalian cells.
[show abstract][hide abstract] ABSTRACT: beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.
[show abstract][hide abstract] ABSTRACT: S-Nitrosylation, the covalent addition of a nitrogen monoxide group to a cysteine thiol, has been shown to modify the function of a broad spectrum of mammalian, plant, and microbial proteins and thereby to convey the ubiquitous influence of nitric oxide on cellular signal transduction and host defense. Accumulating evidence indicates that dysregulated, diminished, or excessive S-nitrosylation may be implicated in a wide range of pathophysiological conditions. A recent study establishes a functional relationship between inhibitory S-nitrosylation of the redox enzyme protein disulfide isomerase (PDI), defects in regulation of protein folding within the endoplasmic reticulum (ER), and neurodegeneration. Further, an examination of human brains afflicted with Parkinson's or Alzheimer's disease supports a causal role for the S-nitrosylation of PDI and consequent ER stress in these prevalent neurodegenerative disorders.
ACS Chemical Biology 08/2006; 1(6):355-8. · 5.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cisplatin (CDDP) is an efficient DNA-damaging antitumor agent employed for the treatment of various human cancers. CDDP activates nuclear as well as cytoplasmatic signaling pathways involved in regulation of the cell cycle, damage repair and programmed cell death. Here we report that CDDP also activates a membrane-integrated protein, the epidermal growth factor receptor (EGFR). We show that EGFR is activated in response to CDDP in various types of cells that overexpress the receptor, including transformed human glioma cells and human breast tumor cells. CDDP-induced EGFR activation requires its kinase activity, as it can be blocked by an EGFR kinase inhibitor or by expression of a kinase dead receptor. We also show that CDDP-induced EGFR activation is independent of receptor ligand. CDDP induces the activation of c-Src, and EGFR activation is blocked by Src-family inhibitor PP1, suggesting that Src kinases mediate CDDP-induced EGFR activation. We propose that EGFR activation in response to CDDP is a survival response, since inhibition of EGFR activation enhances CDDP-induced death. These findings show that signals generated by DNA damage can modulate EGFR activity, and argue that interfering with CDDP-induced EGFR activation in tumor cells might be a useful approach to sensitize these cells to genotoxic agents.
[show abstract][hide abstract] ABSTRACT: Protein kinase B/Akt (PKB) is an anti-apoptotic protein kinase that has strongly elevated activity in human malignancies. We therefore initiated a program to develop PKB inhibitors, "Aktstatins". We screened about 500 compounds for PKB inhibitors, using a radioactive assay and an ELISA assay that we established for this purpose. These compounds were produced as combinatorial libraries, designed using the structure of the selective PKA inhibitor H-89 as a starting point. We have identified a successful lead compound, which inhibits PKB activity in vitro and in cells overexpressing active PKB. The new compound shows reversed selectivity to H-89: In contrast to H-89, which inhibits PKA 70 times better than PKB, the new compound, NL-71-101, inhibits PKB 2.4-fold better than PKA. The new compound, but not H-89, induces apoptosis in tumor cells in which PKB is amplified. We have identified structural features in NL-71-101 that are significant for the specificity and that can be used for future development and optimization of PKB inhibitors.
[show abstract][hide abstract] ABSTRACT: Anticancer therapy is frequently efficient in early stages of the disease, whereas advanced tumors are usually resistant to the same treatments. The molecular basis for this change is not entirely understood. Many anticancer agents are DNA- or cytoskeleton-damaging drugs that show some specificity towards dividing cells. However, recent studies show that these agents also activate stress-signaling cascades that may play a role in eliciting the observed therapeutic effects. We discuss recent findings that suggest that induction of stress signaling in oncogenically transformed cells is integrated into apoptotic pathways. Reactive oxygen species (ROS) and stress-activated protein kinases (SAPKs), which are potentiated in recently transformed cells, emerge as key effectors of cell death. In advanced tumors, however, these agents are downregulated and, consequently, death signaling is suppressed. Such changes in ROS and SAPK activity levels during the course of tumor development may underlie the changes in responsiveness to anticancer therapy.