Browsing by Author "Stamler, Jonathan S"
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Item Open Access EPR spectroscopy of nitrite complexes of methemoglobin.(Inorg Chem, 2010-07-19) Schwab, David E; Stamler, Jonathan S; Singel, David JThe chemical interplay of nitrogen oxides (NO's) with hemoglobin (Hb) has attracted considerable recent attention because of its potential significance in the mechanism of NO-related vasoactivity regulated by Hb. An important theme of this interplay-redox coupling in adducts of heme iron and NO's-has sparked renewed interest in fundamental studies of FeNO(x) coordination complexes. In this Article, we report combined UV-vis and comprehensive electron paramagnetic resonance (EPR) spectroscopic studies that address intriguing questions raised in recent studies of the structure and affinity of the nitrite ligand in complexes with Fe(III) in methemoglobin (metHb). EPR spectra of metHb/NO(2)(-) are found to exhibit a characteristic doubling in their sharper spectral features. Comparative EPR measurements at X- and S-band frequencies, and in D(2)O versus H(2)O, argue against the assignment of this splitting as hyperfine structure. Correlated changes in the EPR spectra with pH enable complete assignment of the spectrum as deriving from the overlap of two low-spin species with g values of 3.018, 2.122, 1.45 and 2.870, 2.304, 1.45 (values for samples at 20 K and pH 7.4 in phosphate-buffered saline). These g values are typical of g values found for other heme proteins with N-coordinated ligands in the binding pocket and are thus suggestive of N-nitro versus O-nitrito coordination. The positions and shapes of the spectral lines vary only slightly with temperature until motional averaging ensues at approximately 150 K. The pattern of motional averaging in the variable-temperature EPR spectra and EPR studies of Fe(III)NO(2)(-)/Fe(II)NO hybrids suggest that one of two species is present in both of the alpha and beta subunits, while the other is exclusive to the beta subunit. Our results also reconfirm that the affinity of nitrite for metHb is of millimolar magnitude, thereby making a direct role for nitrite in physiological hypoxic vasodilation difficult to justify.Item Open Access Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.(PLoS One, 2008-05-07) Yu, Zengli; Li, Ping; Zhang, Mei; Hannink, Mark; Stamler, Jonathan S; Yan, ZhenOxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.Item Open Access Pharmacologic Targeting of Red Blood Cells to Improve Tissue Oxygenation.(Clin Pharmacol Ther, 2017-12-14) Reynolds, James D; Jenkins, Trevor; Matto, Faisal; Nazemian, Ryan; Farhan, Obada; Morris, Nathan; Longphre, John M; Hess, Douglas T; Moon, Richard E; Piantadosi, Claude A; Stamler, Jonathan SDisruption of microvascular blood flow is a common cause of tissue hypoxia in disease, yet no therapies are available that directly target the microvasculature to improve tissue oxygenation. Red blood cells (RBCs) autoregulate blood flow through S-nitroso-hemoglobin (SNO-Hb)-mediated export of nitric oxide (NO) bioactivity. We therefore tested the idea that pharmacological enhancement of RBCs using the S-nitrosylating agent ethyl nitrite (ENO) may provide a novel approach to improve tissue oxygenation. Serial ENO dosing was carried out in sheep (1-400 ppm) and humans (1-100 ppm) at normoxia and at reduced fraction of inspired oxygen (FiO2 ). ENO increased RBC SNO-Hb levels, corrected hypoxia-induced deficits in tissue oxygenation, and improved measures of oxygen utilization in both species. No adverse effects or safety concerns were identified. Inasmuch as impaired oxygenation is a major cause of morbidity and mortality, ENO may have widespread therapeutic utility, providing a first-in-class agent targeting the microvasculature.Item Open Access Protein S-nitrosylation: Methods of Detection and Regulation(2009) Forrester, Michael T.Protein S-nitrosylation--the post-translational modification of cysteine thiols into 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. This doctoral thesis is focused on the 1) analysis of existing methodologies for the detection of protein S-nitrosylation; 2) development of new methodologies for the detection of protein S-nitrosylation and 3) discovery of novel enzymatic mechanisms by which S-nitrosylation is regulated in vivo. The specificity of the biotin switch technique, the mainstay assay for detecting S-nitrosylation, was rigorously assessed and validated. This study was paramount as a response to several published (though poorly grounded) criticisms of the biotin switch technique. Separately presented is a unique resin-based assay for proteomic analysis of S-nitrosylation (dubbed "SNO-RAC"), which is combined with mass spectrometric tools to identify sites of S-nitrosylation in several cellular models (e.g. E. coli, mammalian cells). Other data presented herein demonstrate that the thioredoxin system is regulated, in a negative feedback manner, to control S-nitrosylation and prevent nitrosative stress. This system involves nitric oxide-dependent suppression of an established thioredoxin inhibitor, the thioredoxin interacting protein (Txnip). This, in turn, affords thioredoxin an optimal environment to drive protein denitrosylation and prevent nitrosative stress secondary to endogenous nitric oxide production.
Item Open Access Purification and Characterization of Novel Denitrosylases from Yeast and Mammals(2012) Anand, PuneetS-nitrosylation, the prototypic mechanism of redox-based signal transduction, involves the covalent attachment of a nitrogen monoxide group to a Cys-thiol side chain. S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions and protein stability. The primary focus of my dissertation was to solve a problem of great importance in the field of S-nitrosylation, which is, to identify denitrosylase(s) i.e., enzymes that remove NO groups from S-nitrosothiols. Recent progress in elucidating the cellular regulation of S-nitrosylation has led to the identification of two physiologically relevant denitrosylating activities that remove the NO group from S-nitrosylated substrates. Thioredoxin/thioredoxin reductase (Trx system) functions as an NADPH-dependent denitrosylase across a broad range of S-nitrosylated proteins (SNO-proteins). S-nitroso-glutathione reductase (GSNOR), which is highly conserved across phylogeny, metabolizes GSNO utilizing NADH as a reducing coenzyme, thereby shifting equilibria between GSNO and SNO-proteins. This dissertation describes the discovery of two novel denitrosylases: one from yeast and the other from mammals. Using technique of column chromatography we have purified these novel denitrosylases to homogeneity and have demonstrated a principal contribution of these enzymes towards S-nitrosothiol metabolism.