Publications by authors named "Jonathan S Stamler"

175 Publications

An optimized protocol for isolation of S-nitrosylated proteins from .

STAR Protoc 2021 Jun 19;2(2):100547. Epub 2021 May 19.

Institute for Transformative Molecular Medicine and Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from , suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2021.100547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8164088PMC
June 2021

Reducing acetylated tau is neuroprotective in brain injury.

Cell 2021 May 13;184(10):2715-2732.e23. Epub 2021 Apr 13.

Department of Biomedical Informatics, College of Medicine, Ohio State University, Columbus, OH, USA.

Traumatic brain injury (TBI) is the largest non-genetic, non-aging related risk factor for Alzheimer's disease (AD). We report here that TBI induces tau acetylation (ac-tau) at sites acetylated also in human AD brain. This is mediated by S-nitrosylated-GAPDH, which simultaneously inactivates Sirtuin1 deacetylase and activates p300/CBP acetyltransferase, increasing neuronal ac-tau. Subsequent tau mislocalization causes neurodegeneration and neurobehavioral impairment, and ac-tau accumulates in the blood. Blocking GAPDH S-nitrosylation, inhibiting p300/CBP, or stimulating Sirtuin1 all protect mice from neurodegeneration, neurobehavioral impairment, and blood and brain accumulation of ac-tau after TBI. Ac-tau is thus a therapeutic target and potential blood biomarker of TBI that may represent pathologic convergence between TBI and AD. Increased ac-tau in human AD brain is further augmented in AD patients with history of TBI, and patients receiving the p300/CBP inhibitors salsalate or diflunisal exhibit decreased incidence of AD and clinically diagnosed TBI.
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http://dx.doi.org/10.1016/j.cell.2021.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8491234PMC
May 2021

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress.

EMBO Rep 2021 01 27;22(1):e50500. Epub 2020 Nov 27.

Department of Biology, Tor Vergata University, Rome, Italy.

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function.
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http://dx.doi.org/10.15252/embr.202050500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788447PMC
January 2021

Red Blood Cell-Mediated S-Nitrosohemoglobin-Dependent Vasodilation: Lessons Learned from a β-Globin Cys93 Knock-In Mouse.

Antioxid Redox Signal 2021 04 23;34(12):936-961. Epub 2020 Jul 23.

Institute for Transformative Molecular Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.

Red blood cell (RBC)-mediated vasodilation plays an important role in oxygen delivery. This occurs through hemoglobin actions, at least in significant part, to convert heme-bound nitric oxide (NO) (in tense [T]/deoxygenated-state hemoglobin) into vasodilator S-nitrosothiol (SNO) (in relaxed [R]/oxygenated-state hemoglobin), convey SNO through the bloodstream, and release it into tissues to increase blood flow. The coupling of hemoglobin R/T state allostery, both to NO conversion into SNO and to SNO release (along with oxygen), under hypoxia supports the model of a three-gas respiratory cycle (O/NO/CO). Oxygenation of tissues is dependent on a single, strictly conserved Cys residue in hemoglobin (βCys93). Hemoglobin couples SNO formation/release at βCys93 to O binding/release at hemes ("thermodynamic linkage"). Mice bearing βCys93Ala hemoglobin that is unable to generate SNO-βCys93 establish that SNO-hemoglobin is important for R/T allostery-regulated vasodilation by RBCs that couple blood flow to tissue oxygenation. The model for RBC-mediated vasodilation originally proposed by Stamler in 1996 has been largely validated: SNO-βCys93 forms , dilates blood vessels, and is hypoxia-regulated, and RBCs actuate vasodilation proportionate to hypoxia. Numerous compensations in βCys93Ala animals to alleviate tissue hypoxia (discussed herein) are predicted to preserve vasodilatory responses of RBCs but impair linkage to R/T transition in hemoglobin. This is borne out by loss of responsivity of mutant RBCs to oxygen, impaired blood flow responses to hypoxia, and tissue ischemia in βCys93-mutant animals. SNO-hemoglobin mediates hypoxic vasodilation in the respiratory cycle. This fundamental physiology promises new insights in vascular diseases and blood disorders.
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http://dx.doi.org/10.1089/ars.2020.8153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035927PMC
April 2021

Essential Role of Hemoglobin βCys93 in Cardiovascular Physiology.

Physiology (Bethesda) 2020 07;35(4):234-243

Institute for Transformative Molecular Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio.

The supply of oxygen to tissues is controlled by microcirculatory blood flow. One of the more surprising discoveries in cardiovascular physiology is the critical dependence of microcirculatory blood flow on a single conserved cysteine within the β-subunit (βCys93) of hemoglobin (Hb). βCys93 is the primary site of Hb -nitrosylation [i.e., -nitrosothiol (SNO) formation to produce -nitrosohemoglobin (SNO-Hb)]. Notably, -nitrosylation of βCys93 by NO is favored in the oxygenated conformation of Hb, and deoxygenated Hb releases SNO from βCys93. Since SNOs are vasodilatory, this mechanism provides a physiological basis for how tissue hypoxia increases microcirculatory blood flow (hypoxic autoregulation of blood flow). Mice expressing βCys93A mutant Hb (C93A) have been applied to understand the role of βCys93, and RBCs more generally, in cardiovascular physiology. Notably, C93A mice are unable to effect hypoxic autoregulation of blood flow and exhibit widespread tissue hypoxia. Moreover, reactive hyperemia (augmentation of blood flow following transient ischemia) is markedly impaired. C93A mice display multiple compensations to preserve RBC vasodilation and overcome tissue hypoxia, including shifting SNOs to other thiols on adult and fetal Hbs and elsewhere in RBCs, and growing new blood vessels. However, compensatory vasodilation in C93A mice is uncoupled from hypoxic control, both peripherally (e.g., predisposing to ischemic injury) and centrally (e.g., impairing hypoxic drive to breathe). Altogether, physiological studies utilizing C93A mice are confirming the allosterically controlled role of SNO-Hb in microvascular blood flow, uncovering essential roles for RBC-mediated vasodilation in cardiovascular physiology and revealing new roles for RBCs in cardiovascular disease.
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http://dx.doi.org/10.1152/physiol.00040.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474257PMC
July 2020

A Novel Method to Improve Perfusion of Ex Vivo Pumped Human Kidneys.

Ann Surg 2019 Dec 5. Epub 2019 Dec 5.

Institute for Transformative Molecular Medicine, School of Medicine Case Western Reserve University, Cleveland, OH.

Objective: To determine if addition of the S-nitrosylating agent ethyl nitrite (ENO) to the preservation solution can improve perfusion parameters in pumped human kidneys.

Background: A significant percentage of actively stored kidneys experience elevations in resistance and decreases in flow rate during the ex vivo storage period. Preclinical work indicates that renal status after brain death is negatively impacted by inflammation and reduced perfusion-processes regulated by protein S-nitrosylation. To translate these findings, we added ENO to the preservation solution in an attempt to reverse the perfusion deficits observed in nontransplanted pumped human kidneys.

Methods: After obtaining positive proof-of-concept results with swine kidneys, we studied donated human kidneys undergoing hypothermic pulsatile perfusion deemed unsuitable for transplantation. Control kidneys continued to be pumped a 4°C (ie, standard of care). In the experimental group, the preservation solution was aerated with 50 ppm ENO in nitrogen. Flow rate and perfusion were recorded for 10 hours followed by biochemical analysis of the kidney tissue.

Results: In controls, perfusion was constant during the monitoring period (ie, flow rate remained low and resistance stayed high). In contrast, the addition of ENO produced significant and sustained reductions in resistance and increases in flow rate. ENO-treated kidneys had higher levels of cyclic guanosine monophosphate, potentially explaining the perfusion benefits, and increased levels of interleukin-10, suggestive of an anti-inflammatory effect.

Conclusions: S-Nitrosylation therapy restored the microcirculation and thus improved overall organ perfusion. Inclusion of ENO in the renal preservation solution holds promise to increase the number and quality of kidneys available for transplant.
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http://dx.doi.org/10.1097/SLA.0000000000003702DOI Listing
December 2019

Anaerobic Transcription by OxyR: A Novel Paradigm for Nitrosative Stress.

Antioxid Redox Signal 2020 04 3;32(12):803-816. Epub 2019 Dec 3.

Institute for Transformative Molecular Medicine and Department of Medicine, Case Western Reserve University School of Medicine and University Hospitals Cleveland Medical Center, Cleveland, Ohio.

S-nitrosylation, the post-translational modification by nitric oxide (NO) to form S-nitrosothiols (SNOs), regulates diverse aspects of cellular function, and aberrant S-nitrosylation (nitrosative stress) is implicated in disease, from neurodegeneration to cancer. Essential roles for S-nitrosylation have been demonstrated in microbes, plants, and animals; notably, bacteria have often served as model systems for elucidation of general principles. Recent conceptual advances include the idea of a molecular code through which proteins sense and differentiate S-nitrosothiol (SNO) from alternative oxidative modifications, providing the basis for specificity in SNO signaling. In , S-nitrosylation relies on an enzymatic cascade that regulates, and is regulated by, the transcription factor OxyR under anaerobic conditions. S-nitrosylated OxyR activates an anaerobic regulon of >100 genes that encode for enzymes that both mediate S-nitrosylation and protect against nitrosative stress. Mitochondria originated from endosymbiotic bacteria and generate NO under hypoxic conditions, analogous to conditions in . Nitrosative stress in mitochondria has been implicated in Alzheimer's and Parkinson's disease, among others. Many proteins that are S-nitrosylated in mitochondria are also S-nitrosylated in . Insights into enzymatic regulation of S-nitrosylation in may inform the identification of disease-relevant regulatory machinery in mammalian systems. Using as a model system, in-depth analysis of the anaerobic response controlled by OxyR may lead to the identification of enzymatic mechanisms regulating S-nitrosylation in particular, and hypoxic signaling more generally, providing novel insights into analogous mechanisms in mammalian cells and within dysfunctional mitochondria that characterize neurodegenerative diseases.
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http://dx.doi.org/10.1089/ars.2019.7921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074925PMC
April 2020

Letter by Reynolds et al Regarding Article, "Hemoglobin β93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell".

Circulation 2019 11 4;140(19):e758-e759. Epub 2019 Nov 4.

Institute for Transformative Molecular Medicine (J.D.R., R.T.P., J.S.S.), Department of Medicine, University Hospitals Cleveland Medical Center, Case Western Reserve University School of Medicine, Ohio.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.041389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834231PMC
November 2019

AKR1A1 is a novel mammalian -nitroso-glutathione reductase.

J Biol Chem 2019 11 23;294(48):18285-18293. Epub 2019 Oct 23.

Institute for Transformative Molecular Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44016; Harrington Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, Ohio 44016; Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44016. Electronic address:

Oxidative modification of Cys residues by NO results in -nitrosylation, a ubiquitous post-translational modification and a primary mediator of redox-based cellular signaling. Steady-state levels of -nitrosylated proteins are largely determined by denitrosylase enzymes that couple NAD(P)H oxidation with reduction of -nitrosothiols, including protein and low-molecular-weight (LMW) -nitrosothiols (-nitroso-GSH (GSNO) and -nitroso-CoA (SNO-CoA)). SNO-CoA reductases require NADPH, whereas enzymatic reduction of GSNO can involve either NADH or NADPH. Notably, GSNO reductase (GSNOR, ) accounts for most NADH-dependent GSNOR activity, whereas NADPH-dependent GSNOR activity is largely unaccounted for (CBR1 mediates a minor portion). Here, we purified NADPH-coupled GSNOR activity from mammalian tissues and identified aldo-keto reductase family 1 member A1 (AKR1A1), the archetypal mammalian SNO-CoA reductase, as a primary mediator of NADPH-coupled GSNOR activity in these tissues. Kinetic analyses suggested an AKR1A1 substrate preference of SNO-CoA > GSNO. AKR1A1 deletion from murine tissues dramatically lowered NADPH-dependent GSNOR activity. Conversely, GSNOR-deficient mice had increased AKR1A1 activity, revealing potential cross-talk among GSNO-dependent denitrosylases. Molecular modeling and mutagenesis of AKR1A1 identified Arg-312 as a key residue mediating the specific interaction with GSNO; in contrast, substitution of the SNO-CoA-binding residue Lys-127 minimally affected the GSNO-reducing activity of AKR1A1. Together, these findings indicate that AKR1A1 is a multi-LMW-SNO reductase that can distinguish between and metabolize the two major LMW-SNO signaling molecules GSNO and SNO-CoA, allowing for wide-ranging control of protein -nitrosylation under both physiological and pathological conditions.
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http://dx.doi.org/10.1074/jbc.RA119.011067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885624PMC
November 2019

Role of Nitric Oxide Carried by Hemoglobin in Cardiovascular Physiology: Developments on a Three-Gas Respiratory Cycle.

Circ Res 2020 01 8;126(1):129-158. Epub 2019 Oct 8.

From the Institute for Transformative Molecular Medicine (R.T.P., J.D.R., R.Z., J.S.S.), Case Western Reserve University School of Medicine, OH.

A continuous supply of oxygen is essential for the survival of multicellular organisms. The understanding of how this supply is regulated in the microvasculature has evolved from viewing erythrocytes (red blood cells [RBCs]) as passive carriers of oxygen to recognizing the complex interplay between Hb (hemoglobin) and oxygen, carbon dioxide, and nitric oxide-the three-gas respiratory cycle-that insures adequate oxygen and nutrient delivery to meet local metabolic demand. In this context, it is blood flow and not blood oxygen content that is the main driver of tissue oxygenation by RBCs. Herein, we review the lines of experimentation that led to this understanding of RBC function; from the foundational understanding of allosteric regulation of oxygen binding in Hb in the stereochemical model of Perutz, to blood flow autoregulation (hypoxic vasodilation governing oxygen delivery) observed by Guyton, to current understanding that centers on S-nitrosylation of Hb (ie, S-nitrosohemoglobin; SNO-Hb) as a purveyor of oxygen-dependent vasodilatory activity. Notably, hypoxic vasodilation is recapitulated by native S-nitrosothiol (SNO)-replete RBCs and by SNO-Hb itself, whereby SNO is released from Hb and RBCs during deoxygenation, in proportion to the degree of Hb deoxygenation, to regulate vessels directly. In addition, we discuss how dysregulation of this system through genetic mutation in Hb or through disease is a common factor in oxygenation pathologies resulting from microcirculatory impairment, including sickle cell disease, ischemic heart disease, and heart failure. We then conclude by identifying potential therapeutic interventions to correct deficits in RBC-mediated vasodilation to improve oxygen delivery-steps toward effective microvasculature-targeted therapies. To the extent that diseases of the heart, lungs, and blood are associated with impaired tissue oxygenation, the development of new therapies based on the three-gas respiratory system have the potential to improve the well-being of millions of patients.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034631PMC
January 2020

Author Correction: Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury.

Nature 2019 Jun;570(7759):E23

Institute for Transformative Molecular Medicine, Department of Medicine, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, OH, USA.

Change history: In Fig. 1j of this Letter, one data point was inadvertently omitted from the graph for the acute kidney injury (AKI), double knockout (-/-), S-nitrosothiol (SNO) condition at a nitrosylation level of 25.9 pmol mg and the statistical significance given of P = 0.0221 was determined by Fisher's test instead of P = 0.0032 determined by Tukey's test (with normalization for test-day instrument baseline). Figure 1 and its Source Data have been corrected online.
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http://dx.doi.org/10.1038/s41586-019-1225-0DOI Listing
June 2019

Regulation of MicroRNA Machinery and Development by Interspecies S-Nitrosylation.

Cell 2019 02;176(5):1014-1025.e12

Institute for Transformative Molecular Medicine and Department of Medicine, Case Western Reserve University School of Medicine and University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA; Harrington Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA. Electronic address:

Bioactive molecules can pass between microbiota and host to influence host cellular functions. However, general principles of interspecies communication have not been discovered. We show here in C. elegans that nitric oxide derived from resident bacteria promotes widespread S-nitrosylation of the host proteome. We further show that microbiota-dependent S-nitrosylation of C. elegans Argonaute protein (ALG-1)-at a site conserved and S-nitrosylated in mammalian Argonaute 2 (AGO2)-alters its function in controlling gene expression via microRNAs. By selectively eliminating nitric oxide generation by the microbiota or S-nitrosylation in ALG-1, we reveal unforeseen effects on host development. Thus, the microbiota can shape the post-translational landscape of the host proteome to regulate microRNA activity, gene expression, and host development. Our findings suggest a general mechanism by which the microbiota may control host cellular functions, as well as a new role for gasotransmitters.
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http://dx.doi.org/10.1016/j.cell.2019.01.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559381PMC
February 2019

Improvement in Outcomes After Cardiac Arrest and Resuscitation by Inhibition of S-Nitrosoglutathione Reductase.

Circulation 2019 02;139(6):815-827

Anesthesia Center for Critical Care Research, Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Harvard Medical School (K.H., A.B., Y.M., S.H., E.R., E.M., N.M., A.M., L.B., A.G.H., F.I.), Boston, MA.

Background: The biological effects of nitric oxide are mediated via protein S-nitrosylation. Levels of S-nitrosylated protein are controlled in part by the denitrosylase, S-nitrosoglutathione reductase (GSNOR). The objective of this study was to examine whether GSNOR inhibition improves outcomes after cardiac arrest and cardiopulmonary resuscitation (CA/CPR).

Methods: Adult wild-type C57BL/6 and GSNOR-deleted (GSNOR) mice were subjected to potassium chloride-induced CA and subsequently resuscitated. Fifteen minutes after a return of spontaneous circulation, wild-type mice were randomized to receive the GSNOR inhibitor, SPL-334.1, or normal saline as placebo. Mortality, neurological outcome, GSNOR activity, and levels of S-nitrosylated proteins were evaluated. Plasma GSNOR activity was measured in plasma samples obtained from post-CA patients, preoperative cardiac surgery patients, and healthy volunteers.

Results: GSNOR activity was increased in plasma and multiple organs of mice, including brain in particular. Levels of protein S-nitrosylation were decreased in the brain 6 hours after CA/CPR. Administration of SPL-334.1 attenuated the increase in GSNOR activity in brain, heart, liver, spleen, and plasma, and restored S-nitrosylated protein levels in the brain. Inhibition of GSNOR attenuated ischemic brain injury and improved survival in wild-type mice after CA/CPR (81.8% in SPL-334.1 versus 36.4% in placebo; log rank P=0.031). Similarly, GSNOR deletion prevented the reduction in the number of S-nitrosylated proteins in the brain, mitigated brain injury, and improved neurological recovery and survival after CA/CPR. Both GSNOR inhibition and deletion attenuated CA/CPR-induced disruption of blood brain barrier. Post-CA patients had higher plasma GSNOR activity than did preoperative cardiac surgery patients or healthy volunteers ( P<0.0001). Plasma GSNOR activity was positively correlated with initial lactate levels in postarrest patients (Spearman correlation coefficient=0.48; P=0.045).

Conclusions: CA and CPR activated GSNOR and reduced the number of S-nitrosylated proteins in the brain. Pharmacological inhibition or genetic deletion of GSNOR prevented ischemic brain injury and improved survival rates by restoring S-nitrosylated protein levels in the brain after CA/CPR in mice. Our observations suggest that GSNOR is a novel biomarker of postarrest brain injury as well as a molecular target to improve outcomes after CA.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.032488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361681PMC
February 2019

Molecular recognition of -nitrosothiol substrate by its cognate protein denitrosylase.

J Biol Chem 2019 02 11;294(5):1568-1578. Epub 2018 Dec 11.

Institute for Transformative Molecular Medicine, Case Western Reserve University, Cleveland, Ohio 44106; Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; Harrington Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, Ohio 44106. Electronic address:

Protein -nitrosylation mediates a large part of nitric oxide's influence on cellular function by providing a fundamental mechanism to control protein function across different species and cell types. At steady state, cellular -nitrosylation reflects dynamic equilibria between -nitrosothiols (SNOs) in proteins and small molecules (low-molecular-weight SNOs) whose levels are regulated by dedicated -nitrosylases and denitrosylases. -Nitroso-CoA (SNO-CoA) and its cognate denitrosylases, SNO-CoA reductases (SCoRs), are newly identified determinants of protein -nitrosylation in both yeast and mammals. Because SNO-CoA is a minority species among potentially thousands of cellular SNOs, SCoRs must preferentially recognize this SNO substrate. However, little is known about the molecular mechanism by which cellular SNOs are recognized by their cognate enzymes. Using mammalian cells, molecular modeling, substrate-capture assays, and mutagenic analyses, we identified a single conserved surface Lys (Lys-127) residue as well as active-site interactions of the SNO group that mediate recognition of SNO-CoA by SCoR. Comparing SCoR SCoR HEK293 cells, we identified a SNO-CoA-dependent nitrosoproteome, including numerous metabolic protein substrates. Finally, we discovered that the SNO-CoA/SCoR system has a role in mitochondrial metabolism. Collectively, our findings provide molecular insights into the basis of specificity in SNO-CoA-mediated metabolic signaling and suggest a role for SCoR-regulated -nitrosylation in multiple metabolic processes.
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http://dx.doi.org/10.1074/jbc.RA118.004947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364788PMC
February 2019

Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury.

Nature 2019 01 28;565(7737):96-100. Epub 2018 Nov 28.

Institute for Transformative Molecular Medicine, Department of Medicine, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, OH, USA.

Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood. Nitric oxide-based cellular signalling is generally mediated by protein S-nitrosylation, the oxidative modification of Cys residues to form S-nitrosothiols (SNOs). S-nitrosylation regulates proteins in all functional classes, and is controlled by enzymatic machinery that includes S-nitrosylases and denitrosylases, which add and remove SNO from proteins, respectively. In Saccharomyces cerevisiae, the classic metabolic intermediate co-enzyme A (CoA) serves as an endogenous source of SNOs through its conjugation with nitric oxide to form S-nitroso-CoA (SNO-CoA), and S-nitrosylation of proteins by SNO-CoA is governed by its cognate denitrosylase, SNO-CoA reductase (SCoR). Mammals possess a functional homologue of yeast SCoR, an aldo-keto reductase family member (AKR1A1) with an unknown physiological role. Here we report that the SNO-CoA-AKR1A1 system is highly expressed in renal proximal tubules, where it transduces the activity of eNOS in reprogramming intermediary metabolism, thereby protecting kidneys against acute kidney injury. Specifically, deletion of Akr1a1 in mice to reduce SCoR activity increased protein S-nitrosylation, protected against acute kidney injury and improved survival, whereas this protection was lost when Enos (also known as Nos3) was also deleted. Metabolic profiling coupled with unbiased mass spectrometry-based SNO-protein identification revealed that protection by the SNO-CoA-SCoR system is mediated by inhibitory S-nitrosylation of pyruvate kinase M2 (PKM2) through a novel locus of regulation, thereby balancing fuel utilization (through glycolysis) with redox protection (through the pentose phosphate shunt). Targeted deletion of PKM2 from mouse proximal tubules recapitulated precisely the protective and mechanistic effects of S-nitrosylation in Akr1a1 mice, whereas Cys-mutant PKM2, which is refractory to S-nitrosylation, negated SNO-CoA bioactivity. Our results identify a physiological function of the SNO-CoA-SCoR system in mammals, describe new regulation of renal metabolism and of PKM2 in differentiated tissues, and offer a novel perspective on kidney injury with therapeutic implications.
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http://dx.doi.org/10.1038/s41586-018-0749-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318002PMC
January 2019

Cross Talk Between -Nitrosylation and Phosphorylation Involving Kinases and Nitrosylases.

Circ Res 2018 05;122(11):1485-1487

From the Department of Medicine, Institute for Transformative Molecular Medicine, University Hospitals Cleveland Medical Center (H.-L.Z., C.T.S., J.S.S.)

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http://dx.doi.org/10.1161/CIRCRESAHA.118.313109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978766PMC
May 2018

S-Nitrosylation of β-Arrestins Biases Receptor Signaling and Confers Ligand Independence.

Mol Cell 2018 05;70(3):473-487.e6

Institute for Transformative Molecular Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA; Harrington Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA. Electronic address:

Most G protein-coupled receptors (GPCRs) signal through both heterotrimeric G proteins and β-arrestins (βarr1 and βarr2). Although synthetic ligands can elicit biased signaling by G protein- vis-à-vis βarr-mediated transduction, endogenous mechanisms for biasing signaling remain elusive. Here we report that S-nitrosylation of a novel site within βarr1/2 provides a general mechanism to bias ligand-induced signaling through GPCRs by selectively inhibiting βarr-mediated transduction. Concomitantly, S-nitrosylation endows cytosolic βarrs with receptor-independent function. Enhanced βarr S-nitrosylation characterizes inflammation and aging as well as human and murine heart failure. In genetically engineered mice lacking βarr2-Cys253 S-nitrosylation, heart failure is exacerbated in association with greatly compromised β-adrenergic chronotropy and inotropy, reflecting βarr-biased transduction and β-adrenergic receptor downregulation. Thus, S-nitrosylation regulates βarr function and, thereby, biases transduction through GPCRs, demonstrating a novel role for nitric oxide in cellular signaling with potentially broad implications for patho/physiological GPCR function, including a previously unrecognized role in heart failure.
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http://dx.doi.org/10.1016/j.molcel.2018.03.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940012PMC
May 2018

Distinct roles of resident and nonresident macrophages in nonischemic cardiomyopathy.

Proc Natl Acad Sci U S A 2018 05 30;115(20):E4661-E4669. Epub 2018 Apr 30.

Case Cardiovascular Research Institute, Case Western Reserve University School of Medicine, Cleveland, OH 44106;

Nonischemic cardiomyopathy (NICM) resulting from long-standing hypertension, valvular disease, and genetic mutations is a major cause of heart failure worldwide. Recent observations suggest that myeloid cells can impact cardiac function, but the role of tissue-intrinsic vs. tissue-extrinsic myeloid cells in NICM remains poorly understood. Here, we show that cardiac resident macrophage proliferation occurs within the first week following pressure overload hypertrophy (POH; a model of heart failure) and is requisite for the heart's adaptive response. Mechanistically, we identify Kruppel-like factor 4 (KLF4) as a key transcription factor that regulates cardiac resident macrophage proliferation and angiogenic activities. Finally, we show that blood-borne macrophages recruited in late-phase POH are detrimental, and that blockade of their infiltration improves myocardial angiogenesis and preserves cardiac function. These observations demonstrate previously unappreciated temporal and spatial roles for resident and nonresident macrophages in the development of heart failure.
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http://dx.doi.org/10.1073/pnas.1720065115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960298PMC
May 2018

-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy.

Proc Natl Acad Sci U S A 2018 04 26;115(15):E3388-E3397. Epub 2018 Mar 26.

Danish Cancer Society Research Center, Center for Autophagy, Recycling and Disease, 2100 Copenhagen, Denmark;

-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. -nitrosoglutathione reductase (GSNOR) regulates protein -nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive -nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein -nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.
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http://dx.doi.org/10.1073/pnas.1722452115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899480PMC
April 2018

A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

Mol Cell 2018 02 18;69(3):451-464.e6. Epub 2018 Jan 18.

Institute for Transformative Molecular Medicine and Department of Medicine, Case Western Reserve University School of Medicine and University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA; Harrington Discovery Institute, University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA. Electronic address:

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling.
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http://dx.doi.org/10.1016/j.molcel.2017.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999318PMC
February 2018

Pharmacologic Targeting of Red Blood Cells to Improve Tissue Oxygenation.

Clin Pharmacol Ther 2018 09 17;104(3):553-563. Epub 2018 Jan 17.

Institute for Transformative Molecular Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.

Disruption 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 (FiO ). 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.
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http://dx.doi.org/10.1002/cpt.979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590078PMC
September 2018

S-Nitrosohemoglobin Levels and Patient Outcome After Transfusion During Pediatric Bypass Surgery.

Clin Transl Sci 2018 03 12;11(2):237-243. Epub 2017 Dec 12.

Institute for Transformative Molecular Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.

Banked blood exhibits impairments in nitric oxide (NO)-based oxygen delivery capability, reflected in rapid depletion of S-nitrosohemoglobin (SNO-Hb). We hypothesized that transfusion of even freshly-stored blood used in pediatric heart surgery would reduce SNO-Hb levels and worsen outcome. In a retrospective review (n = 29), the percent of estimated blood volume (% eBV) replaced by transfusion directly correlated with ventilator time and inversely correlated with kidney function; similar results were obtained in a prospective arm (n = 20). In addition, an inverse association was identified between SNO-Hb and postoperative increase in Hb (∆Hb), reflecting the amount of blood retained by the patient. Both SNO-Hb and ∆Hb correlated with the probability of kidney dysfunction and oxygenation-related complications. Further, regression analysis identified SNO-Hb as an inverse predictor of outcome. The findings suggest that SNO-Hb and ∆Hb are prognostic biomarkers following pediatric cardiopulmonary bypass, and that maintenance of red blood cell-derived NO bioactivity might confer therapeutic benefit.
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http://dx.doi.org/10.1111/cts.12530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867013PMC
March 2018

Endothelial cell-surface tissue transglutaminase inhibits neutrophil adhesion by binding and releasing nitric oxide.

Sci Rep 2017 11 23;7(1):16163. Epub 2017 Nov 23.

Hematology/Oncology, Medical University of South Carolina, Charleston, SC, United States.

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface.
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http://dx.doi.org/10.1038/s41598-017-16342-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701052PMC
November 2017

Protein S-Nitrosylation: Determinants of Specificity and Enzymatic Regulation of S-Nitrosothiol-Based Signaling.

Antioxid Redox Signal 2019 04 10;30(10):1331-1351. Epub 2018 Jan 10.

2 Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio.

Significance: Protein S-nitrosylation, the oxidative modification of cysteine by nitric oxide (NO) to form protein S-nitrosothiols (SNOs), mediates redox-based signaling that conveys, in large part, the ubiquitous influence of NO on cellular function. S-nitrosylation regulates protein activity, stability, localization, and protein-protein interactions across myriad physiological processes, and aberrant S-nitrosylation is associated with diverse pathophysiologies. Recent Advances: It is recently recognized that S-nitrosylation endows S-nitroso-protein (SNO-proteins) with S-nitrosylase activity, that is, the potential to trans-S-nitrosylate additional proteins, thereby propagating SNO-based signals, analogous to kinase-mediated signaling cascades. In addition, it is increasingly appreciated that cellular S-nitrosylation is governed by dynamically coupled equilibria between SNO-proteins and low-molecular-weight SNOs, which are controlled by a growing set of enzymatic denitrosylases comprising two main classes (high and low molecular weight). S-nitrosylases and denitrosylases, which together control steady-state SNO levels, may be identified with distinct physiology and pathophysiology ranging from cardiovascular and respiratory disorders to neurodegeneration and cancer.

Critical Issues: The target specificity of protein S-nitrosylation and the stability and reactivity of protein SNOs are determined substantially by enzymatic machinery comprising highly conserved transnitrosylases and denitrosylases. Understanding the differential functionality of SNO-regulatory enzymes is essential, and is amenable to genetic and pharmacological analyses, read out as perturbation of specific equilibria within the SNO circuitry.

Future Directions: The emerging picture of NO biology entails equilibria among potentially thousands of different SNOs, governed by denitrosylases and nitrosylases. Thus, to elucidate the operation and consequences of S-nitrosylation in cellular contexts, studies should consider the roles of SNO-proteins as both targets and transducers of S-nitrosylation, functioning according to enzymatically governed equilibria.
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http://dx.doi.org/10.1089/ars.2017.7403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391618PMC
April 2019

-Nitrosoglutathione Reductase Dysfunction Contributes to Obesity-Associated Hepatic Insulin Resistance via Regulating Autophagy.

Diabetes 2018 02 26;67(2):193-207. Epub 2017 Oct 26.

Department of Anatomy and Cell Biology, Fraternal Order of Eagles Diabetes Research Center, The Pappajohn Biomedical Institute, Carver College of Medicine, University of Iowa, Iowa City, IA

Obesity is associated with elevated intracellular nitric oxide (NO) production, which promotes nitrosative stress in metabolic tissues such as liver and skeletal muscle, contributing to insulin resistance. The onset of obesity-associated insulin resistance is due, in part, to the compromise of hepatic autophagy, a process that leads to lysosomal degradation of cellular components. However, it is not known how NO bioactivity might impact autophagy in obesity. Here, we establish that -nitrosoglutathione reductase (GSNOR), a major protein denitrosylase, provides a key regulatory link between inflammation and autophagy, which is disrupted in obesity and diabetes. We demonstrate that obesity promotes -nitrosylation of lysosomal proteins in the liver, thereby impairing lysosomal enzyme activities. Moreover, in mice and humans, obesity and diabetes are accompanied by decreases in GSNOR activity, engendering nitrosative stress. In mice with a GSNOR deletion, diet-induced obesity increases lysosomal nitrosative stress and impairs autophagy in the liver, leading to hepatic insulin resistance. Conversely, liver-specific overexpression of GSNOR in obese mice markedly enhances lysosomal function and autophagy and, remarkably, improves insulin action and glucose homeostasis. Furthermore, overexpression of -nitrosylation-resistant variants of lysosomal enzymes enhances autophagy, and pharmacologically and genetically enhancing autophagy improves hepatic insulin sensitivity in GSNOR-deficient hepatocytes. Taken together, our data indicate that obesity-induced protein -nitrosylation is a key mechanism compromising the hepatic autophagy, contributing to hepatic insulin resistance.
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http://dx.doi.org/10.2337/db17-0223DOI Listing
February 2018

-Nitrosoglutathione Reductase Deficiency Confers Improved Survival and Neurological Outcome in Experimental Cerebral Malaria.

Infect Immun 2017 09 18;85(9). Epub 2017 Aug 18.

Sandra Rotman Centre for Global Health, University Health Network, University of Toronto, Toronto, ON, Canada

Artesunate remains the mainstay of treatment for cerebral malaria, but it is less effective in later stages of disease when the host inflammatory response and blood-brain barrier integrity dictate clinical outcomes. Nitric oxide (NO) is an important regulator of inflammation and microvascular integrity, and impaired NO bioactivity is associated with fatal outcomes in malaria. Endogenous NO bioactivity in mammals is largely mediated by -nitrosothiols (SNOs). Based on these observations, we hypothesized that animals deficient in the SNO-metabolizing enzyme, -nitrosoglutathione reductase (GSNOR), which exhibit enhanced -nitrosylation, would have improved outcomes in a preclinical model of cerebral malaria. GSNOR knockout (KO) mice infected with ANKA had significantly delayed mortality compared to WT animals ( < 0.0001), despite higher parasite burdens ( < 0.01), and displayed markedly enhanced survival versus the wild type (WT) when treated with the antimalarial drug artesunate (77% versus 38%; < 0.001). Improved survival was associated with higher levels of protein-bound NO, decreased levels of CD4 and CD8 T cells in the brain, improved blood-brain barrier integrity, and improved coma scores, as well as higher levels of gamma interferon. GSNOR KO animals receiving WT bone marrow had significantly reduced survival following ANKA infection compared to those receiving KO bone barrow ( < 0.001). Reciprocal transplants established that survival benefits of GSNOR deletion were attributable primarily to the T cell compartment. These data indicate a role for GSNOR in the host response to malaria infection and suggest that strategies to disrupt its activity will improve clinical outcomes by enhancing microvascular integrity and modulating T cell tissue tropism.
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http://dx.doi.org/10.1128/IAI.00371-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563579PMC
September 2017

Letter by Stamler et al Regarding Article, "Nitrite and -Nitrosohemoglobin Exchange Across the Human Cerebral and Femoral Circulation: Relationship to Basal and Exercise Blood Flow Responses to Hypoxia".

Circulation 2017 06;135(24):e1135-e1136

From Institute for Transformative Molecular Medicine, Case Western Reserve University School of Medicine and University Hospitals Cleveland Medical Center, OH (J.S.S., J.D.R., D.T.H.); Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (J.S.S., D.T.H.); Harrington Discovery Institute, University Hospitals Cleveland Medical Center, OH (J.S.S., J.D.R.); Department of Anesthesiology and Perioperative Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (J.D.R.).

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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.027071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5523964PMC
June 2017

The LargPAD Trial: Phase IIA evaluation of l-arginine infusion in patients with peripheral arterial disease.

J Vasc Surg 2017 07 30;66(1):187-194. Epub 2017 Mar 30.

Institute for Transformative Molecular Medicine, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio.

Objective: Endothelial function is improved by l-arginine (l-arg) supplementation in preclinical and clinical studies of mildly diseased vasculature; however, endothelial function and responsiveness to l-arg in severely diseased arteries is not known. Our objective was to evaluate the acute effects of catheter-directed l-arg delivery in patients with chronic lower extremity ischemia secondary to peripheral arterial disease.

Methods: The study enrolled 22 patients (45% male) with peripheral arterial disease (mean age, 62 years) requiring lower extremity angiography. Endothelium-dependent relaxation of patent but atherosclerotic superficial femoral arteries was measured using a combination of intravascular ultrasound (IVUS) imaging and a Doppler FloWire (Volcano Corporation, Rancho Cordova, Calif) during the infusion of incremental acetylcholine (10 to 10 molar concentration) doses. Patients received 50 mg (n = 3), 100 mg (n = 10), or 500 mg (n = 9) l-arg intra-arterially, followed by repeat endothelium-dependent relaxation measurement (limb volumetric flow). IVUS-derived virtual histology of the culprit vessel was also obtained. Endothelium-independent relaxation was measured using a nitroglycerin infusion. Levels of nitrogen oxides and arginine metabolites were measured by chemiluminescence and mass spectrometry, respectively.

Results: Patients tolerated limb l-arg infusion well. Serum arginine and ornithine levels increased by 43.6% ± 13.0% and 23.2% ± 10.3%, respectively (P < .005), and serum nitrogen oxides increased by 85% (P < .0001) after l-arg infusion. Average vessel area increased by 6.8% ± 1.3% with l-arg infusion (acetylcholine 10; P < .0001). Limb volumetric flow increased in all patients and was greater with l-arg supplementation by 130.9 ± 17.6, 136.9 ± 18.6, and 172.1 ± 24.8 mL/min, respectively, for each cohort. Maximal effects were seen with l-arg at 100 mg (32.8%). Arterial smooth muscle responsiveness to nitroglycerin was intact in all vessels (endothelium-independent relaxation, 137% ± 28% volume flow increase). IVUS-derived virtual histology indicated plaque volume was 14 ± 1.3 mm/cm, and plaque stratification revealed a predominantly fibrous morphology (46.4%; necrotic core, 28.4%; calcium, 17.4%; fibrolipid, 6.6%). Plaque morphology did not correlate with l-arg responsiveness.

Conclusions: Despite extensive atherosclerosis, endothelial function in diseased lower extremity human arteries can be enhanced by l-arg infusion secondary to increased nitric oxide bioactivity. Further studies of l-arg as a therapeutic modality in patients with endothelial dysfunction (ie, acute limb ischemia) are warranted.
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http://dx.doi.org/10.1016/j.jvs.2016.12.127DOI Listing
July 2017

Hemoglobin S-nitrosylation plays an essential role in cardioprotection.

J Clin Invest 2016 12 14;126(12):4654-4658. Epub 2016 Nov 14.

Homeostatic control of tissue oxygenation is achieved largely through changes in blood flow that are regulated by the classic physiological response of hypoxic vasodilation. The role of nitric oxide (NO) in the control of blood flow is a central tenet of cardiovascular biology. However, extensive evidence now indicates that hypoxic vasodilation entails S-nitrosothiol-based (SNO-based) vasoactivity (rather than NO per se) and that this activity is conveyed substantially by the βCys93 residue in hemoglobin. Thus, tissue oxygenation in the respiratory cycle is dependent on S-nitrosohemoglobin. This perspective predicts that red blood cells (RBCs) may play an important but previously undescribed role in cardioprotection. Here, we have found that cardiac injury and mortality in models of myocardial infarction and heart failure were greatly enhanced in mice lacking βCys93 S-nitrosylation. In addition, βCys93 mutant mice exhibited adaptive collateralization of cardiac vasculature that mitigated ischemic injury and predicted outcomes after myocardial infarction. Enhanced myopathic injury and mortality across different etiologies in the absence of βCys93 confirm the central cardiovascular role of RBC-derived SNO-based vasoactivity and point to a potential locus of therapeutic intervention. Our findings also suggest the possibility that RBCs may play a previously unappreciated role in heart disease.
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http://dx.doi.org/10.1172/JCI90425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127672PMC
December 2016

Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1.

PLoS One 2016;11(9):e0163359. Epub 2016 Sep 22.

Cell Biology Program, Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, 44106, United States of America.

Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033456PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163359PLOS
September 2016
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