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Treatment with nitroxyl (HNO) improves cardiac function in failing hearts by inducing release of SR Ca2+-ATPase from phospholamban (PLN), thereby enhancing Ca2+ reuptake. Keceli et al. use 15N-edited NMR spectroscopy to show that HNO achieves this by reversibly modifying PLN cysteines 41 and 46., Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca2+ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca2+ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca2+-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH2) that are less easily reversed by reductants, remain to be determined. Using an 15N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, 15N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

Objectives: This review summarizes the spectroscopic results, which will provide useful suggestions for future research. In addition, the fields that urgently need more information are also advised. Background: Nitrite-NO-cGMP has been considered as an important signaling pathway of NO in human cells. To date, all the four known human molybdenum-containing enzymes, xanthine oxidase, aldehyde oxidase, sulfite oxidase, and mitochondrial amidoxime-reducing component, have been shown to function as nitrite reductases under hypoxia by biochemical, cellular, or animal studies. Various spectroscopic techniques have been applied to investigate the structure and catalytic mechanism of these enzymes for more than 20 years. Methods: We summarize the published data on the applications of UV-vis and EPR spectroscopies, and X-ray crystallography in studying nitrite reductase activity of the four human molybdenum-containing enzymes. Results: UV-vis has provided useful information on the redox active centers of these enzymes. The utilization of EPR spectroscopy has been critical in determining the coordination and redox status of the Mo center during catalysis. Despite the lack of substrate-bound crystal structures of these nitrite reductases, valuable structural information has been obtained by X-ray crystallography. Conclusions: To fully understand the catalytic mechanisms of these physiologically/pathologically important nitrite reductases, structural studies on substrate-redox center interaction are needed.

Objective— KLF15 (Kruppel-like factor 15) has recently been shown to suppress activation of proinflammatory processes that contribute to atherogenesis in vascular smooth muscle, however, the role of KLF15 in vascular endothelial function is unknown. Arginase mediates inflammatory vasculopathy and vascular injury in pulmonary hypertension. Here, we tested the hypothesis that KLF15 is a critical regulator of hypoxia-induced Arg2 (arginase 2) transcription in human pulmonary microvascular endothelial cells (HPMEC). Approach and Results— Quiescent HPMEC express ample amounts of full-length KLF15. HPMECs exposed to 24 hours of hypoxia exhibited a marked decrease in KLF15 protein levels and a reciprocal increase in Arg2 protein and mRNA. Chromatin immunoprecipitation indicated direct binding of KLF15 to the Arg2 promoter, which was relieved with HPMEC exposure to hypoxia. Furthermore, overexpression of KLF15 in HPMEC reversed hypoxia-induced augmentation of Arg2 abundance and arginase activity and rescued nitric oxide (NO) production. Ectopic KLF15 also reversed hypoxia-induced endothelium-mediated vasodilatation in isolated rat pulmonary artery rings. Mechanisms by which hypoxia regulates KLF15 abundance, stability, and compartmentalization to the nucleus in HPMEC were then investigated. Hypoxia triggered deSUMOylation of KLF15 by SENP1 (sentrin-specific protease 1), and translocation of KLF15 from nucleus to cytoplasm. Conclusions— KLF15 is a critical regulator of pulmonary endothelial homeostasis via repression of endothelial Arg2 expression. KLF15 abundance and nuclear compartmentalization are regulated by SUMOylation/deSUMOylation—a hypoxia-sensitive process that is controlled by SENP1. Strategies including overexpression of KLF15 or inhibition of SENP1 may represent novel therapeutic targets for pulmonary hypertension.

The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.

Reactions based on the transfer of one or more electrons from a donor (reductant) to an acceptor (oxidant) are at the basis of several physiologically relevant cellular processes. Both reactive oxygen and nitrogen species (ROS and RNS, respectively) can signal through these reduction/oxidation (redox) reactions, particularly via reversible interaction with highly reactive thiols. Nitroxyl (HNO), the one-electron reduction product of nitric oxide (NO•), is an RNS that in the last few lusters has garnered a lot of attention owing to its pharmacological properties that are quite dissimilar from those exhibited by its sibling NO• or other RNS such as nitrite/nitrate. HNO uniqueness becomes particularly evident in the cardiovascular system. One aim of this chapter is to put the following three fundamental chemical properties of HNO in a cardiovascular physiology and therapeutic perspective: (1) HNO elective, and likely selective thiophilic nature; (2) inertness toward ROS; and (3) modest reactivity with molecular O 2 . With this conceptual framework in mind, here we will first review routes accounting for possible HNO endogenous formation as they may be relevant to govern basal and stress-stimulated cardiac and vascular function. Then, we will provide an updated account of the pharmacological properties of HNO donors in the heart and in vessels, both in vivo and in vitro, under normal and disease conditions, flanking this evidence with additional HNO pharmacological properties that may nicely complement its main cardiovascular actions. We will conclude discussing the perspective of HNO donors as a treatment for heart failure, more specifically for acute decompensated heart failure (ADHF), comparing HNO therapeutic portfolio to current mainstay ADHF therapies.

Recent discoveries of important pharmacological properties have drawn attention to the reactivity of HNO (azanone, nitroxyl) with biologically relevant substrates. Apart from its role in thiol oxidation, HNO has been reported to have nitrosative properties, for example, with tryptophan resulting in N-nitrosotryptophan formation. We have investigated the reactivity of HNO with tryptophan and small peptides containing either tryptophan or both a tryptophan and a cysteine residue. Our results point to the more reactive nature of cysteine towards HNO compared with tryptophan.

Congestive heart failure frequently involves reduced cardiac contractility and slowed muscle relaxation. The reactive nitrogen species HNO (nitroxyl, azanone) has been shown to augment contractility and accelerate relaxation in both normal and failing hearts. In humans, greater than 70% of cytosolic Ca2+ is pumped into the sarco(endo)plasmic reticulum (SR) by SR Ca2+-ATPase (SERCA2a). Phospholamban (PLN) is a small transmembrane protein, which is associated with SERCA2a, and depending on its phosphorylation state, regulates the activity of the Ca2+ pump. Our previous work has shown that HNO modifies PLN and affects SERCA2a activity independent of the β-adrenergic pathway. We have applied our 15N-edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO-derived modifications on PLN. These studies demonstrate that PLN cysteines 46 and 41 are the major sites of HNO reactivity. Furthermore, our results indicate that sulfinamide formation is the major HNO-derived modification in the presence of excess HNO donor, whereas disulfide formation is expected to dominate under physiologically relevant conditions.

Sulfinamide [RS(O)NH(2)] formation is known to occur upon exposure of cysteine residues to nitroxyl (HNO), which has received recent attention as a potential heart failure therapeutic. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including a small organic molecule, peptides, and a protein. Although it has generally been assumed that this thiol to sulfinamide modification is irreversible, we show that sulfinamides can be reduced back to the free thiol in the presence of excess thiol at physiological pH and temperature. We have examined this sulfinamide reduction both in peptides, where a cyclic intermediate analogous to that proposed for asparagine deamidation reactions potentially can contribute, and in a small organic molecule, where the mechanism is restricted to a direct thiolysis. These studies suggest that the contribution from the cyclic intermediate becomes more important in environments with lower dielectric constants. In addition, although sulfinic acid [RS(O)OH] formation is observed upon prolonged incubations in water, reduction of sulfinamides is found to dominate in the presence of thiols. Finally, studies with the cysteine protease, papain, suggest that the reduction of sulfinamide to the free thiol is viable in a protein environment.

Heart failure (HF) is characterized by perturbed myocyte Ca2+ handling. Altered SR Ca2+-ATPase (SERCA2a) activity sizably contributes to impair Ca2+ cycling in the heart, affecting its contraction/relaxation. Strategies to rescue or improve SERCA2a expression/activity are being clinically tested in HF patients. Donors of nitroxyl (HNO) augment cardiac muscle performance in failing hearts, by modulating phospholamban (PLN) and SERCA2a interaction. They do so independently from β-adrenergic activation. In fact, HNO increases SERCA2a activity by promoting redox-dependent PLN oligomerization, thus reducing the amount of monomeric/inhibitory PLN. Yet, the precise sites (cysteines that are modified by HNO in PLN) and the chemical nature of these redox modifications (disulfide bond vs. sulfinamide formation) remain elusive. To fill this gap, we have applied our 15N-edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO-derived modifications on PLN. We found that cysteines 46 and 41 are the sites of HNO action in PLN, and that sulfinamide formation is the major HNO-derived modification when HNO is in excess with respect to thiols. However, when HNO/thiol approach the 1:1 ratio (thus reflecting more physiological conditions), intramolecular disulfide bonds are expected to be the dominant HNO-induced species. To investigate the intramolecular disulfide bond formation further, we have started to employ fluorescence labeling methodologies. Present findings provide more mechanistic insight into the salutary actions of HNO donors that are currently tested for safety and efficacy in patients with acute decompensated HF.

HNO, a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS(O)NH2] is unique to HNO. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including small organic molecules, peptides, and a protein. At physiological pH and temperature, relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS(O)OH]. In addition to utilizing ESI-MS and other spectroscopic methods to study sulfinamide reduction, we have applied 15N-edited 1H-NMR techniques to sulfinamide detection and used this method to explore sulfinamide hydrolysis. We have also investigated the effect of local environment on the reactivity of HNO with C-terminal cysteines. Our findings indicate that the nature of HNO-derived modifications of C-terminal ...

Nitroxyl (HNO), a potential heart failure therapeutic, is known to target cysteine residues to form sulfinamides and/or disulfides. Because HNO-derived modifications may depend on their local environment, we have investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides at physiological pH and temperature. Our findings indicate that the nature of HNO-derived modifications of C-terminal cysteines is affected by the C-terminal carboxylate. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative (RS(O)2NHOH) and a thiosulfonate (RS(O)2SR), presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Moreover, these modifications are formed independent of oxygen.

It is currently not known whether caffeine has an enhancing effect on long-term memory in humans. We used post-study caffeine administration to test its effect on memory consolidation using a behavioral discrimination task. Caffeine enhanced performance 24 h after administration according to an inverted U-shaped dose-response curve; this effect was specific to consolidation and not retrieval. We conclude that caffeine enhanced consolidation of long-term memories in humans.

Our enjoyable collaboration with Dr. Jeffrey P. Froehlich focused on the role of phospholamban (PLN) in HNO-induced enhancement of SR Ca2+-ATPase (SERCA2a) activity. We hypothesized that given HNO thiophilic nature it modifies cysteine residues in PLN transmembrane domain, altering its interaction with SERCA2a, thus enhancing pump activity. When HNO, donated by Angeli's salt (AS), was administered to control isolated myocytes, it enhanced both sarcomere shortening and Ca2+ transient. However, when AS/HNO was applied to PLN-/- ventriculocytes, HNO inotropy was reduced by ≅ 50% (the remaining likely stemming from enhanced myofilament sensitivity to Ca2+). PLN centrality to HNO cardiotropic action was confirmed incubating SR vesicles from WT and PLN-/- mice with AS/HNO to measure ATP-dependent Ca2+ uptake by stopped-flow mixing. In WT, HNO increased Ca2+ uptake rate, but it failed to do so in PLN-/- vesicles. The role of cysteines in PLN emerged from studies using ER microsomes from Sf21 insect cells expressing SERCA2a±PLN (WT or Cys 36-41-46→Ala mutant) where we assessed SERCA2a dephosphorylation, a measure of E2P hydrolysis, i.e. a rate-limiting step of SERCA2a activity. AS/HNO augmented SERCA2a dephosphorylation in ER microsomes co-expressing SERCA2a and WT PLN, but this stimulation was absent in microsomes expressing SERCA2a and Cys 36-41-46→Ala mutant PLN. Thus, Jeff's elegant, creative and passionate approach helped us to show that PLN is essential for HNO-induced faster Ca2+ uptake by SERCA2a, suggesting that HNO action occurs, at least partly, via modifications of critical cysteines in PLN transmembrane domain. In Jeff's view, “forbidden” disulfide bonds in PLN are involved, and one of his legacies for us is unearthing the cysteine pairs that are involved in HNO-induced formation of an intramolecular bond that could distort the conformation of PLN, thus perturbing its interaction with SERCA2a and relieving the inhibition.

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca 2+ by the cardiac SR Ca 2+ pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphoryla- tion) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys f Ala mutant). The results show that activation of SERCA2a dephos- phorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca 2+ pump. Stimulation of the cardiac SR Ca 2+ pump (SERCA2a) 1 in isolated SR vesicles by cAMP/protein kinase A-dependent phosphorylation of phospholamban (PLN) was originally demonstrated by Tada et al. (1). Subsequent studies showed that phosphorylation of PLN at Ser 16 increases the apparent Ca 2+ affinity of SERCA2a (2), the rates of phosphorylation and dephosphorylation (2, 3), and the Vmax of Ca 2+ transport (3, 4). Phosphorylation of Ser 16 induces a change in the conformation of PLN (5, 6), which relieves its inhibition and activates the pump. Recent work with ex- pressed Ca 2+ pump proteins suggests that activation of the cardiac SR Ca 2+ pump following the relief of PLN inhibition involves changes in the kinetic behavior of the Ca 2+ -ATPase consistent with SERCA2a oligomerization (7). These changes include stabilization of the ADP-sensitive phosphoenzyme, E1P, and allosteric activation of dephosphorylation by ATP, resulting in an increased rate of turnover of the ADP- insensitive phosphoenzyme, E2P. We have recently shown that nitroxyl (HNO) donated by Angeli's salt (AS, Na2N2O3) activates the SR Ca 2+ pump in isolated murine cardiac myocytes (8). This effect is inde- pendent of � -adrenergic activation (9) and results from a direct effect of HNO on the SR Ca 2+ pump as evidenced by stimulation of Ca 2+ uptake by HNO in isolated murine heart SR vesicles (8). We hypothesize that HNO stimulates Ca 2+ uptake by covalently modifying critical thiol residues in the SERCA2a pump and/or its regulatory protein, PLN, altering the conformation of these proteins, and relieving the inhibi- tion of the pump. Chemical modification of protein thiols by HNO can proceed by two pathways leading to either (a) the formation of a sulfinamide (RS(O)NH2) when one thiol group is involved or (b) the formation of a disulfide (RSSR) plus hydroxylamine (H2NOH) when two thiols are in the proximity of each other (10, 11). The fact that HNO-induced activation of RyR2 receptors reconstituted in lipid planar bilayers can be reversed by DTT (8) suggests that disulfide bond formation may be important in activating Ca 2+ release. In this study, we investigated the mechanism of activation of the SR Ca 2+ pump by HNO using SERCA2a expressed in the absence or presence of PLN in High Five (HF) insect cell microsomes. To study Ca 2+ pump activation by HNO, we measured the kinetics of dephosphorylation of SERCA2a, which is accelerated by HNO. To test whether thiol residues in PLN are critical for activation, we carried out these experiments in microsomes expressing SERCA2a and null-Cys PLN (PLN(-C)) in which the three transmembrane (TM) domain cysteine residues were replaced with alanine. Immunoblotting with an anti-PLN antibody was used to test whether HNO-induced PLN oligomer formation contributes to the relief of inhibition of SERCA2a by PLN. The results show that PLN is necessary for activation of SERCA2a by HNO and that cysteine residues in the TM domain of PLN play a critical role in PLN-dependent HNO activation of the Ca 2+ pump.

Nat. Neurosci. 17, 201–203; published online 12 January 2014; corrected online 17 January 2014; corrected after print 30 June 2014 In the version of this article initially published, there were errors in the reporting of statistics. In the Figure 1b legend, the asterisked P value was given in the HTML version as *P = 0.

Phospholamban (PLN) is an integral membrane protein that regulates the Ca2+ pump (SERCA2a) in cardiac sarcoplasmic reticulum (CSR). Phosphorylation of PLN in response to β-adrenergic stimulation enhances cardiac inotropy by increasing CSR Ca2+ uptake. Nitroxyl (HNO), a new candidate drug therapy for congestive heart failure, improves overall cardiovascular function by increasing Ca2+ release and re-uptake in CSR through a direct interaction with RyR2 and SERCA2a, respectively. Using insect cell ER microsomes expressing SERCA2a +/− PLN (WT and Cys → Ala mutant) we have shown that activation of SERCA2a by HNO is PLN-dependent and entails covalent modification of PLN cysteines. Although HNO stimulates SERCA2a activity by uncoupling PLN from SERCA2a, the role of the cysteine residues in the activation mechanism is not completely understood. We propose that HNO, a thiol oxidant, modifies one or more of the three PLN cysteine residues (C36, C41, C46), affecting the regulatory potency of PLN toward SERCA2a. Examples include intra-molecular disulfide cross-links within single PLN molecules or inter-molecular disulfide cross-links between PLN molecules or PLN and SERCA2a. To test this hypothesis, we have constructed a series of PLN mutants containing single, double and triple cysteine substitutions (alanine replacing cysteine). Each of these mutant PLNs will be co-expressed with SERCA2a in insect cells and cell microsomes will be treated with Angeli's salt (an HNO donor) to determine which cysteine residue(s) are essential for activation monitored by enzyme assay and fluorescence spectroscopy of SERCA2a. The results show that intermolecular PLN disulfides play a minor role in activation by HNO. Studies with the Cys → Ala mutations will be useful in determining which cysteine pairs in PLN contribute to intramolecular disulfide cross-links leading to the relief of PLN inhibition and SERCA2a activation.

Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by 1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB). Activation increases both the apparent Ca2+ affinity and the Vmax of SERCA2a, requiring a sustained input of free energy during cycling. In collaborative work with Jeffrey Froehlich, MD, and others, we investigated this process by comparing the kinetic and spectroscopic properties of SERCA2a expressed with and without PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Our data suggests PLB modifies the state of oligomerization of SERCA2a, which in the presence of unphosphorylated, inhibitory PLB is predominantly monomeric. The absence of PLB allows SERCA2a molecules to reorient and interact conformationally, resulting in catalytic behavior characteristic of SERCA1 in skeletal SR, which has no PLB. We propose that intermolecular conformational coupling of SERCA2a units increases the catalytic efficiency of the Ca2+ pump (raises Vmax) and may contribute to activation of the cardiac SR Ca2+ pump induced by 1-adrenergic agonists. We have recently applied these insights to the mechanism of SERCA2a stimulation by nitroxyl, HNO, which we propose uncouples PLB from SERCA2a by modification of one or more PLB cysteine residues. Dr. Froehlich's kinetics studies indicated the HNO-treated SERCA2a +PLB sample has the same kinetic properties as SERCA2a in the absence of PLB. SERCA2a coexpressed with a null-cysteine PLB construct showed no change in kinetic properties following treatment with HNO. Our ongoing fluorescence and electron paramagnetic resonance spectroscopic experiments agree, showing that HNO-treated SERCA+PLB samples have physical properties similar to SERCA2a without PLB. Our results support the conclusion that HNO-treatment, at least in part, uncouples PLB from SERCA2a, leading to HNO-stimulated SERCA2a activation. Direct effects of HNO on SERCA2a are also under investigation.