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Summary: Jonathan Stamler’s laboratory investigates the mechanisms of nitric oxide signaling, in studies that provide insights into the redox-based regulation of protein function and the mechanistic basis of complex physiological responses.

Most cellular functions are sensitive to redox state. Sensitivity is conferred by specialized redox centers in proteins whose regulation has been viewed as a simple on-off switch, corresponding to reduced and oxidized states. But studies of nitric oxide (NO) are revealing the components of a precisely regulated mechanism for control of protein function: NO and related redox species target cysteine thiols and transition metal centers with exquisite spatial and temporal resolution, to transduce a panoply of cellular control signals. In particular, S -nitrosylation, the covalent attachment of a nitrogen monoxide group to the thiol side chain of the amino acid cysteine, has emerged as an important mechanism for dynamic post-translational regulation of all major protein classes. S -nitrosylation thereby conveys a large part of the ubiquitous influence of NO on cellular signal transduction and provides a mechanism for redox-based physiological regulation. We are interested in understanding both the molecular mechanisms of NO signaling and the regulation by NO/redox systems of complex physiological responses.

In addressing these questions, we have relied on a number of model systems. Human hemoglobin provides unique examples of post-translational protein modification, including oxidation by small diatomic ligands, which is the essence of redox signaling. We have employed the theory of allostery and thermodynamic linkage to reveal novel, salient features of NO binding to hemoglobin under physiological conditions, and we have extended this analysis to other model redox systems to elucidate the molecular bases of several complex physiological responses.

Blood flow in the microcirculation is regulated by physiological O ₂ gradients that are coupled to vasodilation and vasoconstriction, but the mechanism underlying this fundamental vascular response (which controls O ₂ delivery to tissues) has remained a major unanswered question in vascular physiology. The discovery of a previously unknown S -nitrosothiol (SNO) derivative of hemoglobin that has potent vasodilatory activity has changed the picture. Most significantly, the affinity of cysteine β93 for NO is high in the R (or oxy) structure and low in the T (or deoxy) structure. Thus, deoxygenation is accompanied by an allosteric transition in SNO-hemoglobin (from R to T structure) that promotes release of the NO group. Moreover, the NO liberated upon deoxygenation is transferred to the red blood cell (RBC) membrane protein, anion exchanger AE1. NO transfer to AE1 plays a role in export of NO that subserves RBC-mediated vasodilation—a novel activity of RBCs. Emerging evidence that vasodilation by RBCs may be impaired in disorders characterized by impaired blood flow, including pulmonary hypertension, heart failure, and sickle cell disease, and that blood transfusions may be associated with NO insufficiency and increased mortality, suggests that defective RBC processing of NO is a contributing factor.

These lessons learned from hemoglobin have been applied to other proteins and allosteric regulators. For example, differential regulation of the bacterial transcriptional activator OxyR can be achieved through alternative redox-based modifications of a single critical cysteine, which subserve either graded (cooperative) or maximal (noncooperative) transcriptional responses. By means of this molecular code, OxyR transduces different redox-based modifications into distinct genetic responses. We have also shown (in collaboration with Gerhard Meissner, University of North Carolina at Chapel Hill) that calcium and O ₂ are allosteric regulators of ryanodine receptor/calcium-release channel (RyR) S -nitrosylation. In skeletal muscle, the oxygen-dependent redox state of approximately six RyR cysteines governs the S -nitrosylation of an additional, single cysteine, which occurs only in the low-pO ₂ (reduced) state; the consequent activation of the channel is calmodulin dependent. Thus, O ₂ and calcium concentration determine whether NO binds, and the effect of S -nitrosylation is transduced by calmodulin. Furthermore, we have discovered that contractility of skeletal muscle is similarly regulated by the concerted action of NO/O ₂ . Together with our findings on RBCs, these data suggest the operation of an integrated physiological system: muscle contractility is coupled to blood flow through O ₂ -dependent transduction of NO signals, conveyed by S -nitrosylation, in RBCs (Hb/AE1) and skeletal muscle (RyR).

Specificity of S -nitrosylation within and between proteins is conferred in part by structural motifs. An acid-base motif for S -nitrosylation is found in many proteins (including Hb). A second, related motif (present in OxyR) may subserve S -nitrosylation by S -nitrosoglutathione (GSNO). Cysteines within domains of high relative hydrophobicity (exemplified by RyR) represent a third class of S -nitrosylation motif that may concentrate NO and O ₂ to produce nitrosylating equivalents. Our recent demonstration of NO-dependent protein-protein interactions revealed an additional, major determinant of specificity of S -nitrosylation between proteins. This analysis indicates that NO synthases (NOSs) regulate their own direct interactions with proteins that may serve as substrates for S -nitrosylation or that bind to other proteins that are thereby targeted for S -nitrosylation. In particular, we have shown that NOS associates with procaspase-3 through NO-dependent protein-protein interactions, facilitating S -nitrosylation and inhibition of caspase activity (and explaining the discovery—that Joan Mannick [University of Massachusetts] and I made—that NO inhibits apoptosis). Apoptotic stimuli such as Fas induce denitrosylation of the active-site cysteine of caspase-3, suggesting that additional proteins may associate with caspase to regulate denitrosylation and thus activity.

Genetic evidence for regulated S -nitrosylation/denitrosylation is provided by the studies of Limin Liu (former HHMI Associate), who isolated from Escherichia coli an SNO-consuming activity that is highly conserved throughout phylogeny. We identified this activity as the glutathione-dependent formaldehyde dehydrogenase (GSFDH), and showed that the enzyme's preferred substrate is in fact GSNO. We have knocked out this "GSNO reductaseâ€? (GSNOR) in yeast and mice and find that it controls intracellular levels of both S -nitrosylated proteins and GSNO. Yeast deficient in GSNOR accumulate SNO proteins and are hypersensitive to nitrosative stress. GSNOR ^–/– mice are hypotensive under anesthesia and show increased mortality following endotoxic challenge. Additional cardiovascular, pulmonary, and immune alterations in these animals establish S -nitrosylation of cysteine thiols as a critical mechanism of NO function in heath and disease.

We have discovered additional enzymes that regulate NO and O ₂ homeostasis across a range of organisms, including microbes, worms, and mammals. In bacteria and yeast, the predominant NO-consuming activity was identified as the flavohemoglobin—a protein of previously unknown function. Mutants deficient in this "denitrosylase" are sensitized to NO. In collaboration with Daniel Goldberg (HHMI, Washington University), we have shown that hemoglobin from the nematode Ascaris , which sits at a bridge point in hemoglobin phylogeny (between the microbial hemoglobins that metabolize NO, and the mammalian hemoglobins that deliver it) is an NO-activated "deoxygenase": it utilizes (S)NO to consume and thereby protect from oxygen. These studies offer a new perspective on the molecular evolution of hemoglobin that is based on the presence of signature cysteines, employed for NO processing.

Related findings have been provided by Zhiqiang Chen (HHMI Associate), who discovered that nitroglycerin (NTG) is metabolized in mitochondria by aldehyde dehydogenase (ALDH) to produce NO-based vasodilatory activity. As corollaries, oxidative inhibition of this “nitrate reductase� underlies mechanism-based NTG tolerance, and chronic administration of NTG leads to mitochondrial dysfunction. These findings have solved the 150-year-old mystery surrounding nitroglycerine biotransformation, and may help explain the adverse clinical outcomes associated with nitroglycerine use.

These lines of study have provided a new perspective on proteins such as hemoglobin, ALDH, and GSFDH, which are employed by microorganisms and mammals to protect themselves against NO, O ₂ , and SNO and to enable physiological regulation of NO-based cellular functions.

This work was supported in part by a grant from the National Institutes of Health.

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