Innate and adaptive defense mechanisms protect the respiratory system from attack by microbes. Here, we present evidence that the bitter taste receptor T2R38 regulates the mucosal innate defense of the human upper airway. Utilizing immunofluorescent and live cell imaging techniques in polarized primary human sinonasal cells, we demonstrate that T2R38 is expressed in human upper respiratory epithelium and is activated in response to acyl-homoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Receptor activation regulates calcium-dependent NO production, resulting in stimulation of mucociliary clearance and direct antibacterial effects. Moreover, common polymorphisms of the TAS2R38 gene were linked to significant differences in the ability of upper respiratory cells to clear and kill bacteria. Lastly, TAS2R38 genotype correlated with human sinonasal gram-negative bacterial infection. These data suggest that T2R38 is an upper airway sentinel in innate defense and that genetic variation contributes to individual differences in susceptibility to respiratory infection.

S-nitrosylation, the selective posttranslational modification of protein cysteine residues to form S-nitrosocysteine, is one of the molecular mechanisms by which nitric oxide influences diverse biological functions. In this study, unique MS-based proteomic approaches precisely pinpointed the site of S-nitrosylation in 328 peptides in 192 proteins endogenously modified in WT mouse liver. Structural analyses revealed that S-nitrosylated cysteine residues were equally distributed in hydrophobic and hydrophilic areas of proteins with an average predicted pK a of 10.01 ± 2.1. S-nitrosylation sites were over-represented in α-helices and under-represented in coils as compared with unmodified cysteine residues in the same proteins (χ 2 test, P < 0.02). A quantile-quantile probability plot indicated that the distribution of S-nitrosocysteine residues was skewed toward larger surface accessible areas compared with the unmodified cysteine residues in the same proteins. Seventy percent of the S-nitrosylated cysteine residues were surrounded by negatively or positively charged amino acids within a 6-Å distance. The location of cysteine residues in α-helices and coils in highly accessible surfaces bordered by charged amino acids implies site directed S-nitrosylation mediated by protein-protein or small molecule interactions. Moreover, 13 modified cysteine residues were coordinated with metals and 15 metalloproteins were endogenously modified supporting metalcatalyzed S-nitrosylation mechanisms. Collectively, the endogenous Snitrosoproteome in the liver has structural features that accommodate multiple mechanisms for selective site-directed S-nitrosylation.cysteine modification | nitric oxide | S-nitrosation | posttranslational modification | proteomics C ysteine S-nitrosylation is a reversible and apparently selective posttranslational protein modification that regulates protein activity, localization, and stability within a variety of organs and cellular systems (1-6). Despite the considerable biological importance of this posttranslational modification, significant gaps exist regarding its in vivo specificity and origin. The identification of in vivo S-nitrosylated proteins has indicated that not all reduced cysteine residues and not all proteins with reduced cysteine residues are modified, implying a biased selection. Several biological chemistries have been proposed to account for the S-nitrosylation of proteins in vivo (1,7,8). Broadly, these include (i) oxidative S-nitrosation by higher oxides of NO, (ii) transnitrosylation by small molecular weight NO carriers such as S-nitrosoglutathione or dinitrosyliron complexes, (iii) catalysis by metalloproteins, and (iv) protein-assisted transnitrosation, as elegantly documented for the S-nitrosylation of caspase-3 by S-nitrosothioredoxin (9, 10). With the exception of the protein-assisted transnitrosylation and metalloprotein catalyzed S-nitrosylation, which we presume necessitates protein-protein interaction, the other proposed mechanisms are rather nondiscriminatory unless th...

Background Inorganic nitrate (NO3−), abundant in certain vegetables, is converted to nitrite by bacteria in the oral cavity. Nitrite can be converted to nitric oxide (NO) in the setting of hypoxia. We tested the hypothesis that NO3− supplementation improves exercise capacity in HFpEF via specific adaptations to exercise. Methods Seventeen subjects participated in this randomized, double-blind, cross-over study comparing a single-dose of NO3-rich beetroot juice (NO3−:12.9 mmoles) versus an identical nitrate-depleted placebo. Subjects performed supine-cycle maximal-effort cardiopulmonary exercise tests, with measurements of cardiac output (CO) and skeletal muscle oxygenation. We also assessed skeletal muscle oxidative function. Study endpoints included exercise efficiency (total work/total oxygen consumed), peak VO2, total work performed, vasodilatory reserve, forearm mitochondrial oxidative function, and augmentation index (a marker of arterial wave reflections, measured via radial arterial tonometry). Results Supplementation increased plasma NO-metabolites (median 326 μM versus 10 μM; P=0.0003), peak VO2 (12.6±3.7 vs. 11.6±3.1 mL O2/min/kg; P=0.005), and total work performed (55.6±35.3 vs. 49.2±28.9 kJ; P=0.04). However, efficiency was unchanged. NO3− led to greater reductions in SVR (−42.4±16.6 vs. −31.8±20.3%; P=0.03) and increases in CO (121.2±59.9 vs. 88.7±53.3%; P=0.006) with exercise. NO3− reduced aortic augmentation index (132.2±16.7 vs. 141.4±21.9%, P=0.03) and tended to improve mitochondrial oxidative function. Conclusion NO3− increased exercise capacity in HFpEF by targeting peripheral abnormalities. Efficiency did not change due to parallel increases in total work and VO2. NO3− increased exercise vasodilatory and cardiac output reserves. NO3− also reduced arterial wave reflections, which are linked to left ventricular diastolic dysfunction and remodeling.

Cysteine S-nitrosylation is a posttranslational modification by which nitric oxide regulates protein function and signaling. Studies of individual proteins have elucidated specific functional roles for S-nitrosylation, but knowledge of the extent of endogenous S-nitrosylation, the sites that are nitrosylated, and the regulatory consequences of S-nitrosylation remains limited. We used mass spectrometry-based methodologies to identify 1011 S-nitrosocysteine residues in 647 proteins in various mouse tissues. We uncovered selective S-nitrosylation of enzymes participating in glycolysis, gluconeogenesis, tricarboxylic acid cycle, and oxidative phosphorylation, indicating that this posttranslational modification may regulate metabolism and mitochondrial bioenergetics. S-nitrosylation of the liver enzyme VLCAD (very long acyl-CoA dehydrogenase) at Cys238, which was absent in mice lacking endothelial nitric oxide synthase, improved its catalytic efficiency. These data implicate protein S-nitrosylation in the regulation of β-oxidation of fatty acids in mitochondria.

NO is a versatile free radical that mediates numerous biological functions within every major organ system. A molecular pathway by which NO accomplishes functional diversity is the selective modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, stability, and location. Despite considerable advances with individual proteins, the in vivo biological chemistry, the structural elements that govern the selective S-nitrosylation of cysteine residues, and the potential overlap with other redox modifications are unknown. In this minireview, we explore the functional features of S-nitrosylation at the proteome level and the structural diversity of endogenously modified residues, and we discuss the potential overlap and complementation that may exist with other cysteine modifications.

Overnutrition disrupts circadian metabolic rhythms by mechanisms that are not well understood. Here, we show that diet-induced obesity (DIO) causes massive remodeling of circadian enhancer activity in mouse liver, triggering synchronous high-amplitude circadian rhythms of both fatty acid (FA) synthesis and oxidation. SREBP expression was rhythmically induced by DIO, leading to circadian FA synthesis and, surprisingly, FA oxidation (FAO). DIO similarly caused a high-amplitude circadian rhythm of PPARα, which was also required for FAO. Provision of a pharmacological activator of PPARα abrogated the requirement of SREBP for FAO (but not FA synthesis), suggesting that SREBP indirectly controls FAO via production of endogenous PPARα ligands. The high-amplitude rhythm of PPARα imparted time-of-day-dependent responsiveness to lipid-lowering drugs. Thus, acquisition of rhythmicity for non-core clock components PPARα and SREBP1 remodels metabolic gene transcription in response to overnutrition and enables a chronopharmacological approach to metabolic disorders.

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