Characterization and quantifi cation of endogenous fatty acid nitroalkene metabolites in human urine

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca- 9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 + 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and co-elution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with HgCl2. This reaction increases the level of free nitroalkene fraction >10-fold and displays a KD of 7.5x10-6 M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive non-invasive index of metabolic and inflammatory status.Fil: Salvatore, Sonia Rosana. University of Pittsburgh; Estados UnidosFil: Vitturi, Dario A.. University of Pittsburgh; Estados UnidosFil: Baker, Paul R. S.. University of Pittsburgh; Estados UnidosFil: Bonacci, Gustavo Roberto. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Koenitzer, Jeffrey R.. University of Pittsburgh; Estados UnidosFil: Woodcock, Steven R.. University of Pittsburgh; Estados UnidosFil: Freeman, Bruce A.. University of Pittsburgh; Estados UnidosFil: Schopfer, Francisco J.. University of Pittsburgh; Estados Unido

Modulation of Nitro-fatty acid signaling: prostaglandin reductase-1 is a Nitroalkene reductase

Background: Nitroalkenes are electrophilic anti-inflammatory mediators that signal via Michael addition and are metabolized in vivo. Results: Prostaglandin reductase-1 is identified as a nitroalkene reductase. Conclusion: Prostaglandin reductase-1 reduces fatty acid nitroalkenes to nitroalkanes, inactivating electrophilic reactivity. Significance: A mammalian enzyme is identified that metabolizes fatty acid nitroalkenes in vivo to silence their signaling reactions.Fil: Vitturi, Dario A.. University of Pittsburgh; Estados UnidosFil: Chen, Chen Shan. University of Pittsburgh; Estados UnidosFil: Woodcock, Steven R.. University of Pittsburgh; Estados UnidosFil: Salvatore, Sonia R.. University of Pittsburgh; Estados UnidosFil: Bonacci, Gustavo Roberto. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Koenitzer, Jeffrey R.. University of Pittsburgh; Estados UnidosFil: Stewart, Nicolas A.. University of Pittsburgh; Estados UnidosFil: Wakabayashi, Nobunao. University of Pittsburgh; Estados UnidosFil: Kensler, Thomas W.. University of Pittsburgh; Estados UnidosFil: Freeman, Bruce A.. University of Pittsburgh; Estados UnidosFil: Schopfer, Francisco J.. University of Pittsburgh; Estados Unido

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies

Fatty acid nitroalkenes induce resistance to ischemic cardiac injury by modulating mitochondrial respiration at complex II

Nitro-fatty acids (NO2-FA) are metabolic and inflammatory-derived electrophiles that mediate pleiotropic signaling actions. It was hypothesized that NO2-FA would impact mitochondrial redox reactions to induce tissue-protective metabolic shifts in cells. Nitro-oleic acid (OA-NO2) reversibly inhibited complex II-linked respiration in isolated rat heart mitochondria in a pH-dependent manner and suppressed superoxide formation. Nitroalkylation of Fp subunit was determined by BME capture and the site of modification by OA-NO2 defined by mass spectrometric analysis. These effects translated into reduced basal and maximal respiration and favored glycolytic metabolism in H9C2 cardiomyoblasts as assessed by extracellular H+ and O2 flux analysis. The perfusion of NO2-FA induced acute cardioprotection in an isolated perfused heart ischemia/reperfusion (IR) model as evidenced by significantly higher rate-pressure products. Together these findings indicate that NO2-FA can promote cardioprotection by inducing a shift from respiration to glycolysis and suppressing reactive species formation in the post-ischemic interval.Fil: Koenitzer, Jeffrey. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Bonacci, Gustavo Roberto. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Woodcock, Steven R.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Che, Chen-Shan. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Cantu Medellin, Nadiezhda. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Kelley, Eric E.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Schopfer, Francisco J.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados Unido

Conjugated linoleic acid is a preferential substrate for fatty acid nitration

The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 105 greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO2-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.Fil: Bonacci, Gustavo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Baker, Paul R. S.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Salvatore, Sonia Rosana. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Shores, Darla. University of Pittsburgh; Estados UnidosFil: Khoo, Nicholas K. H.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Koenitzer, Jeffrey R.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Vitturi, Dario A.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Woodcock, Steven R.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Golin-Bisello, Franca. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Cole, Marsha P.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Watkins, Simon. University of Pittsburgh; Estados UnidosFil: St. Croix, Claudette. University of Pittsburgh; Estados UnidosFil: Batthyany, Carlos I.. Instituto Pasteur; Uruguay. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Freeman, Bruce A.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados UnidosFil: Schopfer, Francisco J.. Univeristy Of Pittsburgh. School Of Medicine. Department Of Pharmacology And Chemical Biology; Estados Unido