Singlet molecular oxygen ( 1 O 2 ) has well-established roles in photosynthetic plants, bacteria and fungi 1-3 , but not in mammals. Chemically generated 1 O 2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine 4 , while enzymatic oxidation of tryptophan to N-formylkynurenine is catalyzed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1 5 . Under inflammatory conditions, this hemecontaining enzyme becomes expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure 6 . However, whether indoleamine 2,3-dioxygenase 1 forms 1 O 2 and whether this contributes to blood pressure control is unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1 O 2 . We observed that in the presence of hydrogen peroxide, the enzyme generates 1 O 2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophanderived hydroperoxide acts as a hitherto undiscovered signaling molecule in vivo, which induces arterial relaxation and decreases blood pressure dependent on cysteine residue 42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1 O 2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions. Several small molecules, such as nitric oxide and hydrogen peroxide (H 2 O 2 ) regulate cellular signaling via interaction with proteins containing redox active metals and/or cysteine residues. Of these molecules, nitric oxide, formed from L-arginine by endothelial nitric oxide synthase, is an important regulator of vascular tone 7 . Sustained increases in nitric oxide synthesis by inducible nitric oxide synthase, as observed in pathological settings such as sepsis, are associated with profound hypotension 8 . Paradoxically, inhibitors of the nitric oxide pathway have generally failed to ameliorate severe septic shock 9,10 , suggesting involvement of additional mediators of hypotension.Based on functional similarity with the metabolism of L-arginine by nitric oxide synthase, we 3 reported previously that metabolism of L-tryptophan (Trp) to N-formylkynurenine (NFK) and kynurenine by endothelial indoleamine 2,3-dioxygenase 1 (IDO1) ( Fig. 1a) contributes to the regulation of vascular tone and blood pressure in inflammation 6 . We also showed that commercial kynurenine relaxed pre-constricted arteries, which suggested that kynurenine is an endotheliumderived relaxant factor 6 . Although others have since confirmed these findings 11,12 , we noticed that recently purchased kynurenine no longer caused arterial relaxation, and that HPLC-purified kynurenine and NFK also failed to relax naïve mouse arteries ( Fig. 1b). However, purified Trp relaxed pre-constricted mouse abdominal aortas that expressed IDO1, irrespective of whether IDO1 expression was...

Pilot Project #1—the identification and characterization of human histone H4 proteoforms by top-down MS—is the first project launched by the Consortium for Top-Down Proteomics (CTDP) to refine and validate top-down MS. Within the initial results from seven participating laboratories, all reported the probability-based identification of human histone H4 (UniProt accession P62805) with expectation values ranging from 10−13 to 10−105. Regarding characterization, a total of 74 proteoforms were reported, with 21 done so unambiguously; one new PTM, K79ac, was identified. Inter-laboratory comparison reveals aspects of the results that are consistent, such as the localization of individual PTMs and binary combinations, while other aspects are more variable, such as the accurate characterization of low-abundance proteoforms harboring >2 PTMs. An open-access tool and discussion of proteoform scoring are included, along with a description of general challenges that lie ahead including improved proteoform separations prior to mass spectrometric analysis, better instrumentation performance, and software development.

Abstract-Dysregulated blood pressure control leading to hypertension is prevalent and is a risk factor for several common diseases. Fully understanding blood pressure regulation offers the possibility of developing rationale therapies to alleviate hypertension and associated disease risks. Although hydrogen sulfide (H 2 S) is a well-established endogenous vasodilator, the molecular basis of its blood-pressure lowering action is incompletely understood. H 2 S-dependent vasodilation and blood pressure lowering in vivo was mediated by it catalyzing formation of an activating interprotein disulfide within protein kinase G (PKG) Iα. However, this oxidative activation of PKG Iα is counterintuitive because H 2 S is a thiol-reducing molecule that breaks disulfides, and so it is not generally anticipated to induce their formation. This apparent paradox was explained by H 2 S in the presence of molecular oxygen or hydrogen peroxide rapidly converting to polysulfides, which have oxidant properties that in turn activate PKG by inducing the disulfide. These observations are relevant in vivo because transgenic knockin mice in which the cysteine 42 redox sensor within PKG has been systemically replaced with a redox-dead serine residue are resistant to H 2 S-induced blood pressure lowering. Thus, a primary mechanism by which the reductant molecule H 2 S lowers blood pressure is mediated somewhat paradoxically by the oxidative activation of PKG. (Hypertension. 2014;64:1344-1351.)