2-Oxo-histidine-containing dipeptides are functional oxidation products.

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC-electrospray ionization-tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine-containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H2O2 exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H2O2 cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs.

Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation.

We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3',5'-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.

Computational and Experimental Analyses for Pathogenicity Prediction of ACVRL1 Missense Variants in Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease caused by the defects of ALK1/ACVRL1 receptor signaling. In this study, we evaluated 25 recently identified ACVRL1 missense variants using multiple computational pathogenicity classifiers and experimentally characterized their signal transduction capacity. Three extracellular residue variants showed no detectable cell surface expression and impairment of bone morphogenetic protein 9 (BMP9) responsiveness of SMAD-dependent transcription in luciferase assays. Four variants with amino acid replacement in the motifs essential for the intracellular kinase function lost SMAD-dependent signaling. Most of other variations in the kinase domain also caused marked downregulation of signaling; however, two variants behaved as the wild-type ACVRL1 did, while computational classifiers predicted their functional abnormalities. Three-dimensional structure prediction using the ColabFold program supported the significance of the L45 loop and NANDOR domain of ACVRL1 for its association with SMAD1 and BMPR2, respectively, and the variations in these motifs resulted in the reduction of SMAD signaling. On the other hand, two of the GS domain variants maintained high signal transduction capacity, which did not accord with their computational pathogenicity prediction. These results affirm the requirement of a combinatory approach using computational and experimental analyses to accurately predict the pathogenicity of ACVRL1 missense variants in the HHT patients.

Distribution and quantitative analysis of homoanserine and its 2-oxo derivative in mouse tissues.

Imidazole-containing dipeptides (IDPs), such as carnosine and anserine, are endogenously produced and have been shown to function as antioxidants. Recently, we have characterized the endogenous production of 2-oxo-imidazole-containing dipeptides (2-oxo-IDPs), such as 2-oxo-carnosine, 2-oxo-anserine, and 2-oxo-homocarnosine in mouse tissues, including brain, and demonstrated that 2-oxo-IDPs exhibit higher antioxidant activities than the corresponding IDPs. In this study, we established a highly sensitive, specific, and quantitative method for the detection of the IDP homoanserine and its oxidized derivative 2-oxo-homoanserine via high-performance liquid chromatography tandem mass spectrometry coupled with a stable-isotope dilution method, and quantitatively analyzed its tissue distribution and age-related intra-brain distribution in C57BL/6J mice. The quantitative analysis revealed that homoanserine exists abundantly not only in the mouse brain but also in other tissues, such as the muscle and lungs. Further, we successfully detected the endogenous production of 2-oxo-homoanserine in the mouse brain. The mass spectrometric analysis revealed that homoanserine predominantly exists in the cerebrum and cerebellum and the concentrations in 10-week-old mice were approximately 50-fold higher than those in 1-week-old mice. Accordingly, this is the first study that reports the spatial and temporal expression patterns of homoanserine and its 2-oxo derivative in C57BL/6J mice.

2-Oxo-Imidazole-Containing Dipeptides Play a Key Role in the Antioxidant Capacity of Imidazole-Containing Dipeptides.

There is substantial evidence for the antioxidant functions of imidazole-containing dipeptides (IDPs), including carnosine and anserine, under physiological and pathological conditions in vivo. However, the detailed mechanism underlying the antioxidant functions is still poorly understood. Recently, we discovered the endogenous production of 2-oxo-imidazole-containing dipeptides (2-oxo-IDPs), such as 2-oxo-carnosine and 2-oxo-anserine, as novel derivatives of IDPs in mouse tissues and revealed that the antioxidant capacity of 2-oxo-carnosine was much greater than that of carnosine. However, the antioxidant capacity of 2-oxo-IDPs still remains unclear. In this study, we evaluated 2-oxo-carnosine and 2-oxo-anserine by multiple in vitro assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing/antioxidant power, and oxygen radical absorbance capacity assays in comparison with the corresponding IDPs, carnosine and anserine. All the assays employed herein demonstrated that 2-oxo-carnosine and 2-oxo-anserine exhibited a greater antioxidant capacity than that of the corresponding IDPs. Quantitative high-performance liquid chromatography tandem mass spectrometry revealed that commercial IDPs standards were contaminated with a certain amount of 2-oxo-IDPs, which was correlated with the antioxidant capacity. DPPH radical scavenging assay revealed that the elimination of contaminated 2-oxo-IDPs from the IDPs standards caused a significant decrease in the antioxidant capacity compared to the original IDPs standards. These results suggest that the main driver of the antioxidant capacity of IDPs is 2-oxo-IDPs; accordingly, the conversion of IDPs to 2-oxo-IDPs may be a critical step in the antioxidant functions.

Generation of Rat Monoclonal Antibody to Detect Hydrogen Sulfide and Polysulfides in Biological Samples.

Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective H2S measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples. We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM). We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively. The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM. By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 µM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.

8-Nitro-cGMP Attenuates the Interaction between SNARE Complex and Complexin through S-Guanylation of SNAP-25.

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is the second messenger in nitric oxide/reactive oxygen species redox signaling. This molecule covalently binds to protein thiol groups, called S-guanylation, and exerts various biological functions. Recently, we have identified synaptosomal-associated protein 25 (SNAP-25) as a target of S-guanylation, and demonstrated that S-guanylation of SNAP25 enhanced SNARE complex formation. In this study, we have examined the effects of S-guanylation of SNAP-25 on the interaction between the SNARE complex and complexin (cplx), which binds to the SNARE complex with a high affinity. Pull-down assays and coimmunoprecipitation experiments have revealed that S-guanylation of Cys90 in SNAP-25 attenuates the interaction between the SNARE complex and cplx. In addition, blue native-PAGE followed by Western blot analysis revealed that the amount of cplx detected at a high molecular weight decreased upon 8-nitro-cGMP treatment in SH-SY5Y cells. These results demonstrated for the first time that S-guanylation of SNAP-25 attenuates the interaction between the SNARE complex and cplx.