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Chika Wakita Takuya Maeshima Atsushi Yamazaki Takahiro Shibata Sohei Ito Mitsugu Akagawa Makoto Ojika Junji Yodoi Koji Uchida 《The Journal of biological chemistry》2009,284(42):28810-28822
4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys73 and, to the lesser extent, the active site Cys32. More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys73, whereas Cys32 exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids and has been implicated in the pathogenesis of numerous diseases, including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, post-ischemic reoxygenation injury, and aging (1). The peroxidative breakdown of polyunsaturated fatty acids has also been implicated in the pathogenesis of many types of liver injury and especially in the hepatic damage induced by several toxic substances. Lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them is a variety of different aldehydes (2). The primary products of lipid peroxidation, lipid hydroperoxides, can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals giving rise to the formation of short chain, unesterified aldehydes, or a second class of aldehydes still esterified to the parent lipid. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including protein, leading to disruption of important cellular functions. The important agents that give rise to the modification of protein may be represented by α,β-unsaturated aldehydic intermediates, such as 2-alkenals, 4-hydroxy-2-alkenals, and 4-oxo-2-alkenals (3, 4).4-Hydroxy-2-nonenal (HNE),2 among the reactive aldehydes, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress (2, 5). HNE exerts these effects because of its facile reactivity with biological materials, particularly the sulfhydryl groups of proteins. The reaction of HNE with sulfhydryl groups leads to the formation of thioether adducts that further undergo cyclization to form cyclic hemiacetals (2). Although HNE also forms Michael adducts with the imidazole moiety of histidine residues and the ϵ-amino group of lysine residues (5), the formation of thiol-derived Michael adducts, stabilized as the cyclic hemiacetal, is considered to constitute the main reactivity of HNE, because of the nucleophilic potential of the sulfhydryl group compared with those of the imidazole and amine groups. However, because of the lack of specific and reliable methods for the determination of HNE-cysteine adducts, no study has so far quantitatively demonstrated their formation in proteins.Because HNE generated in lipid peroxidation is a racemic mixture of 4R- and 4S-enantiomers (6), the HNE Michael adducts, possessing three chiral centers at C-2, C-4, and C-5 in the tetrahydrofuran moiety (Fig. 1A), are composed of at least eight isomers. In our previous study (7), we characterized the configurational isomers of an HNE-histidine adduct by NMR spectroscopy and by molecular orbital calculations, and we found that the configuration of the tetrahydrofuran ring could affect the electron delocalization features, which contribute to the stability of the adduct. Moreover, we raised monoclonal antibodies against (R)-HNE- and (S)-HNE-histidine adducts and observed differential cellular distributions of these adducts in vivo. Balogh et al. (8) recently characterized the stereochemical configurations of the HNE-glutathione adduct by NMR experiments in combination with simulated annealing structure determinations. Despite these studies, however, the stereoselectivity of the HNE Michael addition adducts generated in proteins remains to be fully explored. In this study, to gain further structural insight into sulfhydryl modification by the lipid peroxidation product, we characterized the stereochemical configuration of the HNE-N-acetylcysteine adducts by NMR spectroscopy. In addition, we adapted a pyridylamination-based method for fluorescent labeling of the HNE-cysteine adducts, using 2-aminopyridine (2-AP) and sodium cyanoborohydride (NaCNBH3), and successfully analyzed the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Furthermore, using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin (Trx).Open in a separate windowFIGURE 1.Reaction of cysteine residue with HNE. A, formation of the HNE-cysteine Michael adduct, possessing three chiral centers (asterisks). B, reaction of N-acetylcysteine with enantioisomeric HNE. The reactions were performed as described under “Experimental Procedures.” AU, absorbance units. 相似文献
25.
Masakazu Kotoda Tadahiko Ishiyama Kazuha Mitsui Sohei Hishiyama Takashi Matsukawa 《BMC anesthesiology》2017,17(1):168
Background
Ion channels play a crucial role in the development of ischemic brain injury. Recent studies have reported that the blockade of various types of ion channels improves outcomes in experimental stroke models. Amiodarone, one of the most effective drugs for life-threatening arrhythmia, works as a multiple channel blocker and its characteristics cover all four Vaughan-Williams classes. Although it is known that amiodarone indirectly contributes to preventing ischemic stroke by maintaining sinus rhythm in patients with atrial fibrillation, the direct neuroprotective effect of amiodarone has not been clarified. The purpose of this study was to investigate the direct effect of amiodarone on ischemic stroke in mice.Methods
Focal cerebral ischemia was induced via distal permanent middle cerebral artery occlusion (MCAO) in adult male mice. The amiodarone pre-treatment group received 50 mg/kg of amiodarone 1 h before MCAO; the amiodarone post-treatment groups received 50 mg/kg of amiodarone immediately after MCAO; the control group received vehicle only. In addition, the sodium channel opener veratrine and selective beta-adrenergic agonist isoprotelenol were used to elucidate the targeted pathway. Heart rate and blood pressure were monitored perioperatively. Infarct volume analysis was conducted 48 h after MCAO. The body asymmetry test and the corner test were used for neurological evaluation.Results
Amiodarone pre-treatment and post-treatment reduced the heart rate but did not affect the blood pressure. No mice showed arrhythmia. Compared with the control group, the amiodarone pre-treatment group had smaller infarct volumes (8.9?±?2.1% hemisphere [mean?±?SD] vs. 11.2?±?1.4%; P?<?0.05) and improved functional outcomes: lower asymmetric body swing rates (52?±?17% vs. 65?±?18%; P?<?0.05) and fewer left turns (7.1?±?1.2 vs. 8.3?±?1.2; P?<?0.05). In contrast, amiodarone post-treatment did not improve the outcomes after MCAO. The neuroprotective effect of amiodarone pre-treatment was abolished by co-administration of veratrine but not by isoproterenol.Conclusions
Amiodarone pre-treatment attenuated ischemic brain injury and improved functional outcomes without affecting heart rhythm and blood pressure. The present results showed that amiodarone pre-treatment has neuroprotective effects, at least in part, via blocking the sodium channels.26.
Shibata T Kimura Y Mukai A Mori H Ito S Asaka Y Oe S Tanaka H Takahashi T Uchida K 《The Journal of biological chemistry》2011,286(49):42150-42161
Isothiocyanates, membrane-permeable electrophiles that form adducts with thiols, have been suggested to have important medical benefits. Here we shed light on isothiocyanate-thiol conjugates and studied their electrophilic potential transferring an isothiocyanate moiety to cellular proteins. When we examined the effect of sulfhydryl molecules on cellular response induced by 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analog of sulforaphane isolated from broccoli, we observed significant induction of heme oxygenase-1 by 6-HITC even in the presence of N-acetyl-L-cysteine or glutathione (GSH). In addition, the authentic 6-HITC-β-mercaptoethanol (6-HITC-ME) conjugate markedly up-regulated the enzyme expression, suggesting the electrophilic potential of thiolated isothiocyanates. To gain a chemical insight into the cellular response induced by thiolated isothiocyanates, we studied the occurrence of transthiocarbamoylation of sulfhydryl molecules by 6-HITC-ME and observed that, upon incubation of 6-HITC-ME with GSH, a single product corresponding to the GSH conjugate of 6-HITC was generated. To test the functional ability of thiolated isothiocyanates to thiocarbamoylate proteins in living cells, we designed a novel probe, combining an isothiocyanate-reactive group and an alkyne functionality, and revealed that the transthiocarbamoylation of proteins occurred in the cells upon exposure to 6-HITC-ME. The target of thiocarbamoylation included heat shock protein 90 β (Hsp90β), a chaperone ATPase of the Hsp90 family implicated in protein maturation and targeting. To identify the sites of the Hsp90β modification, we utilized nano-LC/MALDI-TOF MS/MS and suggested that a thiol group on the peptide containing Cys-521 reacted with 6-HITC, resulting in a covalent adduct in a 6-HITC-treated recombinant Hsp90β in vitro. The site-selective binding to Cys-521 was supported by in silico modeling. Further study on the thiocarbamoylation of Hsp90β suggested that the formation of 6-HITC-Hsp90β conjugate might cause activation of heat shock factor-1, rapidly signaling a potential heat shock response. These data suggest that thiolated isothiocyanates are an active metabolite that could contribute to cellular responses through transthiocarbamoylation of cellular proteins. 相似文献
27.
Hisa I Inoue Y Hendy GN Canaff L Kitazawa R Kitazawa S Komori T Sugimoto T Seino S Kaji H 《The Journal of biological chemistry》2011,286(11):9787-9796
The mechanisms whereby the parathyroid hormone (PTH) exerts its anabolic action on bone are incompletely understood. We previously showed that inhibition of ERK1/2 enhanced Smad3-induced bone anabolic action in osteoblasts. These findings suggested the hypothesis that changes in gene expression associated with the altered Smad3-induced signaling brought about by an ERK1/2 inhibitor would identify novel bone anabolic factors in osteoblasts. We therefore performed a comparative DNA microarray analysis between empty vector-transfected mouse osteoblastic MC3T3-E1 cells and PD98059-treated stable Smad3-overexpressing MC3T3-E1 cells. Among the novel factors, Tmem119 was selected on the basis of its rapid induction by PTH independent of later increases in endogenous TGF-β. The levels of Tmem119 increased with time in cultures of MC3T3-E1 cells and mouse mesenchymal ST-2 cells committed to the osteoblast lineage by BMP-2. PTH stimulated Tmem119 levels within 1 h as determined by Western blot analysis and immunocytochemistry in MC3T3-E1 cells. MC3T3-E1 cells stably overexpressing Tmem119 exhibited elevated levels of Runx2, osteocalcin, alkaline phosphatase, and β-catenin, whereas Tmem119 augmented BMP-2-induced Runx2 levels in mesenchymal cells. Tmem119 interacted with Runx2, Smad1, and Smad5 in C2C12 cells. In conclusion, we identified a Smad3-related factor, Tmem119, that is induced by PTH and promotes differentiation in mouse osteoblastic cells. Tmem119 is an important molecule in the pathway downstream of PTH and Smad3 signaling in osteoblasts. 相似文献
28.
Hiroaki Asai Hiroshi Fujiwara Sohei Kitazawa Naoto Kobayashi Toshiki Ochi Yukihiro Miyazaki Fumihiro Ochi Yoshiki Akatsuka Sachiko Okamoto Junichi Mineno Kiyotaka Kuzushima Hiroaki Ikeda Hiroshi Shiku Masaki Yasukawa 《Journal of hematology & oncology》2014,7(1):1-4
Because WT1 is expressed in leukemia cells, the development of cancer immunotherapy targeting WT1 has been an attractive translational research topic. However, concern of this therapy still remains, since WT1 is abundantly expressed in renal glomerular podocytes. In the present study, we clearly showed that WT1-specific cytotoxic T lymphocytes (CTLs) certainly exerted cytotoxicity against podocytes in vitro; however, they did not damage podocytes in vivo. This might be due to the anatomical localization of podocytes, being structurally separated from circulating CTLs in glomerular capillaries by an exceptionally thick basement membrane. 相似文献
29.
Kiyoshi Mori Riko Kitazawa Takeshi Kondo Michiko Mori Yasuhiro Hamada Michiru Nishida Yasuhiro Minami Ryuma Haraguchi Yutaka Takahashi Sohei Kitazawa 《PloS one》2014,9(7)
In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These in vitro data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression. 相似文献
30.
T Komatsu M Okao H Miyamoto T Chen S Shinka 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(9):2895-2906
This study characterized totally the effects of early Ag exposure by the suckling route on later specific antibody responses. When mother mice of BALB/c or C57BL/6 strains were injected with deaggregated human gamma-globulin (HGG) immediately after delivery, total amounts of HGG in sera of offspring increased until 2 wk of age. The catabolism of transferred HGG was extremely slow and the half-life was about 3 wk in both strains. Hence, small amounts of Ag in mothers, 0.5 micrograms in C57BL/6 and 50 micrograms in BALB/c, could tolerize their offspring effectively. As these were minimum tolerogenic doses, the strain difference in ease of tolerance induction is apparent already during suckling. The study on timing dependent effects of HGG-specific antiserum on tolerance induction by mothers given 50 micrograms HGG demonstrated that the tolerance is achieved within the 1st wk of lactation in C57BL/6 offspring, but not in BALB/c offspring, and the restoration from the tolerance needs more than 6 wk under circumstances, supposedly, without free Ag. Whereas the tolerance was induced in a dose-dependent manner in each class of antibody, the dissociation of tolerant states between IgM, IgG, and IgE antibody classes was found in C57BL/6 offspring. It is interesting that C57BL/6 offspring were sensitized weakly, but significantly, by mothers given subtolerogenic doses. However, this was not apparent in BALB/c. Thus, the Ag dose and the animal strain are related closely to the consequences of this Ag exposure. The aging of suckling mice within the first 2 wk of life or immunomodulators administered early in life did not seriously affect the consequences. Studies on a cellular basis showed that the tolerance is caused by the selective defect in helper T cell function and the suppressor cell activity is not associated with the mechanisms. This contrasts with other models of oral tolerance. 相似文献