Tasuku Abe, T. Kawarai, Yukihiro Takahashi, K. Konishi
{"title":"Enzymatic kinetics of the quinol peroxidase of an aggressive periodontopathic bacterium","authors":"Tasuku Abe, T. Kawarai, Yukihiro Takahashi, K. Konishi","doi":"10.1093/jb/mvw099","DOIUrl":"https://doi.org/10.1093/jb/mvw099","url":null,"abstract":"Aggregatibacter actinomycetemcomitans is an oral pathogen for aggressive periodontitis, and encodes a triheme c-containing membrane-bound enzyme, quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain. In the previous work, we have characterized recombinant QPO purified from the membrane fraction of Escherichia coli harboring a plasmid containing QPO gene. Irreversible inactivation of QPO by high concentration of H2O2 exhibited pseudo-first order kinetics. Analysis of initial-rate kinetics of QPO may suggest that enzyme catalytic mechanism is explained by a Ping Pong Bi Bi system rather than sequential systems. In addition, the redox reactions of cytochrome c in the presence of several values of [Q1H2]/[Q1] were at equilibrium, and only about 2/3 of the cytochrome c of QPO is reduced at high ratios of [Q1H2]/[Q1]. These results indicated that one of the three heme c moieties of QPO is maintained in an oxidized form even at increased ratios of [Q1H2]/[Q1], suggesting that QPO is reduced in the absence of H2O2 and only two of the three heme c moieties are reduced in the presence of high concentration of the Q1H2. Product inhibition of QPO accorded with our theoretical model for the reaction mechanism. Considered together, the enzymatic kinetics data for QPO confirm the Ping Pong Bi Bi system.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82549242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
K. Tomoo, Y. Miki, H. Morioka, Kiho Seike, T. Ishida, Sadao Ikenishi, K. Miyamoto, T. Hasegawa, A. Yamano, K. Hamada, H. Tsujibo
{"title":"Crystal structure of the solute-binding protein BxlE from Streptomyces thermoviolaceus OPC-520 complexed with xylobiose","authors":"K. Tomoo, Y. Miki, H. Morioka, Kiho Seike, T. Ishida, Sadao Ikenishi, K. Miyamoto, T. Hasegawa, A. Yamano, K. Hamada, H. Tsujibo","doi":"10.1093/jb/mvw097","DOIUrl":"https://doi.org/10.1093/jb/mvw097","url":null,"abstract":"BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 Å resolution. BxlE consists of two globular domains that are linked by two β-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79250708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hiroki Oi, D. Fujita, Yuki Suzuki, H. Sugiyama, Masayuki Endo, Shigeyoshi Matsumura, Y. Ikawa
{"title":"Programmable formation of catalytic RNA triangles and squares by assembling modular RNA enzymes","authors":"Hiroki Oi, D. Fujita, Yuki Suzuki, H. Sugiyama, Masayuki Endo, Shigeyoshi Matsumura, Y. Ikawa","doi":"10.1093/jb/mvw093","DOIUrl":"https://doi.org/10.1093/jb/mvw093","url":null,"abstract":"RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM).","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86488211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Minling Hu, Hanxiao Lin, Li Yang, Yanzhen Cheng, Hua Zhang
{"title":"Interleukin-22 restored mitochondrial damage and impaired glucose-stimulated insulin secretion through down-regulation of uncoupling protein-2 in INS-1 cells.","authors":"Minling Hu, Hanxiao Lin, Li Yang, Yanzhen Cheng, Hua Zhang","doi":"10.1093/jb/mvw084","DOIUrl":"10.1093/jb/mvw084","url":null,"abstract":"<p><p>Defective glucose-stimulated insulin secretion (GSIS) induced by chronic exposure to fatty acids is a hallmark of type 2 diabetes (T2D). Interleukin-22 (IL-22) has been shown to exert beneficial effects on insulin secretion and to protect pancreatic β-cells from stress. Moreover, uncoupling protein-2 (UCP-2) plays a central role in the regulation of GSIS and β-cell dysfunction, whereas the role of UCP-2 in IL-22-enhanced glycemic control under conditions of lipotoxicity remains unclear. In this present study, we investigated the effects of IL-22 on rat insulin-secreting cells (INS-1 cells) and the mechanisms that underlie IL-22 and lipotoxicity-impaired GSIS in vitro. Chronic palmitate (PA) treatment impaired insulin secretion and activated UCP-2 expression in INS-1 cells. Furthermore, in INS-1 cells, both reduced mitochondrial membrane potential (ΔΨm) and impaired GSIS induced by PA treatment were effectively reversed by an inhibitor of UCP-2 (genipin). Additionally, compared with the PA-treated group, INS-1 cells treated with IL-22 down-regulated UCP-2 expression, increased mitochondrial membrane potential, and restored GSIS. Together, our findings indicate that chronic exposure to PA could activate UCP-2, resulting in mitochondrial damage and impaired GSIS in INS-1 cells. We also suggest that IL-22 plays a protective role in this process via the down-regulation of UCP-2.</p>","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jb/mvw084","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83721977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Matsuoka, Akiho Shima, A. Uda, Hirotaka Ezaki, A. Michihara
{"title":"The retinoic acid receptor-related orphan receptor &agr; positively regulates tight junction protein claudin domain-containing 1 mRNA expression in human brain endothelial cells","authors":"H. Matsuoka, Akiho Shima, A. Uda, Hirotaka Ezaki, A. Michihara","doi":"10.1093/jb/mvw092","DOIUrl":"https://doi.org/10.1093/jb/mvw092","url":null,"abstract":"Members of the claudin family play important roles in the formation of tight junctions (TJs) in several tissues. Claudin domain containing 1 (CLDND1) is homologous to this family and localizes to TJs and the cytoplasm when exogenously expressed in cultured epithelial cell lines. Furthermore, serum antibody levels of CLDND1-derived peptides are elevated in patients with cerebral infection, cardiovascular disease or diabetes mellitus as compared to healthy controls. However, CLDND1 transcriptional regulation remains poorly analyzed and most regional transcription factor binding sites remain to be defined. Notably, the CLDND1 promoter contains a putative response element for retinoic acid receptor-related orphan receptor α (RORα), which is involved in the above-mentioned disorders. In this study, we found that Cldnd1 and Rora mRNA levels are correlated in rat tissues and that RORα overexpression in human brain endothelial cells enhanced CLDND1 transcript expression. In addition, siRNA-mediated knockdown of RORα significantly decreased CLDND1 transcription. An electrophoresis mobility shift assay indicated that RORα binds to the identified response element in a sequence-specific manner. Furthermore, luciferase reporter assays confirmed that RORα interacts with the CLDND1 promoter to enhance transcription. Taken together, our findings strongly suggest that CLDND1 is a direct RORα target.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87399591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
T. Nakagawa, Chiharu Suzuki-Nakagawa, A. Watanabe, Eriko Asami, M. Matsumoto, Mami Nakano, A. Ebihara, M. Uddin, F. Suzuki
{"title":"Site-1 protease is required for the generation of soluble (pro)renin receptor","authors":"T. Nakagawa, Chiharu Suzuki-Nakagawa, A. Watanabe, Eriko Asami, M. Matsumoto, Mami Nakano, A. Ebihara, M. Uddin, F. Suzuki","doi":"10.1093/jb/mvw080","DOIUrl":"https://doi.org/10.1093/jb/mvw080","url":null,"abstract":"The extracellular domain of the (pro)renin receptor [(P)RR] is cleaved to generate the soluble form of (P)RR [s(P)RR]. Multiple clinical studies have revealed the association between serum/plasma s(P)RR levels and certain diseases, thereby suggesting a potential role for s(P)RR as a disease biomarker. Here, we investigated whether site-1 protease (S1P) is responsible for cleaving (P)RR to generate s(P)RR. Reduction of endogenous S1P with siRNA attenuated s(P)RR generation in Chinese hamster ovary (CHO) cells exogenously expressing human (P)RR with a C-terminal decahistidine tag [CHO/h(P)RR-10His cells]; conversely, overexpression of S1P by transient transfection increased s(P)RR generation. The S1P inhibitor PF429242 suppressed s(P)RR generation in CHO/h(P)RR-10His and human cervical carcinoma HeLa cells; however, the ADAM inhibitor GM6001 had no effect. The furin inhibitor Dec-RVKR-CMK had no effect on the amount of s(P)RR, but caused a slight increase in the size of the s(P)RR. Moreover, the reversible vesicle-trafficking inhibitor brefeldin A (BFA) enhanced the generation of large-sized s(P)RR; PF429242, but not Dec-RVKR-CMK, suppressed this BFA-induced s(P)RR formation. The size of s(P)RR generated during BFA treatment was reduced after removal of BFA; Dec-RVKR-CMK, but not PF429242, suppressed this conversion. Together, these results suggest that s(P)RR is generated by sequential processing by S1P and furin.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72808497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
S. Funahashi, Yasunori Suzuki, Kiyotaka Nakano, S. Kawai, Masami Suzuki
{"title":"Generation and characterization of monoclonal antibodies against human LGR6","authors":"S. Funahashi, Yasunori Suzuki, Kiyotaka Nakano, S. Kawai, Masami Suzuki","doi":"10.1093/jb/mvw077","DOIUrl":"https://doi.org/10.1093/jb/mvw077","url":null,"abstract":"Leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) is a seven-pass transmembrane protein known to be a marker of stem cells in several organs. To deepen our understanding of the cell biology of LGR6-positive cells, including stem cells, we generated monoclonal antibodies (mAbs) against human LGR6. DNA immunization followed by whole-cell immunization with LGR6-expressing transfectants was performed to obtain mAbs that recognized the native form of LGR6. Hybridomas were screened by flow cytometry using LGR6-transfected cells. Because the molecules of LGR4, LGR5, and LGR6 are 50% homologous at the amino acid level, specificity of the mAbs was confirmed by transfectants expressing LGR4, LGR5, or LGR6. Three LGR6-specific mAbs were generated. Two of the three mAbs (designated 43A6 and 43D10) recognized the large N-terminal extracellular domain of LGR6, and competitively blocked the binding of R-spondin 1, which is known to be the ligand for LGR6. The other mAb, 43A25, recognized the seven-pass transmembrane domain of LGR6, and was able to be used for immunoblot analysis. In addition, mAbs 43A6 and 43D10 detected endogenous expression of LGR6 in cancer cell lines. We expect that our mAbs will contribute to widening our understanding of LGR6-positive cells in humans.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85283491","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Autophagy-independent function of Atg8 in lipid droplet dynamics in yeast.","authors":"Yuichiro Maeda, Masahide Oku, Yasuyoshi Sakai","doi":"10.1093/jb/mvw078","DOIUrl":"10.1093/jb/mvw078","url":null,"abstract":"<p><p>Dynamic features of lipid droplets include growth and degradation of the organelle. Autophagy, a system for the transport of cytoplasmic components to be degraded into the lysosome/vacuole, is regarded to be responsible for the degradation of lipid droplets. Atg8 protein in the yeast Saccharomyces cerevisiae is recruited to membrane structures synthesized during autophagy via a lipidation process. In this study, we report a novel function of Atg8 in lipid droplet dynamics. We found that loss of Atg8 specifically resulted in a decrease in the quantity of lipid droplets in cells at stationary phase. This protein was detected in a lipid droplet fraction independent of its lipidation. Loss of Atg8 hemifusion activity also caused a decrease in the quantity of lipid droplets. Consistent with these results, isolated lipid droplets underwent assembly into large clusters when incubated with Atg8 possessing hemifusion activity. The loss of Atg8 did not reduce the quantity of lipid droplets in a mutant defective in lipolysis, another system for lipid droplet degradation, which strongly suggests the function of Atg8 in antagonizing lipolysis. Together these results indicate a specific function of Atg8 in maintaining the quantity of lipid droplets that is independent of its autophagic function.</p>","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jb/mvw078","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85395014","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ryo Ueta, T. Tezuka, Yosuke Izawa, Sadanori Miyoshi, S. Nagatoishi, K. Tsumoto, Y. Yamanashi
{"title":"The carboxyl-terminal region of Dok-7 plays a key, but not essential, role in activation of muscle-specific receptor kinase MuSK and neuromuscular synapse formation","authors":"Ryo Ueta, T. Tezuka, Yosuke Izawa, Sadanori Miyoshi, S. Nagatoishi, K. Tsumoto, Y. Yamanashi","doi":"10.1093/jb/mvw073","DOIUrl":"https://doi.org/10.1093/jb/mvw073","url":null,"abstract":"As the synapse between a motor neuron and skeletal muscle, the neuromuscular junction (NMJ) is required for muscle contraction. The formation and maintenance of NMJs are controlled by the muscle-specific receptor kinase MuSK. Dok-7 is the essential cytoplasmic activator of MuSK, and indeed mice lacking Dok-7 form no NMJs. Moreover, DOK7 gene mutations underlie DOK7 myasthenia, an NMJ synaptopathy. Previously, we failed to detect MuSK activation in myotubes by Dok-7 mutated in the N-terminal pleckstrin homology (PH) or phosphotyrosine binding (PTB) domain or that lacked the C-terminal region (Dok-7-ΔC). Here, we found by quantitative analysis that Dok-7-ΔC marginally, but significantly, activated MuSK in myotubes, unlike the PH- or PTB-mutant. Purified, recombinant Dok-7-ΔC, but not other mutants, also showed marginal ability to activate MuSK's cytoplasmic portion, carrying the kinase domain. Consistently, forced expression of Dok-7-ΔC rescued Dok-7-deficient mice from neonatal lethality caused by the lack of NMJs, indicating restored MuSK activation and NMJ formation. However, these mice showed only marginal activation of MuSK and died by 3 weeks of age apparently due to an abnormally small number and size of NMJs. Thus, Dok-7's C-terminal region plays a key, but not fully essential, role in MuSK activation and NMJ formation.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86730119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"PRMT-5 converts monomethylarginines into symmetrical dimethylarginines in Caenorhabditis elegans","authors":"Akihiko Kanou, K. Kako, K. Hirota, A. Fukamizu","doi":"10.1093/jb/mvw066","DOIUrl":"https://doi.org/10.1093/jb/mvw066","url":null,"abstract":"The transmethylation to arginine residues of proteins is catalyzed by protein arginine methyltransferases (PRMTs) that form monomethylarginine (MMA), asymmetric (ADMA) and symmetric dimethylarginines (SDMA). Although we previously demonstrated that the generation of ADMA residues in whole proteins is driven by PRMT-1 in Caenorhabditis elegans, much less is known about MMA and SDMA in vivo. In this study, we measured the amounts of different methylarginines in whole protein extracts made from wild-type (N2) C. elegans and from prmt-1 and prmt-5 null mutants using liquid chromatography-tandem mass spectrometry. Interestingly, we found that the amounts of MMA and SDMA are about fourfold higher than those of ADMA in N2 protein lysates using acid hydrolysis. We were unable to detect SDMA residues in the prmt-5 null mutant. In comparison with N2, an increase in SDMA and decrease in MMA were observed in prmt-1 mutant worms with no ADMA, but ADMA and MMA levels were unchanged in prmt-5 mutant worms. These results suggest that PRMT-1 contributes, at least in part, to MMA production, but that PRMT-5 catalyzes the symmetric dimethylation of substrates containing MMA residues in vivo.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82191043","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}