M. Sutoh Yoneyama, Y. Tobisawa, S. Hatakeyama, Misaki Sato, Kiyoshi Tone, Yota Tatara, I. Kakizaki, T. Funyu, M. Fukuda, S. Hoshi, C. Ohyama, S. Tsuboi
Anti-tumour immunity by cytotoxic T-lymphocytes (CTLs) is essential to suppress tumour progression. Cancer cells that evade CTL immunity proliferate in the host, promoting metastasis, but mechanisms underlying this capacity remain unknown. Here we report that bladder cancer cells metastasized to lymph nodes evade CTL immunity by a new mechanism via altered glycosylation. CTLs normally recognize and kill cancer cells presenting antigenic peptides on human leukocyte antigen (HLA) class I. We show bladder cancer cells expressing the O-glycan processing enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) exhibit HLA class I O-glycan modified with poly-N-acetyllactosamine and are highly susceptible to CTL. In those cells, poly-N-acetyllactosamine on HLA class I O-glycan binds galectin-3 to form a cell-surface molecular lattice, enabling efficient cell-surface retention of HLA class I. In contrast, bladder cancer cells in which C2GnT is downregulated show decreased levels of poly-N-acetyllactosamine on HLA class I O-glycans, attenuating lattice formation and reducing the cell-surface half-life of HLA class I. These tumour cells present antigenic peptides less efficiently, thereby evading CTL lysis and facilitating metastasis.
{"title":"A mechanism for evasion of CTL immunity by altered O-glycosylation of HLA class I","authors":"M. Sutoh Yoneyama, Y. Tobisawa, S. Hatakeyama, Misaki Sato, Kiyoshi Tone, Yota Tatara, I. Kakizaki, T. Funyu, M. Fukuda, S. Hoshi, C. Ohyama, S. Tsuboi","doi":"10.1093/jb/mvw096","DOIUrl":"https://doi.org/10.1093/jb/mvw096","url":null,"abstract":"Anti-tumour immunity by cytotoxic T-lymphocytes (CTLs) is essential to suppress tumour progression. Cancer cells that evade CTL immunity proliferate in the host, promoting metastasis, but mechanisms underlying this capacity remain unknown. Here we report that bladder cancer cells metastasized to lymph nodes evade CTL immunity by a new mechanism via altered glycosylation. CTLs normally recognize and kill cancer cells presenting antigenic peptides on human leukocyte antigen (HLA) class I. We show bladder cancer cells expressing the O-glycan processing enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) exhibit HLA class I O-glycan modified with poly-N-acetyllactosamine and are highly susceptible to CTL. In those cells, poly-N-acetyllactosamine on HLA class I O-glycan binds galectin-3 to form a cell-surface molecular lattice, enabling efficient cell-surface retention of HLA class I. In contrast, bladder cancer cells in which C2GnT is downregulated show decreased levels of poly-N-acetyllactosamine on HLA class I O-glycans, attenuating lattice formation and reducing the cell-surface half-life of HLA class I. These tumour cells present antigenic peptides less efficiently, thereby evading CTL lysis and facilitating metastasis.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"10 1","pages":"479–492"},"PeriodicalIF":0.0,"publicationDate":"2016-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80806989","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}
Y. Yoshimura, Aya Araki, Hitomi Maruta, Yoshitaka Takahashi, H. Yamashita
Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.
{"title":"Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix","authors":"Y. Yoshimura, Aya Araki, Hitomi Maruta, Yoshitaka Takahashi, H. Yamashita","doi":"10.1093/jb/mvw067","DOIUrl":"https://doi.org/10.1093/jb/mvw067","url":null,"abstract":"Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"11 1","pages":"279–289"},"PeriodicalIF":0.0,"publicationDate":"2016-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91150661","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}
A. Allegrini, A. Astegno, V. La Verde, P. Dominici
Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours.
{"title":"Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavour applications","authors":"A. Allegrini, A. Astegno, V. La Verde, P. Dominici","doi":"10.1093/jb/mvw079","DOIUrl":"https://doi.org/10.1093/jb/mvw079","url":null,"abstract":"Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"20 1","pages":"349–360"},"PeriodicalIF":0.0,"publicationDate":"2016-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78491642","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}
Aurangazeb Kabir, S. Endo, N. Toyooka, Mayuko Fukuoka, K. Kuwata, Y. Kamatari
Inhibitors of AKR1B10 belonging to the aldo-keto reductase (AKR) superfamily are considered promising candidates for anti-cancer drugs. AKR1B1, a structurally similar isoform of AKR1B10, is involved in glucose metabolism. Thus, selective inhibition of AKR1B10 is required for the development of anti-cancer drugs. In this study, we first compared correlations between melting temperature and the 50% inhibition concentration obtained from differential scanning fluorimetry (DSF) and an enzyme inhibitory experiment, respectively, and a good correlation was found, except for compounds with low solubility. This result indicates that the DSF method is useful for drug screening for the AKR superfamily. We then evaluated their selectivity as inhibitors against all seven major human AKR1 family proteins and found that C18 is most specific for AKR1B10.
{"title":"Evaluation of compound selectivity of aldo-keto reductases using differential scanning fluorimetry","authors":"Aurangazeb Kabir, S. Endo, N. Toyooka, Mayuko Fukuoka, K. Kuwata, Y. Kamatari","doi":"10.1093/jb/mvw063","DOIUrl":"https://doi.org/10.1093/jb/mvw063","url":null,"abstract":"Inhibitors of AKR1B10 belonging to the aldo-keto reductase (AKR) superfamily are considered promising candidates for anti-cancer drugs. AKR1B1, a structurally similar isoform of AKR1B10, is involved in glucose metabolism. Thus, selective inhibition of AKR1B10 is required for the development of anti-cancer drugs. In this study, we first compared correlations between melting temperature and the 50% inhibition concentration obtained from differential scanning fluorimetry (DSF) and an enzyme inhibitory experiment, respectively, and a good correlation was found, except for compounds with low solubility. This result indicates that the DSF method is useful for drug screening for the AKR superfamily. We then evaluated their selectivity as inhibitors against all seven major human AKR1 family proteins and found that C18 is most specific for AKR1B10.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"2 1","pages":"215–222"},"PeriodicalIF":0.0,"publicationDate":"2016-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81480666","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. Kasai, S. Kajimoto, Yuma Ito, T. Saito, K. Yasumoto, M. Tokunaga, K. Sakata-Sogawa, H. Fukumura, K. Sogawa
Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.
{"title":"Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1&agr; and Bcl-xL in living cells","authors":"S. Kasai, S. Kajimoto, Yuma Ito, T. Saito, K. Yasumoto, M. Tokunaga, K. Sakata-Sogawa, H. Fukumura, K. Sogawa","doi":"10.1093/jb/mvw068","DOIUrl":"https://doi.org/10.1093/jb/mvw068","url":null,"abstract":"Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"30 1","pages":"291–296"},"PeriodicalIF":0.0,"publicationDate":"2016-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72957204","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}
M. Hayashi, A. Okada, K. Yamamoto, Tomomi Okugochi, Chika Kusaka, Daizou Kudou, M. Nemoto, J. Inagaki, Yuu Hirose, T. Okajima, T. Tamura, K. Soda, K. Inagaki
l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.
{"title":"Gene cloning, recombinant expression, purification and characterization of L-methionine decarboxylase from Streptomyces sp. 590","authors":"M. Hayashi, A. Okada, K. Yamamoto, Tomomi Okugochi, Chika Kusaka, Daizou Kudou, M. Nemoto, J. Inagaki, Yuu Hirose, T. Okajima, T. Tamura, K. Soda, K. Inagaki","doi":"10.1093/jb/mvw083","DOIUrl":"https://doi.org/10.1093/jb/mvw083","url":null,"abstract":"l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"1 1","pages":"389–398"},"PeriodicalIF":0.0,"publicationDate":"2016-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82275627","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. Kida, Aoi Itoh, A. Kimura, H. Matsuoka, H. Imai, K. Kogure, A. Tokumura, Tamotsu Tanaka
Previously, we detected an unknown sphingophospholipid in cabbage leaves and identified it as phytoceramide-1-phosphate (PC1P). We also found an enzyme activity that produces PC1P by glycosylinositol phosphoceramide (GIPC)-specific hydrolysis in cabbage leaves. To characterize the GIPC-specific phospholipase D (GIPC-PLD) activity, we investigated distributions of GIPC-PLD activity in 25 tissues of 10 plants. In most plants, the GIPC-PLD activity was the highest in roots. Young leaves of cabbage and Welsh onion had higher activities than corresponding aged outer leaves. The GIPC-PLD activities in leaves, stems and roots of mung bean were higher in the sprouting stage than in more mature stages. We also examined the distribution of substrate GIPC and product PC1P and found that GIPC was ubiquitously distributed at 50–280 nmol/g (wet wt) in tissues of plants, whereas PC1P was detectable (3–60 nmol/g wet wt.) only in tissues showing considerable GIPC-PLD activity. These results suggest a possibility that GIPC-PLD activity is involved in plant growth.
{"title":"Distribution of glycosylinositol phosphoceramide-specific phospholipase D activity in plants","authors":"T. Kida, Aoi Itoh, A. Kimura, H. Matsuoka, H. Imai, K. Kogure, A. Tokumura, Tamotsu Tanaka","doi":"10.1093/jb/mvw060","DOIUrl":"https://doi.org/10.1093/jb/mvw060","url":null,"abstract":"Previously, we detected an unknown sphingophospholipid in cabbage leaves and identified it as phytoceramide-1-phosphate (PC1P). We also found an enzyme activity that produces PC1P by glycosylinositol phosphoceramide (GIPC)-specific hydrolysis in cabbage leaves. To characterize the GIPC-specific phospholipase D (GIPC-PLD) activity, we investigated distributions of GIPC-PLD activity in 25 tissues of 10 plants. In most plants, the GIPC-PLD activity was the highest in roots. Young leaves of cabbage and Welsh onion had higher activities than corresponding aged outer leaves. The GIPC-PLD activities in leaves, stems and roots of mung bean were higher in the sprouting stage than in more mature stages. We also examined the distribution of substrate GIPC and product PC1P and found that GIPC was ubiquitously distributed at 50–280 nmol/g (wet wt) in tissues of plants, whereas PC1P was detectable (3–60 nmol/g wet wt.) only in tissues showing considerable GIPC-PLD activity. These results suggest a possibility that GIPC-PLD activity is involved in plant growth.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"1 1","pages":"187–195"},"PeriodicalIF":0.0,"publicationDate":"2016-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89430416","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}
The G72 gene is one of the most susceptible genes to schizophrenia and is contained exclusively in the genomes of primates. The product of the G72 gene modulates the activity of D-amino acid oxidase (DAO) and is a small protein prone to aggregate, which hampers its structural studies. In addition, lack of a known structure of a homologue makes it difficult to use the homology modelling method for the prediction of the structure. Thus, we first developed a hybrid ab initio approach for small proteins prior to the prediction of the structure of G72. The approach uses three known ab initio algorithms. To evaluate the hybrid approach, we tested our prediction of the structure of the amino acid sequences whose structures were already solved and compared the predicted structures with the experimentally solved structures. Based on these comparisons, the average accuracy of our approach was calculated to be ∼5 Å. We then applied the approach to the sequence of G72 and successfully predicted the structures of the N- and C-terminal domains (ND and CD, respectively) of G72. The predicted structures of ND and CD were similar to membrane-bound proteins and adaptor proteins, respectively.
{"title":"Structure models of G72, the product of a susceptibility gene to schizophrenia","authors":"Y. Kato, K. Fukui","doi":"10.1093/jb/mvw064","DOIUrl":"https://doi.org/10.1093/jb/mvw064","url":null,"abstract":"The G72 gene is one of the most susceptible genes to schizophrenia and is contained exclusively in the genomes of primates. The product of the G72 gene modulates the activity of D-amino acid oxidase (DAO) and is a small protein prone to aggregate, which hampers its structural studies. In addition, lack of a known structure of a homologue makes it difficult to use the homology modelling method for the prediction of the structure. Thus, we first developed a hybrid ab initio approach for small proteins prior to the prediction of the structure of G72. The approach uses three known ab initio algorithms. To evaluate the hybrid approach, we tested our prediction of the structure of the amino acid sequences whose structures were already solved and compared the predicted structures with the experimentally solved structures. Based on these comparisons, the average accuracy of our approach was calculated to be ∼5 Å. We then applied the approach to the sequence of G72 and successfully predicted the structures of the N- and C-terminal domains (ND and CD, respectively) of G72. The predicted structures of ND and CD were similar to membrane-bound proteins and adaptor proteins, respectively.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"10 1","pages":"223–230"},"PeriodicalIF":0.0,"publicationDate":"2016-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84041669","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}
An MCM4 mutation detected in human cancer cells from endometrium was characterized. The mutation of G486D is located within MCM-box and the glycine at 486 in human MCM4 is conserved in Saccharomyces cerevisiae MCM4 and Sulfolobus solfataricus MCM. This MCM4 mutation affected human MCM4/6/7 complex formation, since the complex containing the mutant MCM4 protein is unstable and the mutant MCM4 protein is tend to be degraded. It is likely that the MCM4 mutation affects the interaction with MCM7 to destabilize the MCM4/6/7 complex. Cells with abnormal nuclear morphology were detected when the mutant MCM4 was expressed in HeLa cells, suggesting that DNA replication was perturbed in the presence of the mutant MCM4. Role of the conserved amino acid in MCM4 function is discussed.
{"title":"An MCM4 mutation detected in cancer cells affects MCM4/6/7 complex formation","authors":"R. Tatsumi, Y. Ishimi","doi":"10.1093/jb/mvw065","DOIUrl":"https://doi.org/10.1093/jb/mvw065","url":null,"abstract":"An MCM4 mutation detected in human cancer cells from endometrium was characterized. The mutation of G486D is located within MCM-box and the glycine at 486 in human MCM4 is conserved in Saccharomyces cerevisiae MCM4 and Sulfolobus solfataricus MCM. This MCM4 mutation affected human MCM4/6/7 complex formation, since the complex containing the mutant MCM4 protein is unstable and the mutant MCM4 protein is tend to be degraded. It is likely that the MCM4 mutation affects the interaction with MCM7 to destabilize the MCM4/6/7 complex. Cells with abnormal nuclear morphology were detected when the mutant MCM4 was expressed in HeLa cells, suggesting that DNA replication was perturbed in the presence of the mutant MCM4. Role of the conserved amino acid in MCM4 function is discussed.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"24 1","pages":"259–268"},"PeriodicalIF":0.0,"publicationDate":"2016-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91148117","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}
The bacterial flagellar motor is a rotary motor complex composed of various proteins. The motor contains a central rod, multiple ring-like structures and stators. The Na+-driven polar flagellar motor of the marine bacterium Vibrio alginolyticus has a specific ring, called the ‘T-ring’, which consists of two periplasmic proteins, MotX and MotY. The T-ring is essential for assembly of the torque-generating unit, the PomA/PomB stator complex, into the motor. To investigate the role of the T-ring for motor function, we performed random mutagenesis of the motX gene on a plasmid. The isolated MotX mutants showed nonmotile, slow-motile, and up-motile phenotypes by the expression from the plasmid. Deletion analysis indicated that the C-terminal region and the signal peptide in MotX are not always essential for flagellar motor function. We also found that overproduction of MotX caused the delay of growth and aberrant cell shape. MotX might have unexpected roles not only in flagellar motor function but also in cell morphology control.
{"title":"Mutational analysis and overproduction effects of MotX, an essential component for motor function of Na+-driven polar flagella of Vibrio","authors":"N. Takekawa, S. Kojima, M. Homma","doi":"10.1093/jb/mvw061","DOIUrl":"https://doi.org/10.1093/jb/mvw061","url":null,"abstract":"The bacterial flagellar motor is a rotary motor complex composed of various proteins. The motor contains a central rod, multiple ring-like structures and stators. The Na+-driven polar flagellar motor of the marine bacterium Vibrio alginolyticus has a specific ring, called the ‘T-ring’, which consists of two periplasmic proteins, MotX and MotY. The T-ring is essential for assembly of the torque-generating unit, the PomA/PomB stator complex, into the motor. To investigate the role of the T-ring for motor function, we performed random mutagenesis of the motX gene on a plasmid. The isolated MotX mutants showed nonmotile, slow-motile, and up-motile phenotypes by the expression from the plasmid. Deletion analysis indicated that the C-terminal region and the signal peptide in MotX are not always essential for flagellar motor function. We also found that overproduction of MotX caused the delay of growth and aberrant cell shape. MotX might have unexpected roles not only in flagellar motor function but also in cell morphology control.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"72 1","pages":"159–166"},"PeriodicalIF":0.0,"publicationDate":"2016-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75962112","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}
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