During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate-enzyme interaction and thereby accelerating hardening.
在鱼卵受精过程中,卵包膜的硬化是由转谷氨酰胺酶(hTGase)介导的。受精后,转谷氨酰胺酶会进行加工。我们从未受精虹鳟鱼卵和水活化虹鳟鱼卵的提取物中分离出了 hTGase。虹鳟鱼 hTGase(Rt-hTGase)呈 80 kDa 蛋白,其加工形式为 55 kDa。它们的 N 端氨基酸序列几乎相同,表明在 C 端区域进行了加工。它们的特异性活性没有明显差异,表明 C 端加工并没有激活酶本身。我们使用变性引物通过反转录聚合酶链反应(RT-PCR)克隆了 cDNA,然后进行了 RACE-PCR。cDNA 的推导氨基酸序列与因子 XIII 亚基 A(FXIIIA)相似。分子系统发育和基因同源分析清楚地表明,hTGase 是由 FXIIIA 在向长尾目进化过程中复制产生的。Rt-hTGase 的 55 kDa 加工形式主要由根据 cDNA 氨基酸序列预测的酶域组成。据推测,Rt-hTGase 的 C 端结构域可与卵包膜蛋白质结合,加工后的酶可在卵包膜内自由移动,增加底物与酶的相互作用,从而加速硬化。
{"title":"Transglutaminase mediates the hardening of fish egg envelope produced by duplication of factor XIIIA gene during the evolution of Teleostei.","authors":"Shigeki Yasumasu, Miyuki Horie, Mayuko Horie, Kodai Sakuma, Chihiro Sato, Hikari Sato, Taiki Nakajima, Tatsuki Nagasawa, Mari Kawaguchi, Ichiro Iuchi","doi":"10.1093/jb/mvae062","DOIUrl":"https://doi.org/10.1093/jb/mvae062","url":null,"abstract":"<p><p>During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate-enzyme interaction and thereby accelerating hardening.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142288110","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Intratumour heterogeneity has been shown to play a role in the malignant progression of cancer. The clonal evolution in primary cancer has been well studied, however, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumours developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumours. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple colour-labelled organoids of mouse intestinal tumours. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumour cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumours.
{"title":"Neutral selection and clonal expansion during the development of colon cancer metastasis.","authors":"Xuelian Lei, Daisuke Yamamoto, Hirotaka Kitamura, Kenji Kita, Noriyuki Inaki, Kazuhiro Murakami, Mizuho Nakayama, Hiroko Oshima, Masanobu Oshima","doi":"10.1093/jb/mvae044","DOIUrl":"10.1093/jb/mvae044","url":null,"abstract":"<p><p>Intratumour heterogeneity has been shown to play a role in the malignant progression of cancer. The clonal evolution in primary cancer has been well studied, however, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumours developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumours. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple colour-labelled organoids of mouse intestinal tumours. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumour cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumours.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.
鉴于严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)新变种的不断出现,有必要开发新的抑制剂,以提高临床疗效,增加 2019 年冠状病毒疾病联合疗法的选择。由于海洋生物是发现大量生物活性分子的资源,我们构建了一个从冲绳群岛采集的海洋无脊椎动物提取物库。在这项研究中,我们利用这些提取物来鉴定针对 SARS-CoV-2 的抗病毒分子。通过在 VeroE6/TMPRSS2 细胞中进行细胞病理效应(CPE)试验,发现海洋海绵 Theonella swinhoei 的提取物可减少病毒诱导的 CPE。通过柱层析和核磁共振分析,最终确定翁酰胺 A 是提取物中的一种抗病毒化合物。Onnamide A 能抑制 VeroE6/TMPRSS2 细胞中几种 SARS-CoV-2 变体诱导的 CPE 以及感染细胞上清液中病毒的产生。此外,这种化合物还能阻止 SARS-CoV-2 伪病毒的进入。综上所述,这些结果表明昂纳米德 A 能抑制 SARS-CoV-2 感染,部分原因可能与抑制病毒进入有关,有望成为开发抗 SARS-CoV-2 药物的候选先导化合物。
{"title":"Onnamide A suppresses the severe acute respiratory syndrome-coronavirus 2 infection without inhibiting 3-chymotrypsin-like cysteine protease.","authors":"Yasuhiro Hayashi, Nanami Higa, Tetsuro Yoshida, Trianda Ayuning Tyas, Kanami Mori-Yasumoto, Mina Yasumoto-Hirose, Hideki Tani, Junichi Tanaka, Takahiro Jomori","doi":"10.1093/jb/mvae037","DOIUrl":"10.1093/jb/mvae037","url":null,"abstract":"<p><p>Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141081400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In the chloroplast stroma, dynamic pH changes occur from acidic to alkaline in response to fluctuating light conditions. We investigated the pH dependency of the electron transfer reaction of ferredoxin-NADP+ reductase (FNR) with ferredoxin (Fd) isoproteins, Fd1 and Fd2, which are localized in mesophyll cells and bundle sheath cells, respectively, in the leaves of C4 plant maize. The pH-dependent profile of the electron transfer activity with FNR was quite different between Fd1 and Fd2, which was mainly explained by the opposite pH dependency of the Km value of these Fds for FNR. Replacement of the amino acid residue at position of 65 (D65N) and 78 (H78A) between the two Fds conferred different effect on their pH dependency of the Km value. Double mutations of the two residues between Fd1 and Fd2 (Fd1D65N/H78A and Fd2N65D/A78H) led to the mutual exchange of the pH dependency of the electron transfer activity. This exchange was mainly explained by the changes in the pH-dependent profile of the Km values. Therefore, the differences in Asp/Asn at position 65 and His/Ala at position 78 between Fd1 and Fd2 were shown to be the major determinants for their different pH dependency in the electron transfer reaction with FNR.
{"title":"Amino acid residues responsible for the different pH dependency of cell-specific ferredoxins in the electron transfer reaction with ferredoxin-NADP+ reductase from maize leaves.","authors":"Yoko Kimata-Ariga, Hikaru Tanaka, Shunsuke Kuwano","doi":"10.1093/jb/mvae043","DOIUrl":"10.1093/jb/mvae043","url":null,"abstract":"<p><p>In the chloroplast stroma, dynamic pH changes occur from acidic to alkaline in response to fluctuating light conditions. We investigated the pH dependency of the electron transfer reaction of ferredoxin-NADP+ reductase (FNR) with ferredoxin (Fd) isoproteins, Fd1 and Fd2, which are localized in mesophyll cells and bundle sheath cells, respectively, in the leaves of C4 plant maize. The pH-dependent profile of the electron transfer activity with FNR was quite different between Fd1 and Fd2, which was mainly explained by the opposite pH dependency of the Km value of these Fds for FNR. Replacement of the amino acid residue at position of 65 (D65N) and 78 (H78A) between the two Fds conferred different effect on their pH dependency of the Km value. Double mutations of the two residues between Fd1 and Fd2 (Fd1D65N/H78A and Fd2N65D/A78H) led to the mutual exchange of the pH dependency of the electron transfer activity. This exchange was mainly explained by the changes in the pH-dependent profile of the Km values. Therefore, the differences in Asp/Asn at position 65 and His/Ala at position 78 between Fd1 and Fd2 were shown to be the major determinants for their different pH dependency in the electron transfer reaction with FNR.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141305995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Most autophagy-related genes, or ATG genes, have been identified through studies using budding yeast. Although the functions of the ATG genes are well understood, the contributions of individual genes to non-selective and various types of selective autophagy remain to be fully elucidated. In this study, we quantified the activity of non-selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy, endoplasmic reticulum (ER)-phagy and pexophagy in all Saccharomyces cerevisiae atg mutants. Among the mutants of the core autophagy genes considered essential for autophagy, the atg13 mutant and mutants of the genes involved in the two ubiquitin-like conjugation systems retained residual autophagic functionality. In particular, mutants of the Atg8 ubiquitin-like conjugation system (the Atg8 system) exhibited substantial levels of non-selective autophagy, the Cvt pathway and pexophagy, although mitophagy and ER-phagy were undetectable. Atg8-system mutants also displayed intravacuolar vesicles resembling autophagic bodies, albeit at significantly reduced size and frequency. Thus, our data suggest that membranous sequestration and vacuolar delivery of autophagic cargo can occur in the absence of the Atg8 system. Alongside these findings, the comprehensive analysis conducted here provides valuable datasets for future autophagy research.
{"title":"Comprehensive analysis of non-selective and selective autophagy in yeast atg mutants and characterization of autophagic activity in the absence of the Atg8 conjugation system.","authors":"Tamara Ginevskaia, Aleksei Innokentev, Kentaro Furukawa, Tomoyuki Fukuda, Manabu Hayatsu, Shun-Ichi Yamashita, Keiichi Inoue, Shinsuke Shibata, Tomotake Kanki","doi":"10.1093/jb/mvae042","DOIUrl":"10.1093/jb/mvae042","url":null,"abstract":"<p><p>Most autophagy-related genes, or ATG genes, have been identified through studies using budding yeast. Although the functions of the ATG genes are well understood, the contributions of individual genes to non-selective and various types of selective autophagy remain to be fully elucidated. In this study, we quantified the activity of non-selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy, endoplasmic reticulum (ER)-phagy and pexophagy in all Saccharomyces cerevisiae atg mutants. Among the mutants of the core autophagy genes considered essential for autophagy, the atg13 mutant and mutants of the genes involved in the two ubiquitin-like conjugation systems retained residual autophagic functionality. In particular, mutants of the Atg8 ubiquitin-like conjugation system (the Atg8 system) exhibited substantial levels of non-selective autophagy, the Cvt pathway and pexophagy, although mitophagy and ER-phagy were undetectable. Atg8-system mutants also displayed intravacuolar vesicles resembling autophagic bodies, albeit at significantly reduced size and frequency. Thus, our data suggest that membranous sequestration and vacuolar delivery of autophagic cargo can occur in the absence of the Atg8 system. Alongside these findings, the comprehensive analysis conducted here provides valuable datasets for future autophagy research.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141283834","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively, in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids and generated CSA and CSE liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.
{"title":"Chondroitin sulfate liposome: clustering toward high functional efficiency.","authors":"Tatsumasa Shioiri, Jun Tsuchimoto, Kaori Fukushige, Takao Takeuchi, Munekazu Naito, Hideto Watanabe, Nobuo Sugiura","doi":"10.1093/jb/mvae041","DOIUrl":"10.1093/jb/mvae041","url":null,"abstract":"<p><p>Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively, in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids and generated CSA and CSE liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141305996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFβ-ELOB-ELOC-CUL5 (VβBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. Whilst recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VβBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VβBCC complex. These aptamers not only bind to the VβBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VβBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VβBCC complex, offering potential avenues for therapeutic intervention.
{"title":"Selection and characterization of aptamers targeting the Vif-CBFβ-ELOB-ELOC-CUL5 complex.","authors":"Kazuyuki Kumagai, Keisuke Kamba, Takuya Suzuki, Yuto Sekikawa, Chisato Yuki, Michiaki Hamada, Kayoko Nagata, Akifumi Takaori-Kondo, Li Wan, Masato Katahira, Takashi Nagata, Taiichi Sakamoto","doi":"10.1093/jb/mvae040","DOIUrl":"10.1093/jb/mvae040","url":null,"abstract":"<p><p>The viral infectivity factor (Vif) of human immunodeficiency virus 1 forms a complex with host proteins, designated as Vif-CBFβ-ELOB-ELOC-CUL5 (VβBCC), initiating the ubiquitination and subsequent proteasomal degradation of the human antiviral protein APOBEC3G (A3G), thereby negating its antiviral function. Whilst recent cryo-electron microscopy (cryo-EM) studies have implicated RNA molecules in the Vif-A3G interaction that leads to A3G ubiquitination, our findings indicated that the VβBCC complex can also directly impede A3G-mediated DNA deamination, bypassing the proteasomal degradation pathway. Employing the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we have identified RNA aptamers with high affinity for the VβBCC complex. These aptamers not only bind to the VβBCC complex but also reinstate A3G's DNA deamination activity by inhibiting the complex's function. Moreover, we delineated the sequences and secondary structures of these aptamers, providing insights into the mechanistic aspects of A3G inhibition by the VβBCC complex. Analysis using selected aptamers will enhance our understanding of the inhibition of A3G by the VβBCC complex, offering potential avenues for therapeutic intervention.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140915615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Aberrant proteostasis is thought to be involved in the pathogenesis of neurodegenerative diseases. Some proteostasis abnormalities are ameliorated by chaperones. Chaperones are divided into three groups: molecular, pharmacological and chemical. Chemical chaperones intended to alleviate stress in organelles, such as the endoplasmic reticulum (ER), are now being administered clinically. Of the chemical chaperones, 4-phenylbutyrate (4-PBA) has been used as a research reagent, and its mechanism of action includes chaperone effects and the inhibition of histone deacetylase. Moreover, it also binds to the B-site of SEC24 and regulates COPII-mediated transport from the ER. Although its therapeutic effect may not be strong, elucidating the mechanism of action of 4-PBA may contribute to the identification of novel therapeutic targets for neurodegenerative diseases.
蛋白稳态异常被认为与神经退行性疾病的发病机制有关。一些蛋白稳态异常可通过伴侣素得到改善。伴侣分为三类:分子伴侣、药理学伴侣和化学伴侣。化学伴侣旨在减轻细胞器(如内质网)的压力,目前已在临床上使用。在化学伴侣剂中,4-苯基丁酸酯(4-PBA)已被用作研究试剂,其作用机制包括伴侣效应和抑制组蛋白去乙酰化酶。此外,它还能与 SEC24 的 B 位点结合,调节 COPII 介导的从 ER 的转运。虽然 4-PBA 的治疗效果可能不强,但阐明其作用机制可能有助于确定神经退行性疾病的新治疗靶点。
{"title":"Neurodegenerative diseases associated with the disruption of proteostasis and their therapeutic strategies using chemical chaperones.","authors":"Takashi Sugiyama, Hideki Nishitoh","doi":"10.1093/jb/mvae048","DOIUrl":"10.1093/jb/mvae048","url":null,"abstract":"<p><p>Aberrant proteostasis is thought to be involved in the pathogenesis of neurodegenerative diseases. Some proteostasis abnormalities are ameliorated by chaperones. Chaperones are divided into three groups: molecular, pharmacological and chemical. Chemical chaperones intended to alleviate stress in organelles, such as the endoplasmic reticulum (ER), are now being administered clinically. Of the chemical chaperones, 4-phenylbutyrate (4-PBA) has been used as a research reagent, and its mechanism of action includes chaperone effects and the inhibition of histone deacetylase. Moreover, it also binds to the B-site of SEC24 and regulates COPII-mediated transport from the ER. Although its therapeutic effect may not be strong, elucidating the mechanism of action of 4-PBA may contribute to the identification of novel therapeutic targets for neurodegenerative diseases.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141492072","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cytidine diphosphate diacylglycerol (CDP-DAG) is a critical intermediate that is converted to multiple phospholipids in prokaryotes and eukaryotes. In budding yeast, CDP-DAG synthesis from cytidine triphosphate (CTP) and phosphatidic acid (PA) is catalyzed by the membrane-integrated protein Cds1 in the endoplasmic reticulum and the peripheral membrane-bound protein Tam41 in mitochondria. Although a recent study revealed that the fission yeast SpTam41 consists of a nucleotidyltransferase domain and a winged helix domain, forming an active-site pocket for CTP binding between the two domains together with a C-terminal amphipathic helix for membrane association, how CTP and Mg 2+, a most-favoured divalent cation, are accommodated with PA remains obscure. A more recent report by Kimura et al. (J. Biochem. 2022; 171:429-441) solved the crystal structure of FbTam41, a functional ortholog from a Firmicutes bacterium, with CTP-Mg 2+, successfully providing a detailed molecular view of CDP-DAG synthesis. In this commentary, our current understanding of Tam41-mediated reaction is discussed.
{"title":"CDP-DAG synthesis by peripheral membrane-bound Tam41-type enzymes.","authors":"Koji Okamoto","doi":"10.1093/jb/mvae046","DOIUrl":"10.1093/jb/mvae046","url":null,"abstract":"<p><p>Cytidine diphosphate diacylglycerol (CDP-DAG) is a critical intermediate that is converted to multiple phospholipids in prokaryotes and eukaryotes. In budding yeast, CDP-DAG synthesis from cytidine triphosphate (CTP) and phosphatidic acid (PA) is catalyzed by the membrane-integrated protein Cds1 in the endoplasmic reticulum and the peripheral membrane-bound protein Tam41 in mitochondria. Although a recent study revealed that the fission yeast SpTam41 consists of a nucleotidyltransferase domain and a winged helix domain, forming an active-site pocket for CTP binding between the two domains together with a C-terminal amphipathic helix for membrane association, how CTP and Mg 2+, a most-favoured divalent cation, are accommodated with PA remains obscure. A more recent report by Kimura et al. (J. Biochem. 2022; 171:429-441) solved the crystal structure of FbTam41, a functional ortholog from a Firmicutes bacterium, with CTP-Mg 2+, successfully providing a detailed molecular view of CDP-DAG synthesis. In this commentary, our current understanding of Tam41-mediated reaction is discussed.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141426964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
3,3,3-Trifluoro-1,2-propanediol undergoes complete defluorination in two distinct steps: first, the conversion into 3,3,3-trifluoropropionaldehyde catalyzed by adenosylcobalamin (coenzyme B12)-dependent diol dehydratase; second, non-enzymatic elimination of all three fluorides from this aldehyde to afford malonic semialdehyde (3-oxopropanoic acid), which is decarboxylated to acetaldehyde. Diol dehydratase accepts 3,3,3-trifluoro-1,2-propanediol as a relatively poor substrate, albeit without significant mechanism-based inactivation of the enzyme during catalysis. Optical and electron paramagnetic resonance (EPR) spectra revealed the steady-state formation of cob(II)alamin and a substrate-derived intermediate organic radical (3,3,3-trifluoro-1,2-dihydroxyprop-1-yl). The coenzyme undergoes Co-C bond homolysis initiating a sequence of reaction by the generally accepted pathway via intermediate radicals. However, the greater steric size of trifluoromethyl and especially its negative impact on the stability of an adjacent radical centre compared to a methyl group has implications for the mechanism of the diol dehydratase reaction. Nevertheless, 3,3,3-trifluoropropionaldehyde is formed by the normal diol dehydratase pathway, but then undergoes non-enzymatic conversion into acetaldehyde, probably via 3,3-difluoropropenal and malonic semialdehyde.
{"title":"The action of coenzyme B12-dependent diol dehydratase on 3,3,3-trifluoro-1,2-propanediol results in elimination of all the fluorides with formation of acetaldehyde.","authors":"Koichi Mori, Bernard T Golding, Tetsuo Toraya","doi":"10.1093/jb/mvae047","DOIUrl":"10.1093/jb/mvae047","url":null,"abstract":"<p><p>3,3,3-Trifluoro-1,2-propanediol undergoes complete defluorination in two distinct steps: first, the conversion into 3,3,3-trifluoropropionaldehyde catalyzed by adenosylcobalamin (coenzyme B12)-dependent diol dehydratase; second, non-enzymatic elimination of all three fluorides from this aldehyde to afford malonic semialdehyde (3-oxopropanoic acid), which is decarboxylated to acetaldehyde. Diol dehydratase accepts 3,3,3-trifluoro-1,2-propanediol as a relatively poor substrate, albeit without significant mechanism-based inactivation of the enzyme during catalysis. Optical and electron paramagnetic resonance (EPR) spectra revealed the steady-state formation of cob(II)alamin and a substrate-derived intermediate organic radical (3,3,3-trifluoro-1,2-dihydroxyprop-1-yl). The coenzyme undergoes Co-C bond homolysis initiating a sequence of reaction by the generally accepted pathway via intermediate radicals. However, the greater steric size of trifluoromethyl and especially its negative impact on the stability of an adjacent radical centre compared to a methyl group has implications for the mechanism of the diol dehydratase reaction. Nevertheless, 3,3,3-trifluoropropionaldehyde is formed by the normal diol dehydratase pathway, but then undergoes non-enzymatic conversion into acetaldehyde, probably via 3,3-difluoropropenal and malonic semialdehyde.</p>","PeriodicalId":15234,"journal":{"name":"Journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":2.1,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141579802","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}