Saeed Nourmohammadi, Sam W Henderson, Sunita A Ramesh, Andrea J Yool
Aquaporin (AQP) channels found in all domains of life are transmembrane proteins which mediate passive transport of water, glycerol, signaling molecules, metabolites, and charged solutes. Discovery of new classes of ion-conducting AQP channels has been slow, likely reflecting time- and labor-intensive methods required for traditional electrophysiology. Work here defines a sensitive mass-throughput system for detecting AQP ion channels, identified by rescue of cell growth in the K+-transport-defective yeast strain CY162 following genetic complementation with heterologously expressed cation-permeable channels, using the well characterized human AQP1 channel for proof of concept. Results showed AQP1 conferred transmembrane permeability to cations which rescued survival in CY162 yeast. Comprehensive testing showed that growth response properties fully recapitulated AQP1 pharmacological agonist and antagonist profiles for activation, inhibition, dose-dependence, and structure-function relationships, demonstrating validity of the yeast screening tool for AQP channel identification and drug discovery efforts. This method also provided new information on divalent cation blockers of AQP1, pH sensitivity of antagonists, and ion permeability of human AQP6. Site-directed mutagenesis of AQP1 channel regulatory domains confirmed that yeast growth rescue was mediated by the introduced channels. Optical monitoring with a lithium-sensitive photoswitchable probe in living cells independently demonstrated monovalent cation permeability of AQP1 channels in yeast plasma membrane. Ion channel properties of AQP1 expressed in yeast were consistent with those of AQP1 expressed in Xenopus laevis oocyte and K+-transport defective Escherichia coli. Outcomes here establish a powerful new approach for efficient screening of phylogenetically diverse AQPs for yet untested functions as cation channels.
{"title":"Characterization of human aquaporin ion channels in a yeast expression system as a tool for novel ion channel discovery.","authors":"Saeed Nourmohammadi, Sam W Henderson, Sunita A Ramesh, Andrea J Yool","doi":"10.1042/BSR20240542","DOIUrl":"10.1042/BSR20240542","url":null,"abstract":"<p><p>Aquaporin (AQP) channels found in all domains of life are transmembrane proteins which mediate passive transport of water, glycerol, signaling molecules, metabolites, and charged solutes. Discovery of new classes of ion-conducting AQP channels has been slow, likely reflecting time- and labor-intensive methods required for traditional electrophysiology. Work here defines a sensitive mass-throughput system for detecting AQP ion channels, identified by rescue of cell growth in the K+-transport-defective yeast strain CY162 following genetic complementation with heterologously expressed cation-permeable channels, using the well characterized human AQP1 channel for proof of concept. Results showed AQP1 conferred transmembrane permeability to cations which rescued survival in CY162 yeast. Comprehensive testing showed that growth response properties fully recapitulated AQP1 pharmacological agonist and antagonist profiles for activation, inhibition, dose-dependence, and structure-function relationships, demonstrating validity of the yeast screening tool for AQP channel identification and drug discovery efforts. This method also provided new information on divalent cation blockers of AQP1, pH sensitivity of antagonists, and ion permeability of human AQP6. Site-directed mutagenesis of AQP1 channel regulatory domains confirmed that yeast growth rescue was mediated by the introduced channels. Optical monitoring with a lithium-sensitive photoswitchable probe in living cells independently demonstrated monovalent cation permeability of AQP1 channels in yeast plasma membrane. Ion channel properties of AQP1 expressed in yeast were consistent with those of AQP1 expressed in Xenopus laevis oocyte and K+-transport defective Escherichia coli. Outcomes here establish a powerful new approach for efficient screening of phylogenetically diverse AQPs for yet untested functions as cation channels.</p>","PeriodicalId":8926,"journal":{"name":"Bioscience Reports","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11358751/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141787235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-28DOI: 10.1042/BSR-2018-1176_RET
{"title":"Retraction: Nucleus pulposus cell apoptosis is attenuated by CDMP-2 through regulating oxidative damage under the hyper-osmotic environment.","authors":"","doi":"10.1042/BSR-2018-1176_RET","DOIUrl":"10.1042/BSR-2018-1176_RET","url":null,"abstract":"","PeriodicalId":8926,"journal":{"name":"Bioscience Reports","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345781/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142016264","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-28DOI: 10.1042/BSR-2018-1708_RET
{"title":"Retraction: Osteogenic protein-1 attenuates nucleus pulposus cell apoptosis through activating the PI3K/Akt/mTOR pathway in a hyperosmotic culture.","authors":"","doi":"10.1042/BSR-2018-1708_RET","DOIUrl":"10.1042/BSR-2018-1708_RET","url":null,"abstract":"","PeriodicalId":8926,"journal":{"name":"Bioscience Reports","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345783/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142016268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-28DOI: 10.1042/BSR-2017-0718_RET
{"title":"Retraction: Role of p38-MAPK pathway in the effects of high-magnitude compression on nucleus pulposus cell senescence in a disc perfusion culture.","authors":"","doi":"10.1042/BSR-2017-0718_RET","DOIUrl":"10.1042/BSR-2017-0718_RET","url":null,"abstract":"","PeriodicalId":8926,"journal":{"name":"Bioscience Reports","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345787/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142016277","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-28DOI: 10.1042/BSR-2018-0064_RET
{"title":"Retraction: The response of nucleus pulposus cell senescence to static and dynamic compressions in a disc organ culture.","authors":"","doi":"10.1042/BSR-2018-0064_RET","DOIUrl":"10.1042/BSR-2018-0064_RET","url":null,"abstract":"","PeriodicalId":8926,"journal":{"name":"Bioscience Reports","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345669/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142016279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues). Despite its critical role in various cellular processes, the physical structure of TTF1 remains unsolved. Attempts to purify the functional TTF1 protein have been unsuccessful till date. Therefore, we focused on characterizing the Myb domain of this essential protein. We started with predicting a 3-D model of the Myb domain using homology modelling, and ab-initio method. We then determined its stability through MD simulation in an explicit solvent. The model predicted is highly stable, which stabilizes at 200ns. To experimentally validate the computational model, we cloned and expressed the codon optimized Myb domain into a bacterial expression vector and purified the protein to homogeneity. Further, characterization of the protein shows that, Myb domain is predominantly helical (65%) and is alone sufficient to bind the Sal Box DNA. This is the first-ever study to report a complete in silico model of the Myb domain, which is physically characterized. The above study will pave the way towards solving the atomic structure of this essential mammalian protein.
转录终止因子 1(TTF1)是一种多功能哺乳动物蛋白,在 Pol I 介导的转录启动和终止、前 RNA 处理、染色质重塑、DNA 损伤修复和极性复制叉停滞等多种细胞过程中发挥着重要作用。它由两个不同的功能区组成:N 端调节区(1-445 aa)和 C 端催化区(445-859 aa)。位于 C 端的 Myb 结构域是一个保守的 DNA 结合结构域,跨度从 550 到 732 aa(183 个残基)。尽管 TTF1 在各种细胞过程中发挥着关键作用,但其物理结构仍未得到解决。迄今为止,纯化功能性 TTF1 蛋白的尝试尚未成功。因此,我们重点研究了这种重要蛋白质的 Myb 结构域。我们首先使用同源建模和非线性方法预测了 Myb 结构域的三维模型。 然后,我们在显式溶剂中通过 MD 模拟确定了它的稳定性。预测出的模型高度稳定,在 200ns 时稳定下来。为了在实验中验证计算模型,我们将经过密码子优化的 Myb 结构域克隆并表达到细菌表达载体中,并将蛋白质纯化至均一。此外,对蛋白质的表征显示,Myb结构域主要是螺旋结构(65%),仅此就足以与Sal Box DNA结合。这是有史以来第一份报告完整的 Myb 结构域硅模型的研究报告,该模型具有物理特征。上述研究将为解决这一哺乳动物重要蛋白质的原子结构问题铺平道路。
{"title":"Structural characterization of DNA-binding domain of essential mammalian protein TTF 1.","authors":"Gajender Singh, Abhinetra Jagdish Bhopale, Saloni Khatri, Prashant Prakash, Rajnish Kumar, Sukh Mahendra Singh, Samarendra Kumar Singh","doi":"10.1042/BSR20240800","DOIUrl":"10.1042/BSR20240800","url":null,"abstract":"<p><p>Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues). Despite its critical role in various cellular processes, the physical structure of TTF1 remains unsolved. Attempts to purify the functional TTF1 protein have been unsuccessful till date. Therefore, we focused on characterizing the Myb domain of this essential protein. We started with predicting a 3-D model of the Myb domain using homology modelling, and ab-initio method. We then determined its stability through MD simulation in an explicit solvent. The model predicted is highly stable, which stabilizes at 200ns. To experimentally validate the computational model, we cloned and expressed the codon optimized Myb domain into a bacterial expression vector and purified the protein to homogeneity. Further, characterization of the protein shows that, Myb domain is predominantly helical (65%) and is alone sufficient to bind the Sal Box DNA. This is the first-ever study to report a complete in silico model of the Myb domain, which is physically characterized. The above study will pave the way towards solving the atomic structure of this essential mammalian protein.</p>","PeriodicalId":8926,"journal":{"name":"Bioscience Reports","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11358750/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141900877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.
{"title":"Transglutaminase 2-mediated histone monoaminylation and its role in cancer.","authors":"Huapeng Li, Jinghua Wu, Nan Zhang, Qingfei Zheng","doi":"10.1042/BSR20240493","DOIUrl":"10.1042/BSR20240493","url":null,"abstract":"<p><p>Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.</p>","PeriodicalId":8926,"journal":{"name":"Bioscience Reports","volume":null,"pages":null},"PeriodicalIF":3.8,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11345673/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141900878","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}