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The impact of cell states on heterochromatin dynamics. 细胞状态对异染色质动态的影响
IF 4.1 3区 生物学
Biochemical Journal Pub Date : 2024-11-06 DOI: 10.1042/bcj20240139
Abby Trouth,Giovana M B Veronezi,Srinivas Ramachandran
{"title":"The impact of cell states on heterochromatin dynamics.","authors":"Abby Trouth,Giovana M B Veronezi,Srinivas Ramachandran","doi":"10.1042/bcj20240139","DOIUrl":"https://doi.org/10.1042/bcj20240139","url":null,"abstract":"Establishing, maintaining, and removing histone post-translational modifications associated with heterochromatin is critical for shaping genomic structure and function as a cell navigates different stages of development, activity, and disease. Dynamic regulation of the repressive chromatin landscape has been documented in several key cell states - germline cells, activated immune cells, actively replicating, and quiescent cells - with notable variations in underlying mechanisms. Here, we discuss the role of cell states of these diverse contexts in directing and maintaining observed chromatin landscapes. These investigations reveal heterochromatin architectures that are highly responsive to the functional context of a cell's existence and, in turn, their contribution to the cell's stable identity.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142449239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Pyroptotic executioner pore-forming protein GSDMD forms oligomeric assembly and exhibits amyloid-like attributes that could contribute for its pore-forming function. 嗜火刽子手孔道形成蛋白 GSDMD 形成寡聚体集合体,并表现出淀粉样特性,这可能有助于其孔道形成功能。
IF 4.4 3区 生物学
Biochemical Journal Pub Date : 2024-11-06 DOI: 10.1042/BCJ20240416
Shamaita Chatterjee, Tarang Gupta, Gurvinder Kaur, Kausik Chattopadhyay
{"title":"Pyroptotic executioner pore-forming protein GSDMD forms oligomeric assembly and exhibits amyloid-like attributes that could contribute for its pore-forming function.","authors":"Shamaita Chatterjee, Tarang Gupta, Gurvinder Kaur, Kausik Chattopadhyay","doi":"10.1042/BCJ20240416","DOIUrl":"https://doi.org/10.1042/BCJ20240416","url":null,"abstract":"<p><p>Gasdermin D (GSDMD) is the chief executioner of inflammatory cell death or pyroptosis. During pyroptosis, proteolytic processing of GSDMD releases its N-terminal domain (NTD), which then forms large oligomeric pores in the plasma membranes. Membrane pore-formation by NTD allows the release of inflammatory cytokines and causes membrane damage to induce cell death. Structural mechanisms of GSDMD-mediated membrane pore-formation have been extensively studied. However, less effort has been made to understand the physicochemical properties of GSDMD and their functional implications. Here, we explore detailed characterization of the physicochemical properties of mouse GSDMD (mGSDMD), and their implications in regulating the pore-forming function. Our study reveals that mGSDMD shows some of the hallmark features of amyloids, and forms oligomeric assemblies in solution that are critically dependent on the disulphide bond-forming ability of the protein. mGSDMD oligomeric assemblies do not resemble typical amyloid fibrils/aggregates, and do not show resistance to proteolytic degradation that is otherwise observed with the conventional amyloids. Our results further elucidate the essential role of an amyloid-prone region (APR) in the oligomerization and amyloid-like features of mGSDMD. Furthermore, alteration of this APR leads to compromised pore-forming ability and cell-killing activity of NTD released from mGSDMD. Taken together, our study for the first time provides crucial new insights regarding implications of the amyloid-like property of mGSDMD in regulating its pore-forming function, which is an essential requirement for this pyroptotic executioner. To the best of our knowledge, such mode of regulation of mGSDMD-function has not been appreciated so far.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142582029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain. 组蛋白去乙酰化酶 7 通过一种与酶无关的机制激活 6-磷酸葡萄糖酸脱氢酶,该机制涉及 N 端蛋白-蛋白相互作用结构域。
IF 4.4 3区 生物学
Biochemical Journal Pub Date : 2024-11-06 DOI: 10.1042/BCJ20240380
Yizhuo Wang, James E B Curson, Divya Ramnath, Kaustav Das Gupta, Robert C Reid, Denuja Karunakaran, David P Fairlie, Matthew J Sweet
{"title":"Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain.","authors":"Yizhuo Wang, James E B Curson, Divya Ramnath, Kaustav Das Gupta, Robert C Reid, Denuja Karunakaran, David P Fairlie, Matthew J Sweet","doi":"10.1042/BCJ20240380","DOIUrl":"10.1042/BCJ20240380","url":null,"abstract":"<p><p>Histone deacetylase 7 (HDAC7) is a member of the class IIa family of classical HDACs with important roles in cell development, differentiation, and activation, including in macrophages and other innate immune cells. HDAC7 and other class IIa HDACs act as transcriptional repressors in the nucleus but, in some cell types, they can also act in the cytoplasm to modify non-nuclear proteins and/or scaffold signalling complexes. In macrophages, HDAC7 is a cytoplasmic protein with both pro- and anti-inflammatory functions, with the latter activity involving activation of the pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase (6PGD) and the generation of anti-inflammatory metabolite ribulose-5-phosphate. Here, we used ectopic expression systems and biochemical approaches to investigate the mechanism by which HDAC7 promotes 6PGD enzyme activity. We reveal that HDAC7 enzyme activity is not required for its activation of 6PGD and that the N-terminal protein-protein interaction domain of HDAC7 is sufficient to initiate this response. Mechanistically, the N-terminus of HDAC7 increases the affinity of 6PGD for NADP+, promotes the generation of a shorter form of 6PGD, and enhances the formation of higher order protein complexes, implicating its scaffolding function in engagement of the PPP. This contrasts with the pro-inflammatory function of HDAC7 in macrophages, in which it promotes deacetylation of the glycolytic enzyme pyruvate kinase M2 for inflammatory cytokine production.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Epigenetics and alternative splicing in cancer: old enemies, new perspectives. 癌症中的表观遗传学和替代剪接:老对手,新视角。
IF 4.1 3区 生物学
Biochemical Journal Pub Date : 2024-11-06 DOI: 10.1042/bcj20240221
Madhura R Pandkar,Sanjeev Shukla
{"title":"Epigenetics and alternative splicing in cancer: old enemies, new perspectives.","authors":"Madhura R Pandkar,Sanjeev Shukla","doi":"10.1042/bcj20240221","DOIUrl":"https://doi.org/10.1042/bcj20240221","url":null,"abstract":"In recent years, significant strides in both conceptual understanding and technological capabilities have bolstered our comprehension of the factors underpinning cancer initiation and progression. While substantial insights have unraveled the molecular mechanisms driving carcinogenesis, there has been an overshadowing of the critical contribution made by epigenetic pathways, which works in concert with genetics. Mounting evidence demonstrates cancer as a complex interplay between genetics and epigenetics. Notably, epigenetic elements play a pivotal role in governing alternative pre-mRNA splicing, a primary contributor to protein diversity. In this review, we have provided detailed insights into the bidirectional communication between epigenetic modifiers and alternative splicing, providing examples of specific genes and isoforms affected. Notably, succinct discussion on targeting epigenetic regulators and the potential of the emerging field of epigenome editing to modulate splicing patterns is also presented. In summary, this review offers valuable insights into the intricate interplay between epigenetics and alternative splicing in cancer, paving the way for novel approaches to understanding and targeting this critical process.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142449238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
ATP-competitive inhibitors of PI3K enzymes demonstrate an isoform selective dual action by controlling membrane binding. PI3K 酶的 ATP 竞争性抑制剂通过控制膜结合,显示出同工酶选择性的双重作用。
IF 4.4 3区 生物学
Biochemical Journal Pub Date : 2024-11-01 DOI: 10.1042/BCJ20240479
Grace Q Gong, Glenn Robert Masson, Woo-Jeong Jeong Lee, James Mj Dickson, Jackie D Kendall, Manoj K Rathinaswamy, Christina M Buchanan, Martin Middleditch, Brady Owen, Julie A Spicer, Gordon W Rewcastle, William A Denny, John E Burke, Peter R Shepherd, Roger L Williams, Jack U Flanagan
{"title":"ATP-competitive inhibitors of PI3K enzymes demonstrate an isoform selective dual action by controlling membrane binding.","authors":"Grace Q Gong, Glenn Robert Masson, Woo-Jeong Jeong Lee, James Mj Dickson, Jackie D Kendall, Manoj K Rathinaswamy, Christina M Buchanan, Martin Middleditch, Brady Owen, Julie A Spicer, Gordon W Rewcastle, William A Denny, John E Burke, Peter R Shepherd, Roger L Williams, Jack U Flanagan","doi":"10.1042/BCJ20240479","DOIUrl":"https://doi.org/10.1042/BCJ20240479","url":null,"abstract":"<p><p>PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and Biolayer Interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142557052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Exopolysaccharide is detrimental for the symbiotic performance of Sinorhizobium fredii HH103 mutants with a truncated lipopolysaccharide core. 外多糖不利于具有截短脂多糖核心的裂殖单胞菌 HH103 突变体的共生性能。
IF 4.1 3区 生物学
Biochemical Journal Pub Date : 2024-10-25 DOI: 10.1042/bcj20240599
Francisco Fuentes-Romero,Marcello Mercogliano,Stefania De Chiara,Cynthia Alías-Villegas,Pilar Navarro-Gómez,Sebastián Acosta-Jurado,Alba Silipo,Carlos Medina,Miguel-Ángel Rodríguez-Carvajal,Marta S Dardanelli,José-Enrique Ruiz-Sainz,Francisco-Javier López-Baena,Antonio Molinaro,José-María Vinardell,Flaviana Di Lorenzo
{"title":"Exopolysaccharide is detrimental for the symbiotic performance of Sinorhizobium fredii HH103 mutants with a truncated lipopolysaccharide core.","authors":"Francisco Fuentes-Romero,Marcello Mercogliano,Stefania De Chiara,Cynthia Alías-Villegas,Pilar Navarro-Gómez,Sebastián Acosta-Jurado,Alba Silipo,Carlos Medina,Miguel-Ángel Rodríguez-Carvajal,Marta S Dardanelli,José-Enrique Ruiz-Sainz,Francisco-Javier López-Baena,Antonio Molinaro,José-María Vinardell,Flaviana Di Lorenzo","doi":"10.1042/bcj20240599","DOIUrl":"https://doi.org/10.1042/bcj20240599","url":null,"abstract":"The nitrogen-fixing rhizobia-legume symbiosis relies on a complex interchange of molecular signals between the two partners during the whole interaction. On the bacterial side, different surface polysaccharides, such as lipopolysaccharide (LPS) and exopolysaccharide (EPS), might play important roles for the success of the interaction. In a previous work we studied two Sinorhizobium fredii HH103 mutants affected in the rkpK and lpsL genes, which are responsible for the production of glucuronic acid and galacturonic acid, respectively. Both mutants produced an altered LPS, and the rkpK mutant, in addition, lacked EPS. These mutants were differently affected in symbiosis with Glycine max and Vigna unguiculata, with the lpsL mutant showing a stronger impairment than the rkpK mutant. In the present work we have further investigated the LPS structure and the symbiotic abilities of the HH103 lpsL and rkpK mutants. We demonstrate that both strains produce the same LPS, with a truncated core oligosaccharide devoid of uronic acids. We show that the symbiotic performance of the lpsL mutant with Macroptilium atropurpureum and Glycyrrhiza uralensis is worse than that of the rkpK mutant. Introduction of an exoA mutation (which avoids EPS production) in HH103 lpsL improved its symbiotic performance with G. max, M. atropurpureum, and G. uralensis to the level exhibited by HH103 rkpK, suggesting that the presence of EPS might hide the truncated LPS produced by the former mutant.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142490566","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Comparative Analysis of Canine and Human HtrA2 to Delineate Its Role in Apoptosis and Cancer. 比较分析犬和人的 HtrA2 以确定其在细胞凋亡和癌症中的作用
IF 4.1 3区 生物学
Biochemical Journal Pub Date : 2024-10-17 DOI: 10.1042/bcj20240295
Snehal Pandav Mudrale,Shubhankar Dutta,Kalyani Natu,Pradip Chaudhari,Kakoli Bose
{"title":"Comparative Analysis of Canine and Human HtrA2 to Delineate Its Role in Apoptosis and Cancer.","authors":"Snehal Pandav Mudrale,Shubhankar Dutta,Kalyani Natu,Pradip Chaudhari,Kakoli Bose","doi":"10.1042/bcj20240295","DOIUrl":"https://doi.org/10.1042/bcj20240295","url":null,"abstract":"Therapeutically, targeting the pro- and anti-apoptotic proteins has been one of the major approaches behind devising strategies to combat associated diseases. Human high-temperature requirement serine protease A2 (hHtrA2), which induces apoptosis through both caspase-dependent and independent pathways is implicated in several diseases including cancer, ischemic heart diseases, and neurodegeneration, thus making it a promising target molecule. In the recent past, the canine model has gained prominence in the understanding of human pathophysiology that was otherwise limited to the rodent system. Moreover, canine models in cancer research provide an opportunity to study spontaneous tumors as their size, lifespan, and environmental exposure are significantly closer to that of humans compared to laboratory rodents. Therefore, using HtrA2 as a model protein, comparative analysis has been done to revisit the hypothesis that canines might be excellent models for cancer research. We have performed evolutionary phylogenetic analyses that confirm a close relationship between canine and human HtrA2s. Molecular modeling demonstrates structural similarities including orientation of the catalytic triad residues, followed by in silico docking and molecular dynamics simulation studies that identify the potential interacting partners for canine HtrA2 (cHtrA2). In vitro biophysical and protease studies depict similarities in interaction with their respective substrates as well as transient transfection of cHtrA2 in mammalian cell culture shows induction of apoptosis. This work, therefore, promises to open a new avenue in cancer research through the study of spontaneous cancer model systems in canines.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142447951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Yop1 stability and membrane curvature generation propensity are controlled by its oligomerisation interface. Yop1 的稳定性和膜曲率生成倾向受其寡聚界面控制。
IF 4.4 3区 生物学
Biochemical Journal Pub Date : 2024-10-16 DOI: 10.1042/BCJ20240190
Anu V Chandran, Daniel Álvarez, Stefano Vanni, Jason R Schnell
{"title":"Yop1 stability and membrane curvature generation propensity are controlled by its oligomerisation interface.","authors":"Anu V Chandran, Daniel Álvarez, Stefano Vanni, Jason R Schnell","doi":"10.1042/BCJ20240190","DOIUrl":"10.1042/BCJ20240190","url":null,"abstract":"<p><p>The DP1 family of integral membrane proteins stabilize high membrane curvature in the endoplasmic reticulum and phagophores. Mutations in the human DP1 gene REEP1 are associated with Hereditary Spastic Paraplegia type 31 and distal hereditary motor neuropathy. Four missense mutations map to a putative dimerization interface but the impact of these mutations on DP1 structure and tubule formation are unknown. Combining biophysical measurements, functional assays, and computational modeling in the context of the model protein Yop1, we found that missense mutations have variable effects on DP1 dimer structure and in vitro tubulation activity, and provide mechanistic insights into the role of DP1 oligomerisation on membrane curvature stabilization. Whereas the mutations P71L and S75F decreased dimer homogeneity and led to polydisperse oligomerization and impaired membrane curving activity, A72E introduced new polar interactions between subunits that stabilized the Yop1 dimer and allowed robust tubule formation but prevented formation of more highly-curved lipoprotein particles (LPP). The introduction of a BRIL domain to the cytoplasmic loop of A72E rescued LPP formation, consistent with a requirement for dimer splaying in highly curved membranes. These results suggest that the membrane curving activity of DP1 proteins requires both dimer stability and conformational plasticity at the intermolecular interface.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142279994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Adapting to change: resolving the dynamic and dual roles of NCK1 and NCK2. 适应变化:解决 NCK1 和 NCK2 的动态和双重作用。
IF 4.4 3区 生物学
Biochemical Journal Pub Date : 2024-10-16 DOI: 10.1042/BCJ20230232
Valentine Teyssier, Casey R Williamson, Erka Shata, Stephanie P Rosen, Nina Jones, Nicolas Bisson
{"title":"Adapting to change: resolving the dynamic and dual roles of NCK1 and NCK2.","authors":"Valentine Teyssier, Casey R Williamson, Erka Shata, Stephanie P Rosen, Nina Jones, Nicolas Bisson","doi":"10.1042/BCJ20230232","DOIUrl":"10.1042/BCJ20230232","url":null,"abstract":"<p><p>Adaptor proteins play central roles in the assembly of molecular complexes and co-ordinated activation of specific pathways. Through their modular domain structure, the NCK family of adaptor proteins (NCK1 and NCK2) link protein targets via their single SRC Homology (SH) 2 and three SH3 domains. Classically, their SH2 domain binds to phosphotyrosine motif-containing receptors (e.g. receptor tyrosine kinases), while their SH3 domains bind polyproline motif-containing cytoplasmic effectors. Due to these functions being established for both NCK1 and NCK2, their roles were inaccurately assumed to be redundant. However, in contrast with this previously held view, NCK1 and NCK2 now have a growing list of paralog-specific functions, which underscores the need to further explore their differences. Here we review current evidence detailing how these two paralogs are unique, including differences in their gene/protein regulation, binding partners and overall contributions to cellular functions. To help explain these contrasting characteristics, we then discuss SH2/SH3 structural features, disordered interdomain linker regions and post-translational modifications. Together, this review seeks to highlight the importance of distinguishing NCK1 and NCK2 in research and to pave the way for investigations into the origins of their interaction specificity.</p>","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Identification of inhibitors of human ChaC1, a cytoplasmic glutathione degrading enzyme through high throughput screens in yeast. 通过酵母中的高通量筛选鉴定人ChaC1(一种细胞质谷胱甘肽降解酶)的抑制剂。
IF 4.1 3区 生物学
Biochemical Journal Pub Date : 2024-10-16 DOI: 10.1042/bcj20240447
Shradha Suyal,Chinmayee Choudhury,Deepinder Kaur,Anand K Bachhawat
{"title":"Identification of inhibitors of human ChaC1, a cytoplasmic glutathione degrading enzyme through high throughput screens in yeast.","authors":"Shradha Suyal,Chinmayee Choudhury,Deepinder Kaur,Anand K Bachhawat","doi":"10.1042/bcj20240447","DOIUrl":"https://doi.org/10.1042/bcj20240447","url":null,"abstract":"The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. Synthetic substrate analogs were also not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":null,"pages":null},"PeriodicalIF":4.1,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142436178","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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