Biochemical Journal最新文献_第9页

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":"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.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"1569-1584"},"PeriodicalIF":4.4,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11555707/pdf/","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":"OA","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":"12 1","pages":"1497-1518"},"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

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":"5 1","pages":""},"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":"31 1","pages":""},"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

Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain. 丝胶蛋白 A 通过肌球蛋白轻链的磷酸化调节血小板形状的改变和收缩力的产生。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-10-17 DOI: 10.1042/BCJ20240114

Felix Hong, Molly Y Mollica, Kalyan Golla, Enoli De Silva, Nathan J Sniadecki, José A López, Hugh Kim

{"title":"Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain.","authors":"Felix Hong, Molly Y Mollica, Kalyan Golla, Enoli De Silva, Nathan J Sniadecki, José A López, Hugh Kim","doi":"10.1042/BCJ20240114","DOIUrl":"10.1042/BCJ20240114","url":null,"abstract":"Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"1395-1410"},"PeriodicalIF":4.4,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11555712/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142071920","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}

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":"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.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"1437-1448"},"PeriodicalIF":4.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11555649/pdf/","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":"OA","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":"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.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":"481 20","pages":"1411-1435"},"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

Revamped role for approved drug: integrative computational and biophysical analysis of saquinavir's peptidyl arginine deiminase 4 inhibition for rheumatoid arthritis. 获批药物的新角色：萨喹那韦对类风湿关节炎的 PAD4 抑制作用的综合计算和生物物理分析。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-10-16 DOI: 10.1042/BCJ20240366

Indhumathi Thirugnanasambandham, Srikanth Jupudi, Parikshit Roychowdhury, Veera Venkata Satyanarayana Reddy Karri, Subba Rao V Madhunapantula, Sachin Kumar Singh, Vetriselvan Subramaniyan, Gowthamarajan Kuppusamy

{"title":"Revamped role for approved drug: integrative computational and biophysical analysis of saquinavir's peptidyl arginine deiminase 4 inhibition for rheumatoid arthritis.","authors":"Indhumathi Thirugnanasambandham, Srikanth Jupudi, Parikshit Roychowdhury, Veera Venkata Satyanarayana Reddy Karri, Subba Rao V Madhunapantula, Sachin Kumar Singh, Vetriselvan Subramaniyan, Gowthamarajan Kuppusamy","doi":"10.1042/BCJ20240366","DOIUrl":"10.1042/BCJ20240366","url":null,"abstract":"The pursuit of novel therapeutics is a complex and resource-intensive endeavor marked by significant challenges, including high costs and low success rates. In response, drug repositioning strategies leverage existing FDA-approved compounds to predict their efficacy across diverse diseases. Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in protein citrullination, a process implicated in the autoimmune pathogenesis of rheumatoid arthritis (RA). Targeting PAD4 has thus emerged as a promising therapeutic approach. This study employs computational and enzyme inhibition strategies to identify potential PAD4-targeting compounds from a library of FDA-approved drugs. In silico docking analyses validated the binding interactions and orientations of screened compounds within PAD4's active site, with key residues such as ASP350, HIS471, ASP473, and CYS645 participating in crucial hydrogen bonding and van der Waals interactions. Molecular dynamics simulations further assessed the stability of top compounds exhibiting high binding affinities. Among these compounds, Saquinavir (SQV) emerged as a potent PAD4 inhibitor, demonstrating competitive inhibition with a low IC50 value of 1.21 ± 0.04 µM. In vitro assays, including enzyme kinetics and biophysical analyses, highlighted significant changes in PAD4 conformation upon SQV binding, as confirmed by circular dichroism spectroscopy. SQV induced localized alterations in PAD4 structure, effectively occupying the catalytic pocket and inhibiting enzymatic activity. These findings underscore SQV's potential as a therapeutic candidate for RA through PAD4 inhibition. Further validation through in vitro and in vivo studies is essential to confirm SQV's therapeutic benefits in autoimmune diseases associated with dysregulated citrullination.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"1379-1393"},"PeriodicalIF":4.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142279993","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":"10 1","pages":"1475-1495"},"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

CDS2 expression regulates de novo phosphatidic acid synthesis. CDS2 的表达调控 PA 的从头合成。

IF 4.4 3区生物学

Biochemical Journal Pub Date : 2024-10-16 DOI: 10.1042/BCJ20240456

Daniel M Collins, Vishnu Janardan, David Barneda, Karen E Anderson, Izabella Niewczas, Diane Taylor, Danye Qiu, Henning J Jessen, Andrea F Lopez-Clavijo, Simon Walker, Padinjat Raghu, Jonathan Clark, Len R Stephens, Phillip T Hawkins

{"title":"CDS2 expression regulates de novo phosphatidic acid synthesis.","authors":"Daniel M Collins, Vishnu Janardan, David Barneda, Karen E Anderson, Izabella Niewczas, Diane Taylor, Danye Qiu, Henning J Jessen, Andrea F Lopez-Clavijo, Simon Walker, Padinjat Raghu, Jonathan Clark, Len R Stephens, Phillip T Hawkins","doi":"10.1042/BCJ20240456","DOIUrl":"10.1042/BCJ20240456","url":null,"abstract":"CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the 'PI cycle', leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.","PeriodicalId":8825,"journal":{"name":"Biochemical Journal","volume":" ","pages":"1449-1473"},"PeriodicalIF":4.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11555650/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142279992","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}

引用次数: 0