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Variation in type IV pilus stability modulates DNA-uptake and biofilm formation. IV型菌毛稳定性的变化调节dna摄取和生物膜的形成。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-06 DOI: 10.1016/j.jbc.2025.110787
Yafan Yu,Rabab Mahdi,Ahmad Al-Hilfy Leon,Nam Vo,Reese Lofgren,Jean Luc Mutabazi,Kurt H Piepenbrink
{"title":"Variation in type IV pilus stability modulates DNA-uptake and biofilm formation.","authors":"Yafan Yu,Rabab Mahdi,Ahmad Al-Hilfy Leon,Nam Vo,Reese Lofgren,Jean Luc Mutabazi,Kurt H Piepenbrink","doi":"10.1016/j.jbc.2025.110787","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110787","url":null,"abstract":"Type IV pili are helical filaments composed of protein subunits which are produced by numerous taxa of bacteria, including Acinetobacter. Type IV pili are extended out from the cell by extension enzyme complexes, which extract subunits from the membrane and insert them into the base of the filament, but can also be retracted by reverse rotation catalyzed by a retraction enzyme. Type IV pili have diverse functions, including twitching motility and DNA-uptake, which require retraction, and host adhesion and bacterial aggregation, which do not. Acinetobacter bacteria, including International Clone I (IC-I) and International Clone II (IC-II) strains, show variable phenotypes in assays of type IV pilus-dependent functions. Here, we show this variation is the result of differentiation of type IV pilus subtypes in Acinetobacter, which we defined based on the sequence of the major subunit, PilA. These subtypes show variable efficiency in pilus retraction between pilus subtypes, and from that, a differential balance between retraction-dependent and retraction-independent functions. In both naturally-occurring pilA variants from the IC-I and IC-II groups and isogenic strains complemented with IC-I or IC-II pilA, the IC-I pilus subtype promotes greater twitching motility and DNA-uptake while the IC-II pilus subtype promotes biofilm formation while showing reduced capacity for DNA-uptake and twitching motility, similar to a retraction-deficient mutant and consistent with the hypothesis that pilus retraction of the IC-II pilus is naturally deficient. This defect in retraction was sufficient to increase the level of piliation on the cell surface when we compared the yields of T4P sheared from the cell surface from IC-I pilA and IC-II pilA complements in an isogenic background. Complementation with IC-II pilA results in greater levels of surface PilA per cell than equivalent complementation with an IC-I pilA gene. Additionally, direct comparisons of pilus stability between type IV pili isolated from IC-I pilA and IC-II pilA complements show greater thermostability for the IC-II pili, supporting the hypothesis that pilus stability can impede retraction and increase piliation.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"82 1","pages":"110787"},"PeriodicalIF":4.8,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145246785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
The non-conserved integrin cytoplasmic region determines integrin subtype-specific characteristics by modulating talin1 binding kinetics. 非保守的整合素细胞质区域通过调节talin1结合动力学来决定整合素亚型特异性特征。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-06 DOI: 10.1016/j.jbc.2025.110793
Naoyuki Kondo,Kenji Fukui,Yuji Kamioka,Yoshihiro Ueda,Yoshiki Ikeda,Taiju Matsushita,Ryo Yazaki,Tatsuo Kinashi
{"title":"The non-conserved integrin cytoplasmic region determines integrin subtype-specific characteristics by modulating talin1 binding kinetics.","authors":"Naoyuki Kondo,Kenji Fukui,Yuji Kamioka,Yoshihiro Ueda,Yoshiki Ikeda,Taiju Matsushita,Ryo Yazaki,Tatsuo Kinashi","doi":"10.1016/j.jbc.2025.110793","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110793","url":null,"abstract":"Talin governs integrin adhesion by binding to the cytoplasmic tail (CT) of integrin β subunits, but the effects of integrin subtype-specific variations on talin interactions remain unclear. Here, we identify a non-conserved region within the integrin-CT, termed the WN linker, that modulates talin1 binding kinetics and integrin adhesiveness. Single-molecule imaging in live lymphocytes revealed that talin1 bound more strongly to β2 than β7 integrin, with higher off-rates in β7 in vivo. This difference was due to the unique NND sequence in the β2 WN linker compared with KQDS in the β7 WN linker. Structural and biochemical analyses showed that NND established a tighter interaction with talin, whereas KQDS bent, narrowing the interaction area and weakening the interaction. Substituting the NND sequence in β2 with KQDS impaired inside-out signaling- and ligand binding-induced conformational activation of LFA1. Multiple sequence alignment and single-molecule binding analyses revealed that the NND sequence is highly conserved only in mammalian β2 integrins, and that the second asparagine in NND, a residue absent in non-mammalian β2 integrins and other integrins, plays a key role in talin1 binding. Parallel observations in β3 integrins reinforced the pivotal role of the WN linker in modulating integrin-talin affinity. These observations highlight the WN linker as a novel regulator of integrin-talin binding strength and adhesiveness diversity.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"61 1","pages":"110793"},"PeriodicalIF":4.8,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145246789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Sirtuin 6 is a histone delactylase. Sirtuin 6是一种组蛋白去乙酰化酶。
IF 4 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-06 DOI: 10.1016/j.jbc.2025.110795
Garrison A Nickel, Nicholas J Pederson, Faheem, Zhenyu Yang, Jack Bulf, Katharine L Diehl
{"title":"Sirtuin 6 is a histone delactylase.","authors":"Garrison A Nickel, Nicholas J Pederson, Faheem, Zhenyu Yang, Jack Bulf, Katharine L Diehl","doi":"10.1016/j.jbc.2025.110795","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110795","url":null,"abstract":"<p><p>Histone lactylation (Kla) is a post translational modification (PTM) that is derived from metabolic lactate. Histone Kla has been extensively studied in the field of inflammation resolution and macrophage polarization but has also been implicated in diverse cellular processes including differentiation, various wound repair phenotypes, and oncogenesis in several cancer models. While mechanistic connections between histone Kla and transcriptional changes have been studied in very limited contexts, general mechanistic details describing how regulation of gene expression by histone Kla occurs are scarce. It is hypothesized that histone Kla may be installed either through nonenzymatic means or by acetyltransferases like p300, and it is known that Class I HDACs and Sirtuins 1-3 can remove histone Kla. Here, we identified histone delactylase activity of the deacylase enzyme Sirtuin 6 (Sirt6), a member of the Class III HDAC family known to have roles in regulating metabolic homeostasis. We characterized the ability of Sirt6 to delactylate histones in vitro and in a mammalian cell culture model. We identified H3K9 and H3K18, canonical histone sites of Sirt6-catalyzed deacetylase activity, as sites of its delactylase activity. We also demonstrated that Sirt6 and the Class I HDACs exhibit some degree of non-overlapping delactylase activity, suggesting that they represent different cellular axes of regulating gene expression via controlling levels of histone Kla.</p>","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":" ","pages":"110795"},"PeriodicalIF":4.0,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251137","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
In vivo analysis of Drosophila chondroitin sulfate biosynthetic genes. 果蝇硫酸软骨素生物合成基因的体内分析。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-06 DOI: 10.1016/j.jbc.2025.110783
Tomomi Izumikawa,Ayano Moriya,Eriko Nakato,Kako Yamamoto,Raiki Sano,Takuya Akiyama,Akiko Kinoshita-Toyoda,Hidenao Toyoda,Hiroshi Nakato
{"title":"In vivo analysis of Drosophila chondroitin sulfate biosynthetic genes.","authors":"Tomomi Izumikawa,Ayano Moriya,Eriko Nakato,Kako Yamamoto,Raiki Sano,Takuya Akiyama,Akiko Kinoshita-Toyoda,Hidenao Toyoda,Hiroshi Nakato","doi":"10.1016/j.jbc.2025.110783","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110783","url":null,"abstract":"Chondroitin sulfate (CS) is an evolutionarily conserved class of glycosaminoglycans and is found in most animal species. Previous studies of CS-deficient Drosophila models, Chondroitin sulfate synthase (Chsy) and Chondroitin polymerizing factor (Chpf) mutants, demonstrated the importance of CS in structural integrity of the basement membrane and organ shape maintenance. However, biosynthetic mechanisms of Drosophila CS remain to be elucidated. To investigate the CS biosynthesis in Drosophila, we generated mutants for two additional biosynthetic enzyme genes, CS N-acetylgalactosaminyltransferase (Csgalnact) and CS 4-O sulfotransferase (C4st), using CRISPR/Cas9 mutagenesis. Csgalnact null mutants show moderate changes in CS biosynthesis, including reduced CS in the larval brain and altered CS chain length. We found that this gene is dispensable for normal viability and morphogenesis. On the other hand, C4st mutants show more severe defects, including a high level of lethality and a folded wing phenotype. The C4st mutation not only eliminates CS sulfation but increases production of unsulfated chondroitin, suggesting the existence of a compensatory feedback mechanism. Both Csgalnact and C4st mutants show impaired adult negative geotaxis behavior, consistent with CSPGs' roles in the neuromuscular systems. Our study revealed unique and poorly understood features of invertebrate CS biosynthesis and provides novel in vivo toolsets to investigate CSPG functions in development.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"15 1","pages":"110783"},"PeriodicalIF":4.8,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145246758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Cyanophycinase is required for heterotrophy in cyanobacteria. 蓝藻酶是异养蓝藻所必需的。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-06 DOI: 10.1016/j.jbc.2025.110791
Éva Kiss,Martin Moos,Jan Mareš,Stanislav Opekar,Lenka Tomanová,Paulina Duhita Anindita,Martin Lukeš,Petra Urajová,Roman Sobotka
{"title":"Cyanophycinase is required for heterotrophy in cyanobacteria.","authors":"Éva Kiss,Martin Moos,Jan Mareš,Stanislav Opekar,Lenka Tomanová,Paulina Duhita Anindita,Martin Lukeš,Petra Urajová,Roman Sobotka","doi":"10.1016/j.jbc.2025.110791","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110791","url":null,"abstract":"Cyanophycin is a biopolymer of arginine and aspartate, and it is found in various prokaryotes. Two key enzymes of cyanophycin metabolism are cyanophycin synthase (CphA) producing cyanophycin, and cyanophycinase (CphB) catalysing the first step of cyanophycin degradation. CphB is a well conserved enzyme found in the majority of cyanobacteria, and ubiquitous amongst those that are known to perform heterotrophy besides their primary photosynthetic lifestyle. Unlike in diazotrophs, where CphB is connected to the mobilization of fixed nitrogen, the importance of this enzyme remains elusive in non-diazotrophs, such as the model cyanobacterium Synechocystis sp. PCC 6803. The Synechocystis ΔcphB deletion strain does not accumulate cyanophycin and shows no photoautotrophic growth defect. However, we show here that ΔcphB is not able to proliferate heterotrophically, although the CphA-less strain exhibits no obvious defect under heterotrophic conditions. Metabolomics profiling revealed that ΔcphB failed to upregulate the biosynthesis of arginine and displayed missregulated carbon and nucleoside metabolisms. These suggest that CphB is needed for the activation of the arginine pathway, which appeared to be crucial for balancing the nitrogen and carbon ratio during the acclimation to heterotrophy. On the other hand, the interaction of CphB with the Arg biosynthetic enzyme, acetylornithine aminotransferase, stimulated the hydrolysis of cyanophycin in an in vitro assay. These data, together with the metabolic profiles of ΔcphB, imply that the catabolism of cyanophycin and the biosynthesis of Arg are mutually co-regulated metabolic pathways.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"213 1","pages":"110791"},"PeriodicalIF":4.8,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145246759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
The deubiquitinase OTUD4 suppresses TAK1 kinase-dependent NF-κB signaling and inflammation. 去泛素酶OTUD4抑制TAK1激酶依赖的NF-κB信号传导和炎症。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-06 DOI: 10.1016/j.jbc.2025.110784
Zhaohui Liu,Xiaolong Wang,Lihui Wu,Li Min,Ying Sheng,Zhiming Sun,Yunfang Deng,Lin Miao,Yue Liu,Jiabing Li,Yu Zhao
{"title":"The deubiquitinase OTUD4 suppresses TAK1 kinase-dependent NF-κB signaling and inflammation.","authors":"Zhaohui Liu,Xiaolong Wang,Lihui Wu,Li Min,Ying Sheng,Zhiming Sun,Yunfang Deng,Lin Miao,Yue Liu,Jiabing Li,Yu Zhao","doi":"10.1016/j.jbc.2025.110784","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110784","url":null,"abstract":"Chronic inflammation contributes to the development of many cancers, including non-small cell lung cancer (NSCLC), and is characterized by persistent activation of pro-inflammatory NF-κB signaling. The mechanisms that restrain NF-κB signaling remain incompletely defined. Here, we identify the deubiquitinase OTUD4 as a suppressor of tumor necrosis factor (TNF)-induced NF-κB activation and chronic inflammation. OTUD4 interacts with core components of the TAK1 signalosome, including TAK1, TAB1, and TAB3, and removes K63-linked polyubiquitin chains from substrates within this complex, such as TAK1 and TAB3, thereby reducing TNF-induced NF-κB signaling. A histidine-centered loop (His loop) in the catalytic domain is required for this K63 linkage specificity. The tumor-associated OTUD4 H148Y missense variant (c.442C>T, p.H148Y), located within this loop, retains TAK1 binding but abolishes intrinsic deubiquitinase activity toward both K63- and K48-linked chains and is associated with sustained NF-κB activation and increased pro-inflammatory cytokine expression. Collectively, these results reveal a mechanism that suppresses TNF-induced NF-κB signaling and links OTUD4 dysfunction to inflammation-driven oncogenesis, including NSCLC.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"33 1","pages":"110784"},"PeriodicalIF":4.8,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145246788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Microphysiological Systems as a Pillar of the Human Exposome Project. 微生理系统作为人体暴露计划的支柱。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-04 DOI: 10.1016/j.jbc.2025.110782
Fenna Sillé,Lena Smirnova,Thomas Hartung
{"title":"Microphysiological Systems as a Pillar of the Human Exposome Project.","authors":"Fenna Sillé,Lena Smirnova,Thomas Hartung","doi":"10.1016/j.jbc.2025.110782","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110782","url":null,"abstract":"The Human Exposome Project (HEP) aims to decode how lifelong environmental exposures shape health and disease, complementing genomic insights with a systems-level understanding of external influences. Achieving this vision requires experimental platforms that move beyond the limitations of animal models, which often lack human relevance and mechanistic resolution. Microphysiological systems (MPS)-including organoids and organs-on-chips derived from human stem cells-offer such an opportunity. These engineered models recapitulate human tissue architecture and function under controlled conditions, enabling direct study of exposure-response relationships at the cellular and organ level. In this review, we outline how MPS can serve as a foundation for exposome research by bridging epidemiological observations with mechanistic biology. We describe applications ranging from air pollutant toxicity to food contaminants, endocrine disruptors, and nanomaterials, highlighting how MPS integrated with omics technologies and artificial intelligence (AI) can reveal pathways of injury, identify biomarkers, and support the development of digital twins to simulate exposure-disease trajectories. We also discuss frameworks for validation, quality assurance, and transparent reporting, which are essential for reproducibility and regulatory acceptance. Finally, we consider ethical issues such as donor rights, data sovereignty, and equitable access, underscoring the importance of anticipatory governance. Together, MPS represent more than alternatives to animal testing-they are strategic enablers of a human-relevant, AI-empowered exposome science. By anchoring statistical associations in mechanistic data, MPS can accelerate translation into public health policies that are predictive, preventive, and personalized.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"10 1","pages":"110782"},"PeriodicalIF":4.8,"publicationDate":"2025-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145235866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly. E3泛素连接酶SPRYD3-MYCBP2(PAM)调节有丝分裂细胞命运和USP11泛素化,控制纺锤体组装。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-04 DOI: 10.1016/j.jbc.2025.110785
Alexandra Rita Turi da Fonte Dias,Ingrid Hoffmann
{"title":"The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly.","authors":"Alexandra Rita Turi da Fonte Dias,Ingrid Hoffmann","doi":"10.1016/j.jbc.2025.110785","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110785","url":null,"abstract":"MYCBP2 (PAM) is a large signalling hub that plays a key role in various processes, including neuronal connectivity and growth, cell division, and protein ubiquitination. Together with the substrate specificity factor FBXO45, MYCBP2 forms an E3 ligase complex that is involved in mitotic cell fate decision. During extended mitotic arrest caused by anti-microtubule drugs, cells may either experience cell death or escape mitosis through mitotic slippage. E3 ligase mediated ubiquitination is antagonized by deubiquitinating enzymes (DUBs). In this study, we show that despite their opposing activities, DUB-E3 ligase complexes can form and cooperate. We identify an E3 ligase complex consisting of MYCBP2 and a new substrate specificity factor, SPRYD3. Interestingly, SPRYD3-MYCBP2 promotes bipolar spindle formation by facilitating non-canonical ubiquitination on the DUB USP11 cysteine 318. We find that this process promotes bipolar spindle formation and mitotic slippage in presence of microtubule targeting drugs.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"49 1","pages":"110785"},"PeriodicalIF":4.8,"publicationDate":"2025-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145235865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
A systematic comparison of in-house prepared transfection reagents in the delivery of mRNA or DNA to a wide range of cultured cells. 系统比较内部制备的转染试剂在mRNA或DNA传递到广泛的培养细胞中的作用。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-10-03 DOI: 10.1016/j.jbc.2025.110742
Ravi Ojha,Emilia Timin,Leonora Szirovicza,Wujun Xu,Jussi Hepojoki
{"title":"A systematic comparison of in-house prepared transfection reagents in the delivery of mRNA or DNA to a wide range of cultured cells.","authors":"Ravi Ojha,Emilia Timin,Leonora Szirovicza,Wujun Xu,Jussi Hepojoki","doi":"10.1016/j.jbc.2025.110742","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110742","url":null,"abstract":"Transfection is a fundamental molecular biology technique, enabling gene editing, protein expression, and vaccine development. However, transfection efficiency and cytotoxicity vary widely between reagent and cell type, necessitating optimization. Commercial reagents such as Lipofectamine 2000 and FuGENE HD are widely used for their high efficiency, but they are expensive and the efficiency can associate with cytotoxicity. In-house alternatives such as linear polyethylenimine (PEI; 25 kDa and 40 kDa) and cationic lipids-1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-combined with dioleoylphosphatidylethanolamine (DOPE) offer cost-effective options, but their performance across diverse cell types and nucleic acid type (RNA or DNA) remains insufficiently characterized. This motivated us to systematically evaluate transfection efficiency, cytotoxicity, and complex stability of these in-house reagents (DOPE:DOTAP and DOPE:DOTMA tested at molar ratios of 0.5:1, 1:1, and 2:1) over a broad range of reagent to nucleic acid ratios, using plasmid DNA and mRNA encoding mCherry. We performed transfections across 14 cell lines derived from human, monkey, frog, snake, and rodent tissues. We utilized automated fluorescence microscopy for quantifying transfection efficiency, luminescence-based viability assays for cytotoxicity, and studied complex stability during storage at 4°C (0, 4, and 24 hours) through transfection. Results revealed cell line-dependent differences in transfection efficiency, and showed in-house cationic lipid formulations to have a high mRNA transfection efficiency with low cytotoxicity. Lipofectamine 2000 and PEI 40k formed the most stable DNA complexes, but with higher cytotoxicity. This study provides a comprehensive reference for selecting customizable, cost-effective transfection reagents for specific cell and nucleic acid types.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"3 1","pages":"110742"},"PeriodicalIF":4.8,"publicationDate":"2025-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145228993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Evidence of an activity-enhancing conformational shift in Arabidopsis thaliana Plant Cysteine Oxidase 4 induced by binding of substrate or substrate-mimics. 拟南芥植物半胱氨酸氧化酶4结合底物或底物模拟物诱导的增强活性的构象转变的证据。
IF 4.8 2区 生物学
Journal of Biological Chemistry Pub Date : 2025-09-29 DOI: 10.1016/j.jbc.2025.110770
R Latter,J C J Hintzen,L M N Shah,D M Gunawardana,R M Sher,M D White,J Mecinović,J L P Benesch,E Flashman
{"title":"Evidence of an activity-enhancing conformational shift in Arabidopsis thaliana Plant Cysteine Oxidase 4 induced by binding of substrate or substrate-mimics.","authors":"R Latter,J C J Hintzen,L M N Shah,D M Gunawardana,R M Sher,M D White,J Mecinović,J L P Benesch,E Flashman","doi":"10.1016/j.jbc.2025.110770","DOIUrl":"https://doi.org/10.1016/j.jbc.2025.110770","url":null,"abstract":"Plant cysteine oxidases (PCOs) are a family of O2-dependent, thiol dioxygenase enzymes that help to coordinate plant responses to flooding by determining the stability of hypoxia-related transcription factors, Group VII Ethylene Response Factors (ERF-VIIs). Under normoxia, PCOs use O2 to catalyse the oxidation of the Nt-Cys of ERF-VIIs. The resultant N-degron proceeds along the Cys/Arg N-degron pathway for degradation. Conversely, hypoxic conditions such as those experienced during flooding, decrease PCO activity, stabilising ERF-VIIs which proceed to upregulate hypoxia responsive genes, enabling adaptations to submergence. PCOs are a target for improving plant flood tolerance. Inhibition of PCOs may prepare plants for submergence by promoting upregulation of hypoxia responsive genes. Previous work identified small molecule inhibitors of AtPCO4 and demonstrated that their use as a pre-treatment improved seedling tolerance to subsequent anoxia exposure. In this work, the pursuit of a peptide-based inhibition approach led to the development of ERF-VII derived peptidomimetics with modified N-termini. Upon testing in vitro, the peptidomimetics unexpectedly enhanced, rather than inhibited, AtPCO4 activity. Furthermore, anaerobic preincubation of PCOs with substrate itself was found to induce a similar response. Hydrogen-deuterium exchange mass spectrometry indicated that preincubation with peptidomimetic induces a conformational change in PCO structure. This suggests that substrate binding under anaerobic conditions could promote \"conformational priming\" of AtPCO4, where flexible regions within or surrounding the active site adopt a conformation that favours enhanced enzymatic activity. These data are the first evidence for dynamic movement of PCO structures and may be of significance for post-hypoxia PCO activity in planta.","PeriodicalId":15140,"journal":{"name":"Journal of Biological Chemistry","volume":"76 1","pages":"110770"},"PeriodicalIF":4.8,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145203466","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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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