Biochimica et biophysica acta. General subjects最新文献_第9页

The polyphenols phloretin and quercetin are potent horseradish peroxidase (HRP) inhibitors 多酚类根皮素和槲皮素是有效的辣根过氧化物酶（HRP）抑制剂。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-08-01 Epub Date: 2025-06-13 DOI: 10.1016/j.bbagen.2025.130833

Ben Faerman , Olivia Chalifoux , Marek Michalak , Luis B. Agellon , Ryan J. Mailloux

The discovery of horseradish peroxidase (HRP) has been highly advantageous because its unique chemistry can be applied to diagnostic tools, including the detection of oxidative distress markers like hydrogen peroxide (H₂O₂) in various experimental systems. Here, we made the surprising and compelling finding that the flavonoids, phloretin and quercetin, which are usually described in the literature as potent antioxidants, strongly inhibit the activity of HRP. Using the amplex ultrared (AUR) assay, we discovered that phloretin at a concentration as low as 50 μM abolishes the detection of H₂O₂ production by isolated liver mitochondria oxidizing pyruvate and malate. Phloretin also nullified the detection of H₂O₂ produced by liver mitochondria oxidizing succinate or dihydroorotate. Moreover, phloretin at 100 μM completely abolished the direct detection of H₂O₂ by AUR and quenched the detection of purified xanthine oxidase (XO) activity, but did not interfere with dichlorodihydrofluorescein diacetate (H₂-DCFDA) or dihydroethidine (DHE) fluorescent assays. Dose response assays revealed quercetin is a more potent inhibitor for HRP when compared to phloretin. Indeed, quercetin abolished resorufin fluorescence in AUR assays in the nM range whereas phloretin had no effect when detecting H₂O₂ in vitro or when it is formed by isolated liver mitochondria or cultured Huh-7 hepatoma and Mia-PaCa2 cells. Collectively, our findings demonstrate phloretin and quercetin, and potentially other polyphenols, potently interfere with HRP-dependent assays, which have strong implications for designing experiments that interrogate the antioxidant potential of flavonoids. Our results also indicate phloretin and quercetin could be applied as controls for HRP reporter assays.

辣根过氧化物酶（HRP）的发现是非常有利的，因为它独特的化学性质可以应用于诊断工具，包括在各种实验系统中检测氧化应激标志物，如过氧化氢（H2O2）。在这里，我们做出了令人惊讶和令人信服的发现，黄酮类化合物，根皮素和槲皮素，通常在文献中被描述为有效的抗氧化剂，强烈抑制HRP的活性。利用紫外红外（AUR）分析，我们发现低至50 μM的根皮素可以消除分离的肝脏线粒体氧化丙酮酸和苹果酸产生H2O2的检测。根皮素也使肝脏线粒体氧化琥珀酸盐或二氢乙酸盐产生的H2O2检测无效。此外，100 μM下的根皮素完全消除了AUR对H2O2的直接检测，猝灭了纯化黄嘌呤氧化酶（XO）活性的检测，但对二氯二氢荧光素（H2-DCFDA）或二氢乙胺（DHE）的荧光检测不产生干扰。剂量反应试验显示，槲皮素是一个更有效的抑制剂HRP时，与根皮素。事实上，槲皮素在nM范围内的AUR检测中消除了间苯二酚荧光，而根皮素在检测体外h2o2或分离的肝线粒体或培养的Huh-7肝癌和Mia-PaCa2细胞形成的h2o2时没有作用。总的来说，我们的研究结果表明，根皮素和槲皮素以及潜在的其他多酚类物质可能会干扰酶依赖性测定，这对设计黄酮类化合物抗氧化潜力的实验具有重要意义。我们的结果还表明，根皮素和槲皮素可以作为HRP报告检测的对照。

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引用次数: 0

Nitric oxide in plant stress: Rewilding and restoring signaling for enhancing plant growth and development 植物胁迫中的一氧化氮：重新野生和恢复促进植物生长和发育的信号

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-08-01 Epub Date: 2025-06-20 DOI: 10.1016/j.bbagen.2025.130837

Sumreen Amin Shah , Awdhesh Kumar Mishra , Abdul Rehaman , Sumit G. Gandhi , Arif Tasleem Jan

Plants, represented as a complex system, are continuously exposed to environmental conditions that affect their growth and development, and sometimes their survival. Being sessile, they complete their life cycle under the influence of varied environmental constraints (biotic and abiotic), that adversely affect the produce's quality and productivity. Plants have evolved several defense strategies orchestrated through phytohormones that play a pivotal role in conferring resistance to stress. Nitric oxide (NO), an endogenously produced gaseous hormone, has emerged as a saviour in plant's response to different stresses. It plays an active role in the growth and development of plants, from seed dormancy and germination to growth, differentiation, flowering, fruiting, and ripening, besides affecting key metabolic processes such as photosynthesis. Endogenous production of NO and its interaction with phytohormones across different signaling cascades helps in alleviating the cellular damage caused by free radicals during drought, salinity, and other stresses. It contributes to stress resilience by inducing the synthesis of stress hormones such as ethylene (ET), which help plants to withstand adverse environmental constraints by minimizing the damage caused by different stresses. Exogenous application of NO exerts protective effects against different stresses by breaking seed dormancy and modulating germination, enhancing acquisition of mineral nutrients, photosynthetic functioning, production of antioxidant enzymes capable of neutralizing free radicals, and maintaining membrane integrity. These multifaceted roles of NO underscore its significance in plant stress tolerance. The present study offers valuable insights into NO production methods, involvement in growth and development, and a mechanistic view of its role in alleviating different stresses. In the current scenario, continued research into NO signaling mechanisms and cross-talk with other pathways seems essential for harnessing its potential in developing crops with enhanced resilience to environmental challenges.

植物作为一个复杂的系统，不断地暴露在影响其生长发育，有时甚至影响其生存的环境条件下。它们是不稳定的，在各种环境约束（生物和非生物）的影响下完成其生命周期，这对农产品的质量和生产力产生不利影响。植物已经进化出几种防御策略，这些策略是通过植物激素来协调的，这些激素在赋予植物抵抗压力的能力中起着关键作用。一氧化氮（NO）是一种内源性气体激素，在植物对不同胁迫的反应中起着救星的作用。它在植物的生长发育过程中起着积极的作用，从种子休眠、萌发到生长、分化、开花、结果、成熟，并影响光合作用等关键代谢过程。内源性NO的产生及其通过不同的信号级联与植物激素的相互作用有助于减轻自由基在干旱、盐度和其他胁迫下造成的细胞损伤。它通过诱导乙烯（ET）等应激激素的合成，帮助植物抵御不利的环境约束，最大限度地减少不同胁迫造成的损害，从而有助于抗逆性。外源施用NO通过打破种子休眠和调节萌发、增强矿质营养物质的获取、促进光合作用、产生能够中和自由基的抗氧化酶和维持膜完整性等方式对不同胁迫产生保护作用。一氧化氮在植物抗逆性中的重要作用。本研究对NO的产生方法、参与生长和发育以及其在缓解不同应激中的作用提供了有价值的见解。在目前的情况下，对NO信号机制的持续研究以及与其他途径的相互作用似乎对于利用其潜力开发具有增强环境适应能力的作物至关重要。

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引用次数: 0

Drought stress induces variation in DNA methylation pattern in a genotype-dependent manner in chickpea 干旱胁迫诱导鹰嘴豆DNA甲基化模式以基因型依赖的方式发生变化。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-08-01 Epub Date: 2025-06-21 DOI: 10.1016/j.bbagen.2025.130836

Khushboo Gupta, Rohini Garg

When plants are exposed to harsh environmental conditions, such as extreme temperatures or drought, certain genes are turned on or off. This process can be controlled by a chemical modification to their DNA called methylation. Here, we examined the impact of DNA methylation during drought stress on two chickpea genotypes, ICC 1882 (drought sensitive, DS) and ICC 4958 (drought tolerant, DT) chickpea genotypes via whole-genome bisulfite sequencing. A higher degree of hypomethylation in the DT genotype and more hypermethylation in the DS genotype were observed. A positive correlation was observed between CG methylation with genes and CHH methylation with TEs. Functional annotation of differentially methylated regions associated with differentially expressed genes revealed distinct pathways enriched in DS, such as enrichment of genes involved in root development, telomere maintenance, ion transport, and regulation of gene expression, while pathways like apoptosis, silencing by miRNAs, programmed cell death and carotenoid metabolic processes were enriched in DT genotype. Further, small RNA distribution and non-CWA context methylation density in TEs suggested the role of the RdDM pathway in mediating CHH hypermethylation in transposable elements. Overall, we observed distinct genes are differentially expressed and differentially methylated under drought stress in sensitive and tolerant genotypes.

当植物暴露在恶劣的环境条件下，比如极端温度或干旱，某些基因就会开启或关闭。这个过程可以通过一种叫做甲基化的化学修饰来控制。通过亚硫酸盐全基因组测序，研究了干旱胁迫下DNA甲基化对两种鹰嘴豆基因型ICC 1882（干旱敏感型，DS）和ICC 4958（干旱耐旱型，DT）的影响。DT基因型的低甲基化程度较高，DS基因型的高甲基化程度较高。CG甲基化与基因呈正相关，CHH甲基化与TEs呈正相关。对与差异表达基因相关的差异甲基化区域的功能注释显示，DS中富集了不同的途径，如参与根发育、端粒维持、离子运输和基因表达调控的基因，而DT基因型中富集了凋亡、mirna沉默、细胞程序性死亡和类胡萝卜素代谢过程等途径。此外，te中较小的RNA分布和非cwa上下文甲基化密度表明RdDM途径在介导转座元件CHH超甲基化中的作用。总的来说，我们观察到在干旱胁迫下，敏感型和耐受性基因型中不同基因的差异表达和差异甲基化。

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引用次数: 0

Neu1 sialidase regulates heterospecific social interaction in zebrafish via D1 dopamine receptor Neu1唾液酸酶通过D1多巴胺受体调节斑马鱼的异种社会交往。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-08-01 Epub Date: 2025-07-04 DOI: 10.1016/j.bbagen.2025.130839

Sumomo Tsuji , Asami Ikeda , Yurina Kubo , Toshiki Hyodo , Mika Ishii , Masaharu Komatsu , Kazuhiro Shiozaki

Neu1 sialidase catalyzes the removal of sialic acids from oligosaccharides and glycoproteins in lysosomes and plasma membranes. Recently, the association between Neu1 and psychiatric disorders, such as manic depression and schizophrenia, has attracted attention. neu1^−/− zebrafish (Neu1-KO) exhibit low anxiety, low aggressiveness, and increased social interaction with unfamiliar conspecific and heterospecific groups; however, the underlying mechanisms of action remain unclear. This study investigated alterations in monoamine levels in the Neu1-KO zebrafish brain and their significance in the unique behavioral response toward heterospecifics. The dopamine (DA) and serotonin (5-HT) levels were significantly elevated in the brains of Neu1-KO zebrafish compared with those of wild-type (WT) zebrafish, accompanied by a decrease in noradrenaline (NE). Immunohistochemical (IHC) analysis revealed increased numbers of DA and 5-HT neurons in the Neu1-KO zebrafish brain. Behavioral analysis revealed that treatment with a D1 receptor antagonist significantly suppressed heterospecific interactions in Neu1-KO zebrafish, whereas treatment with D2 and 5-HT receptor antagonists did not. IHC showed that polysialic acid (PSA), a known regulator of DA neuronal function, was predominantly distributed in the hypothalamus of zebrafish, with markedly enhanced signals in Neu1-KO zebrafish. These findings elucidate the role of Neu1 sialidase in regulating social interaction behaviors via DA neurons, potentially as a mechanism for mitigating risks in social environments.

Neu1唾液酸酶催化从溶酶体和质膜中的低聚糖和糖蛋白中去除唾液酸。最近，Neu1与精神疾病（如躁狂抑郁症和精神分裂症）之间的联系引起了人们的关注。neu1-/-斑马鱼（neu1- ko）表现出低焦虑、低攻击性，与不熟悉的同种和异种群体的社会互动增加；然而，其潜在的作用机制尚不清楚。本研究研究了Neu1-KO斑马鱼大脑中单胺水平的变化及其在对异种鱼的独特行为反应中的意义。与野生型（WT）斑马鱼相比，Neu1-KO斑马鱼脑内多巴胺（DA）和血清素（5-HT）水平显著升高，同时去甲肾上腺素（NE）降低。免疫组织化学（IHC）分析显示，Neu1-KO斑马鱼大脑中DA和5-HT神经元数量增加。行为学分析显示，D1受体拮抗剂显著抑制了Neu1-KO斑马鱼的异源相互作用，而D2和5-HT受体拮抗剂则没有作用。免疫组化结果显示，聚唾液酸（PSA）是一种已知的DA神经元功能调节剂，主要分布在斑马鱼的下丘脑，在Neu1-KO斑马鱼中信号明显增强。这些发现阐明了Neu1唾液酸酶在通过DA神经调节社会互动行为中的作用，可能作为一种减轻社会环境风险的机制。

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引用次数: 0

USP33-mediated stabilization of c-Myc drives glycolytic reprogramming and promotes ovarian cancer progression usp33介导的c-Myc稳定驱动糖酵解重编程并促进卵巢癌进展。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-08-01 Epub Date: 2025-06-16 DOI: 10.1016/j.bbagen.2025.130830

Dejia Chen , Yue Zhao , Xiaobo Zhang , Xiaocheng Shi , Yiming Liu , Ge Lou

Ovarian cancer (OC) is one of the most lethal gynecological malignancies, characterized by late-stage presentation, high recurrence rates, and a lack of effective early diagnostic markers. Recent evidence suggests that deubiquitinating enzymes (DUBs) play pivotal roles in tumor development and metabolic reprogramming. Here, we identify and characterize the function of the deubiquitinase USP33 in regulating c-Myc stability and glycolytic metabolism in OC. Through quantitative PCR (qPCR) and Western blot analyses, we show that USP33 is significantly upregulated in both OC tissues and cell lines compared to normal controls. Functional assays reveal that USP33 knockdown markedly inhibits cell proliferation, migration, and invasion while promoting apoptosis. Metabolically, USP33 silencing reduces glucose uptake, lactate production, and the extracellular acidification rate, consistent with downregulation of key glycolytic enzymes (LDHA, GLUT1, and PKM2). Mechanistically, co-immunoprecipitation and ubiquitination assays demonstrate that USP33 interacts with and deubiquitinates c-Myc at K48-linked chains, thereby stabilizing c-Myc protein levels and enhancing its transcriptional activity. Moreover, c-Myc overexpression rescues the inhibitory effects of USP33 knockdown on both glycolysis and malignant phenotypes. Clinically, high USP33 expression correlates with poor prognosis, suggesting that the USP33–c-Myc axis may serve as both a prognostic biomarker and a potential therapeutic target. Taken together, our findings highlight a critical role for USP33 in OC pathogenesis by mediating c-Myc-driven glycolytic reprogramming, and they provide new insights for developing targeted treatment strategies aimed at disrupting this pathway.

卵巢癌（OC）是最致命的妇科恶性肿瘤之一，其特点是晚期出现，复发率高，缺乏有效的早期诊断标志物。最近的证据表明，去泛素化酶（DUBs）在肿瘤的发展和代谢重编程中起着关键作用。在这里，我们确定并表征了去泛素酶USP33在OC中调节c-Myc稳定性和糖酵解代谢的功能。通过定量PCR （qPCR）和Western blot分析，我们发现与正常对照相比，USP33在OC组织和细胞系中均显著上调。功能分析显示，USP33敲低显著抑制细胞增殖、迁移和侵袭，同时促进细胞凋亡。在代谢方面，USP33沉默降低了葡萄糖摄取、乳酸生成和细胞外酸化速率，这与关键糖酵解酶（LDHA、GLUT1和PKM2）的下调一致。机制上，共免疫沉淀和泛素化实验表明，USP33与k48链上的c-Myc相互作用并使其去泛素化，从而稳定c-Myc蛋白水平并增强其转录活性。此外，c-Myc过表达恢复了USP33敲低对糖酵解和恶性表型的抑制作用。临床上，USP33高表达与不良预后相关，提示USP33-c- myc轴既可作为预后生物标志物，也可作为潜在的治疗靶点。综上所述，我们的研究结果强调了USP33通过介导c- myc驱动的糖酵解重编程在OC发病机制中的关键作用，并为开发旨在破坏这一途径的靶向治疗策略提供了新的见解。

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引用次数: 0

Mechanism of alkaloid-based inhibition of aldose reductase: Computational perspectives and experimental validations 基于生物碱的醛糖还原酶抑制机制：计算视角和实验验证

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-08-01 Epub Date: 2025-07-09 DOI: 10.1016/j.bbagen.2025.130841

Emadeldin M. Kamel , Saleh Maodaa , Sarah I. Othman , Adil Abalkhail , Faris F. Aba Alkhayl , Al Mokhtar Lamsabhi

Excessive aldose reductase activity drives the polyol-pathway damage that underlies diabetic cataract, neuropathy and nephropathy, yet few safe, potent AR inhibitors have reached the clinic. Here we integrated virtual screening, atomistic simulation and enzymology to evaluate six natural alkaloids—calycanthine, rutaecarpine, glaucine, sparteine, berbamine and tetrandrine—as prospective AR antagonists. A 2500-compound AutoDock Vina screen singled out these scaffolds for high predicted affinity (≤ − 7.0 kcal mol⁻¹), chemotype diversity and favorable in silico developability. Docking located all ligands within the catalytic cleft; 200-ns MD trajectories plus free-energy landscapes revealed that rutaecarpine and the bis-benzylisoquinolines tetrandrine and berbamine clamp the anion-binding and specificity pockets simultaneously, collapsing conformational space into a single deep basin. MM/PBSA analysis ranked tetrandrine highest (ΔG_total = −35.8 ± 2.5 kcal mol⁻¹) followed by rutaecarpine (−23.0 ± 1.3 kcal mol⁻¹) and berbamine (−19.4 ± 2.7 kcal mol⁻¹); per-residue decomposition highlighted Phe122, Trp219 and Leu300 as recurring hot-spots. In vitro, the same hierarchy emerged: tetrandrine inhibited recombinant human AR with an IC₅₀ of 1.56 ± 0.23 μM, outperforming quercetin (2.37 ± 0.27 μM), while rutaecarpine and berbamine yielded IC₅₀ values of 4.84 ± 0.81 and 7.35 ± 0.78 μM, respectively. Lineweaver–Burk and Michaelis–Menten plots demonstrated non-competitive inhibition, aligning with the MD-inferred pocket-clamping mechanism. ADMET profiling identified rutaecarpine as the most balanced lead (Lipinski-compliant, moderate hERG/CYP risk), whereas tetrandrine's hERG liability and low solubility call for scaffold refinement. This study validates bis-benzylisoquinoline and indolo-quinazolinone frameworks as privileged AR inhibitory chemotypes and showcases an end-to-end computational–experimental pipeline that rapidly converts ethnopharmacological molecules into mechanistically characterized leads for managing diabetic complications.

过度的醛糖还原酶活性导致多元醇通路损伤，而多元醇通路损伤是糖尿病性白内障、神经病变和肾病的基础，但很少有安全、有效的AR抑制剂进入临床。在这里，我们综合了虚拟筛选、原子模拟和酶学来评估六种天然生物碱——花青素、芦果卡松、青氨酸、sparteine、小檗碱和粉防己碱——作为潜在的AR拮抗剂。2500个化合物的AutoDock Vina筛选筛选出这些支架具有高预测亲和力（≤- 7.0 kcal mol - 1），化学型多样性和良好的硅显影性。对接定位所有配体在催化裂孔内；200-ns MD轨迹和自由能景观显示，rutaecarpine和双苄基异喹啉粉防己碱和小檗碱同时夹住阴离子结合和特异性口袋，将构象空间塌陷成一个单一的深盆地。MM/PBSA分析显示粉防己碱最高（ΔGtotal =−35.8±2.5 kcal mol−1），其次是胡卡松碱（−23.0±1.3 kcal mol−1）和小檗胺（−19.4±2.7 kcal mol−1）；残基分解突出显示Phe122、Trp219和Leu300是反复出现的热点。在体外，出现了相同的层次结构：粉防己碱抑制重组人AR的IC₅₀值为1.56±0.23 μM，优于槲皮素（2.37±0.27 μM），而芦卡松和小檗胺的IC₅₀值分别为4.84±0.81和7.35±0.78 μM。Lineweaver-Burk和Michaelis-Menten图显示非竞争性抑制，与md推断的口袋夹紧机制一致。ADMET分析确定rutaecarpine是最平衡的铅（Lipinski-compliant，中度hERG/CYP风险），而粉防己碱的hERG敏感性和低溶解度需要支架改进。本研究验证了双苄基异喹啉和吲哚-喹唑啉酮框架是优越的AR抑制化学型，并展示了端到端计算实验管道，可快速将民族药理学分子转化为机制特征的先导物，用于管理糖尿病并发症。

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引用次数: 0

EP300-mediated H3K18la regulation of METTL3 promotes macrophage ferroptosis and atherosclerosis through the m6A modification of SLC7A11 ep300介导的H3K18la调控METTL3通过m6A修饰SLC7A11促进巨噬细胞铁凋亡和动脉粥样硬化。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-08-01 Epub Date: 2025-06-28 DOI: 10.1016/j.bbagen.2025.130838

Jingquan Chen , Zongrong Liu , Zhujun Yue , Qiang Tan , Hongshun Yin , Haifei Wang , Zhilong Chen , Yanbing Zhu , Jianghua Zheng

Macrophages, as the primary immune cell population in atherosclerosis (AS), exhibit complex pathogenic mechanisms that are not fully elucidated. This study aims to explore the interplay between histone lactylation and methyltransferase-like protein 3 (METTL3)-mediated m6A modification and their potential mechanisms in AS. We demonstrate that METTL3 is highly expressed in macrophages in both in vivo and in vitro models of atherosclerosis, and myeloid cell-specific deletion of METTL3 attenuates the progression of atherosclerosis. Furthermore, the accumulation of lactate levels in macrophages promotes METTL3 expression through EP300-mediated histone H3 lysine 18 lactylation (H3K18la) binding to the METTL3 promoter site. We found that METTL3-mediated m6A modifications are enriched in solute carrier family 7 member 11 (SLC7A11) and accelerate its mRNA degradation in a YTH domain family member 2 (YTHDF2)-dependent manner, thereby promoting ferroptosis in macrophages. Additionally, lactate stimulation downregulates SLC7A11 through the METTL3/YTHDF2 pathway, further promoting ferroptosis. Overall, during AS, lipid peroxidation induces an increase in lactate levels within macrophages, which enhances METTL3 expression through EP300-mediated H3K18la. This further accelerates the degradation of SLC7A11 mRNA via the YTHDF2-dependent m6A modification pathway, inducing ferroptosis in macrophages. This discovery provides new insights into the mechanisms of macrophage function in AS and offers a theoretical basis for the development of therapies for AS.

巨噬细胞作为动脉粥样硬化（as）的主要免疫细胞群，表现出复杂的致病机制，目前尚未完全阐明。本研究旨在探讨组蛋白乳酸化与甲基转移酶样蛋白3 （METTL3）介导的m6A修饰之间的相互作用及其在AS中的潜在机制。我们证明，在体内和体外动脉粥样硬化模型中，METTL3在巨噬细胞中高表达，髓细胞特异性缺失METTL3可以减缓动脉粥样硬化的进展。此外，巨噬细胞中乳酸水平的积累通过ep300介导的组蛋白H3赖氨酸18乳酸化（H3K18la）结合METTL3启动子位点促进METTL3的表达。我们发现mettl3介导的m6A修饰富集于溶质载体家族7成员11 （SLC7A11）中，并以依赖于YTH结构域家族成员2 （YTHDF2）的方式加速其mRNA降解，从而促进巨噬细胞铁凋亡。此外，乳酸刺激通过METTL3/YTHDF2途径下调SLC7A11，进一步促进铁下垂。总体而言，AS期间，脂质过氧化诱导巨噬细胞内乳酸水平升高，从而通过ep300介导的H3K18la增强METTL3表达。这进一步通过ythdf2依赖的m6A修饰途径加速SLC7A11 mRNA的降解，诱导巨噬细胞铁凋亡。这一发现为巨噬细胞在AS中的功能机制提供了新的见解，并为AS治疗的发展提供了理论基础。

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引用次数: 0

Hybrid virtual screening identifies dipyrazole carboxamide derivatives as novel direct InhA inhibitors with antitubercular activity 混合虚拟筛选鉴定了二吡唑羧酰胺衍生物作为具有抗结核活性的新型直接InhA抑制剂。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-07-01 Epub Date: 2025-05-26 DOI: 10.1016/j.bbagen.2025.130827

Auradee Punkvang , Bongkochawan Pakamwong , Naruedon Phusi , Paptawan Thongdee , Kampanart Chayajarus , Jidapa Sangswan , Kanjana Pangjit , Khomson Suttisintong , Jiraporn Leanpolchareanchai , Poonpilas Hongmanee , Pitak Santanirand , James Spencer , Adrian J. Mulholland , Sanya Sureram , Prasat Kittakoop , Pornpan Pungpo

Direct inhibitors of M. tuberculosis enoyl-acyl carrier protein reductase (M. tuberculosis InhA) remain effective against variants with mutations associated with isoniazid resistance. In our previous study, structure-based virtual screening was employed to discover such inhibitors. However, most identified hits exhibited limited antimycobacterial activity, with minimum inhibitory concentration (MIC) values of >100 μg/mL. To address this challenge, we refined our virtual screening strategy by integrating ligand- and structure-based virtual screening approaches. The efficacy of this hybrid virtual screening approach was validated through biological assays measuring MIC and half-maximal inhibitory concentration (IC₅₀) for the inhibition of M. tuberculosis growth and InhA activity, respectively. Among 14 identified hits, compounds 3 and 10, classified as dipyrazole carboxamide derivatives, were validated as promising lead candidates, with MIC values of 25 and 50 μg/mL and IC₅₀ values of 10.60 ± 0.56 and 5.08 ± 0.30 μM, respectively. The relatively low hit-to‑lead conversion rate (14 %) is ascribed to our observation that nine of the identified hits, including compounds 3 and 10, showed some level of precipitation in the MIC assay medium. Molecular dynamics simulations show that the dipyrazole carboxamide moiety in compounds 3 and 10 forms essential hydrogen bonds with nicotinamide adenine dinucleotide (oxidized form) (NAD⁺) in the InhA binding pocket. Notably, both compounds 3 and 10 exhibit favorable safety profiles, with no toxicity observed in Caco-2 cells at concentrations up to 100 μg/mL. Consequently, we believe that these compounds present promising starting points for further lead optimization and development of novel antitubercular agents.

结核分枝杆菌烯酰酰基载体蛋白还原酶（结核分枝杆菌InhA）的直接抑制剂对与异烟肼耐药性相关的突变变体仍然有效。在我们之前的研究中，采用基于结构的虚拟筛选来发现此类抑制剂。然而，大多数已鉴定的命中表现出有限的抗真菌活性，最低抑制浓度（MIC）值为bbb100 μg/mL。为了应对这一挑战，我们通过整合基于配体和结构的虚拟筛选方法来改进我们的虚拟筛选策略。通过测定MIC和半最大抑制浓度（IC50）分别对结核分枝杆菌生长和InhA活性的抑制，验证了这种混合虚拟筛选方法的有效性。在鉴定的14个命中点中，化合物3和10被认定为二吡唑类carboxamide衍生物，其MIC值分别为25和50 μg/mL， IC50值分别为10.60 ± 0.56和5.08 ± 0.30 μM。相对较低的hit-to - lead转化率（14 %）归因于我们的观察，其中9个已确定的hit，包括化合物3和10，在MIC测定培养基中显示出一定程度的沉淀。分子动力学模拟表明，化合物3和10中的二吡唑羧酰胺部分在InhA结合口袋中与烟酰胺腺嘌呤二核苷酸（氧化形式）（NAD+）形成必需的氢键。值得注意的是，化合物3和10都表现出良好的安全性，在浓度高达100 μg/mL的Caco-2细胞中没有观察到毒性。因此，我们相信这些化合物为进一步优化和开发新型抗结核药物提供了有希望的起点。

{"title":"Hybrid virtual screening identifies dipyrazole carboxamide derivatives as novel direct InhA inhibitors with antitubercular activity","authors":"Auradee Punkvang , Bongkochawan Pakamwong , Naruedon Phusi , Paptawan Thongdee , Kampanart Chayajarus , Jidapa Sangswan , Kanjana Pangjit , Khomson Suttisintong , Jiraporn Leanpolchareanchai , Poonpilas Hongmanee , Pitak Santanirand , James Spencer , Adrian J. Mulholland , Sanya Sureram , Prasat Kittakoop , Pornpan Pungpo","doi":"10.1016/j.bbagen.2025.130827","DOIUrl":"10.1016/j.bbagen.2025.130827","url":null,"abstract":"<div><div>Direct inhibitors of <em>M. tuberculosis</em> enoyl-acyl carrier protein reductase (<em>M. tuberculosis</em> InhA) remain effective against variants with mutations associated with isoniazid resistance. In our previous study, structure-based virtual screening was employed to discover such inhibitors. However, most identified hits exhibited limited antimycobacterial activity, with minimum inhibitory concentration (MIC) values of >100 μg/mL. To address this challenge, we refined our virtual screening strategy by integrating ligand- and structure-based virtual screening approaches. The efficacy of this hybrid virtual screening approach was validated through biological assays measuring MIC and half-maximal inhibitory concentration (IC<sub>50</sub>) for the inhibition of <em>M. tuberculosis</em> growth and InhA activity, respectively. Among 14 identified hits, compounds <strong>3</strong> and <strong>10</strong>, classified as dipyrazole carboxamide derivatives, were validated as promising lead candidates, with MIC values of 25 and 50 μg/mL and IC<sub>50</sub> values of 10.60 ± 0.56 and 5.08 ± 0.30 μM, respectively. The relatively low hit-to‑lead conversion rate (14 %) is ascribed to our observation that nine of the identified hits, including compounds <strong>3</strong> and <strong>10</strong>, showed some level of precipitation in the MIC assay medium. Molecular dynamics simulations show that the dipyrazole carboxamide moiety in compounds <strong>3</strong> and <strong>10</strong> forms essential hydrogen bonds with nicotinamide adenine dinucleotide (oxidized form) (NAD<sup>+</sup>) in the InhA binding pocket. Notably, both compounds <strong>3</strong> and <strong>10</strong> exhibit favorable safety profiles, with no toxicity observed in Caco-2 cells at concentrations up to 100 μg/mL. Consequently, we believe that these compounds present promising starting points for further lead optimization and development of novel antitubercular agents.</div></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":"1869 8","pages":"Article 130827"},"PeriodicalIF":2.8,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144172635","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

Decoding the calcium signal: Structural insights into CBL-CIPK pathway in plants 解码钙信号：植物CBL-CIPK通路的结构见解。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-07-01 Epub Date: 2025-05-13 DOI: 10.1016/j.bbagen.2025.130819

Subhash Chandra Bihani , Tarushi , Ashish Kumar Srivastava

Calcium (Ca²⁺) signaling in plants is a major pathway in transducing diverse environmental stimuli. Calcineurin B-like proteins (CBLs) are one of the unique groups of Ca²⁺ sensors that transduce the Ca²⁺ signals by interacting with plant-specific protein kinases known as CBL-interacting protein kinases (CIPKs). In recent years, structure-function studies have provided key insights into the molecular basis of CBL-CIPK signaling and their interactions with the target proteins. The crystal structures of CBL2 and CBL4 have elucidated the architecture of non-canonical EF hands and provided the rationale for Ca²⁺ binding by CBLs. The molecular basis of interaction of the regulatory domain of CIPKs with CBLs has been established, providing rationale for CBL-mediated activation of CIPKs. The molecular mechanism of fine regulation of CIPK activity under non-stressed conditions and full activation under stressed conditions has been established using crystal structures of CIPK23 and CIPK24. Recently, high-resolution CryoEM structures of Arabidopsis and rice SOS1 led to a comprehensive understanding of its regulation and ion transport mechanism. In this review, major advances in understanding the structural basis of Ca²⁺ sensing by CBLs, molecular determinants of CIPK activation, and subsequent phosphorylation of target proteins are discussed. Remaining questions that need to be answered for a holistic understanding of the CBL-CIPK network are also discussed.

植物钙（Ca2+）信号是多种环境刺激信号转导的主要途径。钙调磷酸酶b样蛋白（CBLs）是一种独特的Ca2+传感器，通过与植物特异性蛋白激酶（称为cbll相互作用蛋白激酶（CIPKs））相互作用来转导Ca2+信号。近年来，结构-功能研究为了解CBL-CIPK信号的分子基础及其与靶蛋白的相互作用提供了重要的见解。CBL2和CBL4的晶体结构阐明了非规范EF手的结构，并为CBLs结合Ca2+提供了理论依据。CIPKs调控域与CBLs相互作用的分子基础已经建立，为CBLs介导的CIPKs激活提供了理论基础。利用CIPK23和CIPK24的晶体结构，建立了非应力条件下CIPK活性精细调控和应力条件下完全活化的分子机制。近年来，拟南芥和水稻SOS1的高分辨率CryoEM结构使人们对其调控和离子转运机制有了全面的了解。在这篇综述中，主要的进展，了解Ca2+传感的CBLs的结构基础，CIPK活化的分子决定因素，以及随后的磷酸化靶蛋白进行了讨论。本文还讨论了为全面理解CBL-CIPK网络而需要回答的剩余问题。

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引用次数: 0

Dual generation of stereo- and linear-specific monoclonal antibodies through B-cell receptors by DNA and cell immunization for therapeutic applications 双代立体和线性特异性单克隆抗体通过b细胞受体通过DNA和细胞免疫治疗应用。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. General subjects

Pub Date : 2025-07-01 Epub Date: 2025-05-22 DOI: 10.1016/j.bbagen.2025.130822

Chiho Miyamae , Yushi Isozaki , Kanta Tsumoto , Masahiro Tomita

The optimized stereospecific targeting (SST) technique features selective generation of conformation-specific monoclonal antibodies against membranous proteins with high specificity after DNA and cell immunization. This technology consists of two critical steps, which are specific selection of sensitized B lymphocytes by antigen-expressing myeloma cells through B-cell receptors (BCRs) and selective fusion of B cell-myeloma cell complexes by electrical pulses to produce hybridoma cells secreting stereospecific monoclonal antibodies. Here we were able to verify the critical step for the selection of B lymphocytes by intact antigen-expressing myeloma cells by a double-label immunofluorescence analysis. Interestingly, the cell complex was a single attachment. Furthermore, we newly found the new progress that the optimized SST technique offered dual production of anti-intact and anti-linear specific monoclonal antibodies against a human ephrin type-A receptor 2 (hEphA2). The optimized SST technique may be useful for producing not only stereospecific monoclonal antibodies, but also primary-specific monoclonal antibodies based on the selection of sensitized B lymphocytes by the target intact antigen through BCRs. It would elicit more advanced medical applications by generating dual monoclonal antibodies against the intact antigen.

优化后的立体特异性靶向（SST）技术在DNA和细胞免疫后选择性地产生针对膜蛋白的构象特异性单克隆抗体，具有高特异性。该技术包括两个关键步骤，即抗原表达的骨髓瘤细胞通过B细胞受体（bcr）特异性选择致敏的B淋巴细胞，以及通过电脉冲选择性融合B细胞-骨髓瘤细胞复合物以产生分泌立体特异性单克隆抗体的杂交瘤细胞。在这里，我们能够通过双标记免疫荧光分析验证完整抗原表达骨髓瘤细胞选择B淋巴细胞的关键步骤。有趣的是，细胞复合体是一个单独的附着体。此外，我们还发现了新的进展，即优化后的SST技术可以双重生产抗完整型和抗线性型特异性单克隆抗体，用于抗人ephrin - a型受体2 （hEphA2）。优化后的SST技术不仅可用于生产立体特异性单克隆抗体，还可用于生产基于靶完整抗原通过bcr选择致敏B淋巴细胞的初级特异性单克隆抗体。通过产生针对完整抗原的双单克隆抗体，将引发更先进的医学应用。

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引用次数: 0