Biochimie最新文献_第4页

Both single mutation and molecular conjugation on the mutant prevent calcitonin from forming amyloid fibrils 突变体的单突变和分子偶联均可阻止降钙素形成淀粉样原纤维。

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-03-01 Epub Date: 2025-11-29 DOI: 10.1016/j.biochi.2025.11.012

Chia-Chi Chang , Govindan Sivakumar , Meng-Chieh Huang , Yun-Hsuan Chen , Shan-Rong Wu , Yun-Ju Lai , Chian-Hui Lai , Ling-Hsien Tu

Human calcitonin (hCT) is a peptide hormone made up of 32 amino acids, produced by the parafollicular cells of the thyroid gland. Its main role is to decrease blood calcium levels by inhibiting osteoclast activity and reducing calcium reabsorption in the kidneys and intestines. This characteristic positions hCT as a potential treatment for bone-related conditions, such as osteoporosis and Paget's disease. However, hCT has a strong tendency to form amyloid fibrils, which can lead to a loss of its biological function. This research aims to stabilize hCT's conformation through molecular conjugation to prevent its aggregation. We used a cross-linking tool with a boronic ester structure (NPC) to react with the side-chain amino groups of two residues in hCT, effectively "locking" its structure. hCT can be "unlocked" using hydrogen peroxide, returning it to its original form. To support this experimental design, we synthesized two hCT variants, hCT-Q14K and hCT-F22K. Our results showed that both variants successfully conjugated with NPC, and the conjugation could be easily removed with hydrogen peroxide within 30 min. NPC-conjugated hCT did not form amyloid as revealed by transmission electron microscopy. Moreover, thioflavin-T fluorescence kinetic studies and dynamic light scattering measurement demonstrated that the aggregation tendencies of hCT-F22K were significantly lower than those of hCT, while still maintaining their biological activity, suggesting this design is feasible. This approach may significantly aid in the development of calcitonin drugs.

人降钙素（hCT）是一种由32个氨基酸组成的肽激素，由甲状腺的滤泡旁细胞产生。它的主要作用是通过抑制破骨细胞活性和减少肾和肠内钙的重吸收来降低血钙水平。这一特点使hCT成为骨相关疾病的潜在治疗方法，如骨质疏松症和佩吉特病。然而，hCT具有形成淀粉样原纤维的强烈倾向，这可能导致其生物学功能的丧失。本研究旨在通过分子偶联稳定hCT的构象，防止其聚集。我们使用具有硼酯结构（NPC）的交联工具与hCT中两个残基的侧链氨基反应，有效地“锁定”了其结构。hCT可以用过氧化氢“解锁”，使其恢复原始形态。为了支持这一实验设计，我们合成了两种hCT变体，hCT- q14k和hCT- f22k。我们的研究结果表明，这两个变体都成功地与NPC结合，并且在30分钟内可以很容易地用双氧水去除结合。透射电镜显示，npc共轭的hCT未形成淀粉样蛋白。此外，硫黄素- t荧光动力学研究和动态光散射测量表明，hCT- f22k的聚集倾向明显低于hCT，但仍保持其生物活性，表明该设计是可行的。这种方法可能对降钙素药物的开发有很大的帮助。

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引用次数: 0

Giardia duodenalis: Base excision repair pathway enzyme FEN1 carries out catalytic activities pertaining to NER pathway 十二指肠贾第虫：碱基切除修复途径酶FEN1具有与NER途径相关的催化活性。

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-03-01 Epub Date: 2025-12-24 DOI: 10.1016/j.biochi.2025.12.010

Ulises Omar García-Lepe, Sofía Gabriela Tomás-Morales, María Teresa Izaguirre-Hernández, María Luisa Bazán-Tejeda, Rosa María Bermúdez-Cruz

Giardia duodenalis is a binuclear protozoan that causes intestinal infection in humans and animals. The life cycle of G. duodenalis is comprised by 2 stages: trophozoite (vegetative, ploidy: 4 N) and cyst (infective, ploidy: 8–16 N) and the transition from one to another requires a precise coordination as well as the support of the DNA repair machinery. While DNA homologous recombination DNA repair has recently been characterized, NER and BER are pathways that had not been explored. Most of the structure specific enzymes (SSE) participate in a variety of processes like DNA replication stress, DNA adduct repair and Holliday junction processing. In an effort to explore these kinds of enzymes in G. duodenalis, a minimalist parasite, we aimed at characterizing the Fen1 enzyme by cloning its gene to study its catalytic properties (binding and nuclease) using flap and bubble DNA substrates. Unexpectedly, we found that GdFen-1 is able to cleave bubble DNA, then to shed light on which domains of this enzyme are responsible for this activity, giardial acid block and a portion of a cap region were substituted by their human counterparts, and while acid block substitution did not affect this activity, the modification in the cap region did. The possible implications of these findings are addressed.

十二指肠贾第虫是一种双核原生动物，可引起人类和动物肠道感染。十二指肠棘豆的生命周期分为两个阶段：滋养体（营养体，倍性：4N）和囊体（感染体，倍性：8-16N），从一个阶段过渡到另一个阶段需要精确的协调和DNA修复机制的支持。虽然DNA同源重组DNA修复最近已被表征，但NER和BER是尚未探索的途径。大多数结构特异性酶（SSE）参与DNA复制胁迫、DNA加合物修复、Holliday结加工等多种过程。为了在极简寄生虫G. duodenalis中探索这些酶，我们旨在通过克隆Fen1酶基因来研究其催化特性（结合酶和核酸酶），并利用皮瓣和气泡DNA底物研究其催化特性。出乎意料的是，我们发现GdFen-1能够切割气泡DNA，然后阐明该酶的哪个结构域负责这种活性，gi心包酸块和一部分帽区被它们的人类对应物取代，虽然酸块取代不影响这种活性，但帽区修饰会影响这种活性。讨论了这些发现可能产生的影响。

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引用次数: 0

Mechanistic insights into NOBA hydrolysis by viper venom secreted phospholipase A2 毒蛇毒液分泌的磷脂酶A2水解NOBA的机理

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-03-01 Epub Date: 2025-12-05 DOI: 10.1016/j.biochi.2025.12.003

Ana Rita Calixto , Roberto Pinto , Maciej Spiegel , Maria João Ramos , Pedro Alexandrino Fernandes

Snake envenoming remains a major global health challenge, particularly in tropical and subtropical regions. Among the key toxic components of snake venom, secreted phospholipases A₂ (sPLA₂) play a crucial role by hydrolysing cell membrane phospholipids, leading to membrane disruption and severe toxic effects such as inflammation, neurotoxicity, and myotoxicity.

To study sPLA₂ catalytic activity, synthetic soluble substrates like 4-nitro-3-octanoyloxy benzoic acid (NOBA) are widely used in experimental assays, in alternative to membrane phospholipids. However, it is questionable whether mechanistic conclusions taken with small, soluble substrates can be extrapolated to true cell-membrane substrates.

Here, we employed QM/MM calculations to investigate the catalytic mechanism of sPLA₂ using NOBA as a substrate. Our focus was on a sPLA₂ from Bothrops asper. However, the high conservation of sPLA₂ active sites suggests our conclusions should be generalisable to the sPLA₂ of most snake species.

The results reveal a mixed single-water/assisted water mechanism. First, a water molecule, deprotonated by His₄₇, performs a nucleophilic attack on NOBA's carbonyl carbon, with a free energy barrier of 12.8 kcal/mol, resembling the single-water pathway. The collapse of the tetrahedral intermediate and protonation of the leaving group involves two water molecules, resembling the assisted-water pathway. This mixed pathway highlights the catalytic versatility of sPLA₂ and offers new insights into its enzymatic activity with synthetic substrates. Importantly, our finding support NOBA as a valid and suitable substrate for studying the chemical component of sPLA₂ toxicity, even though it does not account for the equally important membrane-binding component of the toxicity mechanism.

蛇中毒仍然是一个重大的全球卫生挑战，特别是在热带和亚热带地区。在蛇毒的关键毒性成分中，分泌磷脂酶A2 （sPLA2）通过水解细胞膜磷脂发挥关键作用，导致细胞膜破坏和严重的毒性作用，如炎症、神经毒性和肌毒性。为了研究sPLA2的催化活性，合成可溶性底物如4-硝基-3-辛烷氧基苯甲酸（NOBA）被广泛用于实验分析，以替代膜磷脂。然而，用小的可溶性底物得出的机械结论是否可以外推到真正的细胞膜底物，这是值得怀疑的。在这里，我们采用QM/MM计算来研究NOBA作为底物的sPLA2的催化机理。我们的重点是Bothrops aspper的sPLA2。然而，sPLA2活性位点的高度保守性表明我们的结论应该适用于大多数蛇种的sPLA2。结果显示单水/助水混合机理。首先，一个被His47去质子化的水分子对NOBA的羰基碳进行亲核攻击，自由能垒为12.8 kcal/mol，类似于单水途径。四面体中间体的坍塌和离去基的质子化涉及两个水分子，类似于辅助-水途径。这种混合途径突出了sPLA2的催化多功能性，并为其与合成底物的酶活性提供了新的见解。重要的是，我们的发现支持NOBA作为研究sPLA2毒性化学成分的有效和合适的底物，即使它不能解释毒性机制中同等重要的膜结合成分。

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引用次数: 0

Dietary modulation of purine metabolism and uric acid homeostasis in cancer patients with an ileostomy 回肠造口术后癌症患者嘌呤代谢和尿酸稳态的饮食调节。

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-03-01 Epub Date: 2026-01-05 DOI: 10.1016/j.biochi.2026.01.002

Kamil Aleksander Sobieszek , Jakub Frankowski , Mateusz Labudda

Nutritional management in cancer patients with an ileostomy poses specific challenges due to impaired nutrient absorption, elevated metabolic demands, and the need to control serum urate levels. This review examines the biochemical and clinical relevance of plant-based foods in addressing these issues, with particular emphasis on purine content, digestibility, and metabolic outcomes. Current evidence shows that legumes, soy products, nuts, seeds, whole grains, vegetables, fruits, and fortified foods generally contain low to moderate purine levels and are well tolerated by ileostomy patients. Incorporating these foods into individualized dietary plans supports adequate protein and micronutrient intake, enhances tissue repair, and reduces the risk of gout flares without compromising gastrointestinal function. Moreover, plant-derived bioactive compounds and antioxidants may mitigate inflammation and oxidative stress associated with cancer progression and hyperuricemia. Collectively, a carefully designed plant-based diet can meet the nutritional needs of cancer patients with an ileostomy while contributing to effective gout management and improved metabolic homeostasis.

由于营养吸收受损、代谢需求升高以及控制血清尿酸水平的需要，回肠造口术后癌症患者的营养管理面临着特殊的挑战。这篇综述探讨了植物性食物在解决这些问题方面的生化和临床相关性，特别强调嘌呤含量、消化率和代谢结果。目前的证据表明，豆类、豆制品、坚果、种子、全谷物、蔬菜、水果和强化食品通常含有低至中等水平的嘌呤，并且对回肠造口患者耐受良好。将这些食物纳入个性化的饮食计划有助于摄入足够的蛋白质和微量营养素，增强组织修复，并在不损害胃肠道功能的情况下降低痛风发作的风险。此外，植物源性生物活性化合物和抗氧化剂可能减轻与癌症进展和高尿酸血症相关的炎症和氧化应激。总的来说，精心设计的植物性饮食可以满足回肠造口癌患者的营养需求，同时有助于有效的痛风管理和改善代谢稳态。

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引用次数: 0

Inhibiting catalytic activity of Plasmodium falciparum aspartate protease plasmepsin V: A biochemical approach to malaria intervention 抑制恶性疟原虫天冬氨酸蛋白酶Plasmepsin V的催化活性：一种生物化学方法干预疟疾。

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-03-01 Epub Date: 2025-12-13 DOI: 10.1016/j.biochi.2025.12.006

Anitadevi K. Prajapati , Yogesh Kumar , Sinjan Choudhary

Plasmepsin V, an aspartate protease, is crucial for the survival of the malarial parasite Plasmodium falciparum, making it a promising target for antimalarial drug development. In this study, the quinoline-based drugs amodiaquine (AQ) and clioquinol (CQ) are repurposed to inhibit the catalytic activity of PfPlmV. Fluorescence quenching assays suggest that both AQ and CQ exhibit moderate binding affinity for PfPlmV. Temperature-dependent thermodynamic analyses indicate the involvement of both H-bonding/electrostatic interactions and hydrophobic interactions in the binding of AQ/CQ with PfPlmV. Isothermal titration calorimetry (ITC) indicates that these interactions are both enthalpically and entropically favourable. Molecular docking studies show that AQ binds directly to the substrate-binding site of PfPlmV, engaging catalytic dyad residues Asp118 and Asp365, whereas CQ binds to PfPlmV but does not directly interact with the catalytic dyad. Molecular dynamics simulations also corroborated the docking results, revealing that AQ forms a more conformationally stable and hydrophobic interactions driven complex with PfPlmV compared to CQ. Further, the enzyme kinetics studies using fluorogenic substrate demonstrated that AQ efficiently inhibits the catalytic activity of PfPlmV with an IC₅₀ value of (0.42 ± 0.04) μM via competitive inhibition mode. In contrast, CQ fails to bind at the catalytic site and does not exhibit any inhibitory effect on PfPlmV activity. These mechanistic insights will lay the foundations for new biochemical approaches to develop targeted therapies aimed at malaria disease.

Plasmepsin V是一种天冬氨酸蛋白酶，对疟疾寄生虫恶性疟原虫的存活至关重要，使其成为抗疟疾药物开发的一个有希望的靶点。在本研究中，喹啉类药物阿莫地喹（AQ）和氯喹诺（CQ）被用于抑制PfPlmV的催化活性。荧光猝灭实验表明，AQ和CQ对PfPlmV具有中等的结合亲和力。温度相关的热力学分析表明，AQ/ CQ与PfPlmV的结合涉及h键/静电相互作用和疏水相互作用。等温滴定量热法（ITC）表明，这些相互作用在焓和熵上都是有利的。分子对接研究表明，AQ直接与PfPlmV的底物结合位点结合，与催化二偶体Asp118和Asp365残基结合，而CQ与PfPlmV结合，但不直接与催化二偶体相互作用。分子动力学模拟也证实了对接结果，表明与CQ相比，AQ与PfPlmV形成了更稳定的构象和疏水相互作用驱动的复合物。此外，利用荧光底物进行的酶动力学研究表明，AQ通过竞争抑制模式有效抑制PfPlmV的催化活性，IC50值为（0.42±0.04）μM。相比之下，CQ不能在催化位点结合，对PfPlmV活性没有任何抑制作用。这些机制见解将为开发针对疟疾疾病的靶向治疗的新生化方法奠定基础。

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引用次数: 0

Sodium benzoate, a common food preservative-induced Insulin resistance and atrophy in C2C12 myotubes 苯甲酸钠是一种常见的食品防腐剂，可诱导胰岛素抵抗和C2C12肌管萎缩。

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-02-01 Epub Date: 2025-11-18 DOI: 10.1016/j.biochi.2025.11.009

Onkar Sharma , Anita Dua , Sanjeev Gupta , Elisha Injeti , Ashwani Mittal

Sodium benzoate (SB), a widely used food preservative, has been reported to possess several therapeutic benefits including treating neurodegenerative disorders as well as negative effects such as altering glucose homeostasis. However, limited attention has been given to its role in inducing insulin resistance (IR). Therefore, current study was designed to investigate the relationship between SB and insulin resistance using C2C12 myotubes and to explore the possible underlying mechanisms. Myotubes were exposed to SB (20 mM) for 24 h and glucose consumption and uptake assays along with confocal microscopy (GLUT4 translocation) were used to evaluate IR. Oxidative stress indicators i.e. reactive oxygen species (ROS), lipid peroxidation (LPO), and reduced glutathione (GSH) levels were also measured. Myotubes morphology along with atrophic markers (calpain activity) were evaluated. Data show that in C2C12 myotubes, SB treatment reduced glucose consumption and uptake in a dose-dependent manner. SB inducing IR by disrupting insulin-mediated GLUT4 translocation in cultured myotubes. Alteration in oxidative stress-related markers (i.e. elevated ROS and LPO levels, reduced GSH) were also observed in SB-treated myotubes. Furthermore, decrease in the fusion index, length, and diameter of myotubes along with upregulation of calpain activity and decrease in muscle protein were also observed. Study concludes that SB exposure not only induced IR but also caused atrophy and oxidative stress in the C2C12 myotubes.

苯甲酸钠（SB）是一种广泛使用的食品防腐剂，据报道具有多种治疗益处，包括治疗神经退行性疾病以及改变葡萄糖稳态等负面影响。然而，对其在诱导胰岛素抵抗（IR）中的作用关注有限。因此，本研究旨在通过C2C12肌管研究SB与胰岛素抵抗的关系，并探讨可能的潜在机制。肌管暴露于SB （20mM）中24小时，葡萄糖消耗和摄取测定以及共聚焦显微镜（GLUT4易位）用于评估IR。氧化应激指标，即活性氧（ROS）、脂质过氧化（LPO）和还原性谷胱甘肽（GSH）水平也被测量。观察肌管形态及萎缩标志物（钙蛋白酶活性）。数据显示，在C2C12肌管中，SB治疗以剂量依赖的方式降低了葡萄糖消耗和摄取。SB通过破坏培养肌管中胰岛素介导的GLUT4易位诱导IR。在sb处理的肌管中也观察到氧化应激相关标志物的改变（即ROS和LPO水平升高，GSH降低）。此外，还观察到融合指数、肌管长度和直径减少，钙蛋白酶活性上调，肌肉蛋白减少。研究表明SB暴露不仅可诱导IR，还可引起C2C12肌管萎缩和氧化应激。

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引用次数: 0

Design of competitive inhibitory peptides for 3-hydroxy-3-Methylglutaryl coenzyme A reductase 3-羟基-3-甲基戊二酰辅酶A还原酶竞争性抑制肽的设计

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-02-01 Epub Date: 2025-11-01 DOI: 10.1016/j.biochi.2025.10.017

Valeriy V. Pak , Shomansur Sh Sagdullaev , Aleksandr V. Pak

The effectiveness of statins in preventing hypercholesterolemia and associated cardiovascular diseases has been confirmed for a long time. Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). This enzyme plays a key role in the reaction that represents the rate-limiting step in cholesterol biosynthesis. Numerous studies on the properties of food-derived peptides have revealed various bioactivities that influence human health, including the modulation of endogenous cholesterol levels. Ongoing studies have shown that some peptides function using the same mechanism as statins and can be considered another class of compounds for HMGR inhibition.To date, the competitive inhibition of HMGR by peptides has been confirmed for 36 of them. The half-maximal inhibitory concentration (IC₅₀) of the most active food-derived peptide was found to be 12.8 μM, which is significantly lower than statin activity. This review presents approaches for modeling peptides to enhance their activity. Analysis of the peptides' physicochemical characteristics revealed the potential to design a more active peptide by adjusting a single parameter. The most active designed peptide exhibited 700 times the activity of an isolated peptide found in food. These studies demonstrate the potential of using peptides to develop nutraceuticals or drugs that prevent hypercholesterolemia.

他汀类药物预防高胆固醇血症及相关心血管疾病的有效性早已得到证实。他汀类药物是3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）的竞争性抑制剂。这种酶在代表胆固醇生物合成限速步骤的反应中起着关键作用。对食物来源多肽特性的大量研究揭示了影响人类健康的各种生物活性，包括调节内源性胆固醇水平。正在进行的研究表明，一些肽的作用机制与他汀类药物相同，可以被认为是抑制HMGR的另一类化合物。迄今为止，已证实其中36种多肽对HMGR有竞争性抑制作用。食源性肽的半最大抑制浓度（IC50）为12.8 μM，明显低于他汀类药物的活性。本文综述了建立多肽模型以增强其活性的方法。对多肽理化特性的分析揭示了通过调整单个参数来设计更有活性的多肽的潜力。设计出的活性最高的肽的活性是食品中分离肽活性的700倍。这些研究证明了利用多肽开发预防高胆固醇血症的营养品或药物的潜力。

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引用次数: 0

A Trichomonas vaginalis C2-XYPPX-repeat protein with a structured C2 domain displaying dampened flexibility upon binding calcium 阴道毛滴虫C2- xyppx -重复蛋白，具有结构化的C2结构域，在结合钙时显示受湿的灵活性。

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-02-01 Epub Date: 2025-11-22 DOI: 10.1016/j.biochi.2025.11.011

Garry W. Buchko , Lijun Liu , Kevin P. Battaile , Justin K. Craig , Elizabeth K. Harmon , Wesley C. Van Voorhis , Peter J. Myler , Scott Lovell

C2 domains are ubiquitous membrane-binding modules of ∼130 residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification. The genome of Trichomonas vaginalis, the most common, non-viral, sexually transmitted human pathogen, encodes eight genes that contain a N-terminal C2 module linked to a XYPPX-repeat domain of more than four XYPPX repeats (C2-XYPPX). While the function of the XYPPX-repeat domain remains unknown, its multiple association with C2 domains in T. vaginalis suggests it is important. The C2 domain from one of these C2-XYPPX-repeat proteins, Tv-C2-1, was structurally and physically characterized using X-ray crystallography and NMR spectroscopy. The crystal structure for Tv-C2-1 shows that this domain shares a fold common to all C2 domains, a compact Greek-key motif composed of eight anti-parallel β-strands in the type-2 topology. An NMR chemical shift perturbation study with Ca²⁺ showed that Tv-C2-1 bound two Ca²⁺ atoms primarily via two loops (loop-1 and loop-3) on the predicted calcium binding face of the protein with K_ds of 58.0 ± 0.1 μM and 232 ± 6 μM. Estimations of the overall rotational correlation time, τ_c, in the apo (11.1 ns) and Ca²⁺-bound (9.2 ns) state suggests the protein becomes more compact upon Ca²⁺ binding, consistent with a decrease in dynamics in loop-3 and marginally in loop-1 suggested by amide ¹⁵N heteronuclear steady-state {¹H}-¹⁵N NOEs. Showing Tv-C2-1 binds calcium and adopts a compact Greek-key motif structure, two primary features of C2 domains, suggests understanding the function of the XYPPX-repeat domain may be warranted.

C2结构域是真核生物中普遍存在的约130个残基的膜结合模块，通常与参与膜运输和脂质修饰的蛋白质相关。阴道毛滴虫是最常见的非病毒性性传播的人类病原体，其基因组编码8个基因，这些基因包含一个n端C2模块，该模块与超过4个XYPPX重复序列的XYPPX重复结构域（C2-XYPPX）相连。虽然XYPPX-repeat结构域的功能尚不清楚，但它与阴道T. C2结构域的多重关联表明它是重要的。其中一个C2- xyppx -repeat蛋白Tv-C2-1的结构域通过x射线晶体学和核磁共振光谱进行了结构和物理表征。Tv-C2-1的晶体结构表明，该结构域具有与所有C2结构域相同的褶皱，这是一个紧凑的希腊键基序，由8条反平行的β-链组成，具有2型拓扑结构。对Ca2+的核磁共振化学位移摄动研究表明，Tv-C2-1主要通过蛋白钙结合面上的两个环（loop-1和loop-3）结合两个Ca2+原子，Kds分别为58.0±0.1 μM和232±6 μM。在载脂蛋白（11.1 ns）和Ca2+结合（9.2 ns）状态下的总体旋转相关时间τc的估计表明，蛋白质在Ca2+结合时变得更加紧密，这与酰胺15N异核稳态{1H}-15N NOEs所表明的环-3和环-1的动力学减少一致。显示Tv-C2-1结合钙并采用紧凑的希腊键基序结构，这是C2结构域的两个主要特征，表明了解XYPPX-repeat结构域的功能可能是有必要的。

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引用次数: 0

Are Traditional mutant controls sufficient to identify true RNA G-quadruplex binding proteins? 传统的突变控制是否足以识别真正的RNA g -四重结合蛋白？

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-02-01 Epub Date: 2025-11-01 DOI: 10.1016/j.biochi.2025.10.018

Marc-Antoine Turcotte, Louise Dao Josépha Crespo, Jean-Pierre Perreault

G-quadruplexes (G4s) are stable non-canonical RNA structures involved in various regulatory processes, whose recognition by G4-binding proteins (G4BPs) is often studied using affinity purification and biochemical validation. A critical factor in validating true G4BPs lies in the choice of negative controls, which are frequently limited to simple G-to-A point mutations. Here, we reassess the classification of Guanine Nucleotide-Binding Protein-Like 1 (GNL1), previously identified as an RNA G4BP (rG4BP), by employing 7-deazaguanine (7dG) substitutions and salt variation strategies. Using fluorescence assays with N-methyl mesoporphyrin IX (NMM) and electrophoretic mobility shift assays (EMSAs), we show that GNL1 binds wild-type and 7dG-modified RNAs with comparable affinities. Additional experiments using potassium and lithium ions further that GNL1 binding is independent of G4 structure. Finally, truncation of PRKN RNA to its G4 core significantly reduced GNL1 binding, indicating that the protein does not interact with the folded G4 itself. These results collectively demonstrate that GNL1 binds guanine-containing sequences rather than true G4s, and that its prior classification as a G4BP was likely due to insufficient controls. Our findings highlight the importance of using robust negative controls, such as 7dG substitution and ionic condition modulation, in the accurate identification of bona fide G4BPs.

g -四plex （G4s）是参与多种调控过程的稳定非规范RNA结构，其被g4结合蛋白（g4bp）识别的研究经常使用亲和纯化和生化验证。验证真正g4bp的一个关键因素在于阴性对照的选择，阴性对照通常仅限于简单的G-to-A点突变。在这里，我们重新评估了鸟嘌呤核苷酸结合蛋白样1 （GNL1）的分类，以前鉴定为RNA G4BP (rG4BP)，采用7-去氮鸟嘌呤（7dG）取代和盐变化策略。通过n -甲基间卟啉IX （NMM）荧光分析和电泳迁移迁移分析（EMSAs），我们发现GNL1结合野生型和7dg修饰的rna具有相当的亲和力。另外使用钾离子和锂离子的实验进一步证明了GNL1的结合与G4结构无关。最后，将PRKN RNA截断至其G4核心显著降低了GNL1的结合，表明该蛋白不与折叠的G4本身相互作用。这些结果共同表明，GNL1结合的是含有鸟嘌呤的序列，而不是真正的G4s，而且其先前被分类为G4BP可能是由于控制不足。我们的研究结果强调了使用强大的阴性对照，如7dG取代和离子条件调制，在准确鉴定真正的g4bp中的重要性。

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引用次数: 0

Paracrine effect of fibroblasts on the proliferation and differentiation of bovine satellite cells in vitro 成纤维细胞对体外牛卫星细胞增殖分化的旁分泌作用。

IF 3 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimie

Pub Date : 2026-02-01 Epub Date: 2025-11-15 DOI: 10.1016/j.biochi.2025.11.006

Karolina Zygmunt , Kacper Żukowski , Katarzyna Piórkowska , Grzegorz Smołucha , Alicja Wierzbicka , Wojciech Witarski

The co-culture of fibroblasts and muscle cells is essential for obtaining cultured meat using in vitro techniques. While fibroblasts are known to promote myogenesis through cell-cell interactions, their paracrine effects and associated transcriptomic changes remain unknown. This study aimed to investigate the paracrine effect of fibroblasts on myogenesis, focusing on transcriptome profiling. Muscle satellite cells (isolated enzymatically) and fibroblasts (isolated through the explant culture) were co-cultured in 0.4 μm transwell plates for 5 days of proliferation and 24 and 72 h of differentiation. RNA-Seq and protein analysis (Western Blot, ELISA, immunofluorescence) were used to assess changes in myogenic marker expression. RNA-Seq revealed changes in many genes involved in myogenesis, such as upregulation of EGR1, IL6, and SOCS3 and downregulation of ITGA7. ELISA showed significantly higher MyHC levels at the proliferation stage in the co-culture group (p = 0.0183), with no significant differences at differentiation stages. To summarize, fibroblasts promote early myogenic differentiation and could modulate the extent of myogenic differentiation.

成纤维细胞和肌肉细胞的共培养是获得体外培养肉的必要条件。虽然已知成纤维细胞通过细胞间相互作用促进肌肉发生，但它们的旁分泌作用和相关的转录组变化仍不清楚。本研究旨在探讨成纤维细胞对肌肉发生的旁分泌作用，重点是转录组分析。将肌卫星细胞（酶解分离）和成纤维细胞（外植体培养分离）在0.4 μm transwell板中共培养5 d，分别分化24和72 h。采用RNA-Seq和蛋白分析（Western Blot， ELISA，免疫荧光）评估肌源性标志物表达的变化。RNA-Seq揭示了许多参与肌生成的基因的变化，如EGR1、IL6和SOCS3的上调和ITGA7的下调。ELISA结果显示，共培养组细胞增殖期MyHC水平显著升高（p=0.0183），分化期MyHC水平差异无统计学意义（p=0.0183）。综上所述，成纤维细胞促进早期成肌分化，并可调节成肌分化的程度。

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引用次数: 0