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Urineprint of high-altitude: Insights from analyses of urinary biomarkers and bio-physical-chemical features of extracellular vesicles 高海拔地区的尿指纹:通过分析尿液生物标志物和细胞外囊泡的生物物理化学特征获得启示。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-14 DOI: 10.1016/j.bpc.2024.107351
Serena Pilato , Simona Mrakic-Sposta , Vittore Verratti , Carmen Santangelo , Stefano di Giacomo , Samanta Moffa , Antonella Fontana , Tiziana Pietrangelo , Federica Ciampini , Sofia Bonan , Pamela Pignatelli , Carmine Noce , Pietro di Profio , Michele Ciulla , Danilo Bondi , Fabrizio Cristiano
Humans exposed to altitude hypoxia experience dysfunctions of the urinary system. As a non-invasive, easily manageable and informative biological sample, urine represents a relevant matrix for detecting clinical impairments of urinary system, as well as alterations of other systems and extracellular vesicles (EVs) biology during high-altitude expeditions. Nevertheless, gaps exist in the comprehensive assessment of dysfunction, molecular burden and EVs biology due to high-altitude acute exposure. This study aimed to find a biophysical and biochemical signature of urinary EVs for hypoxia-induced changes in urinary function, putatively accompanied by an oxinflammatory burden. Urine samples of 15 participants were sampled at low and high-altitude during an Alpine project (7 women and 8 men, aged 24-to-63 years and with BMI 17.93-to-30.76 kg/m2) and analysed for: creatinin and albumin, lipid peroxidation, IL6, NO derivatives; atomic force microscopy and Raman spectroscopy were carried out after urinary EVs were isolated through sucrose-gradient ultracentrifugation. Albumin-to-creatinin ratio increased at high altitude, as did IL6 and 8-isoprostane. AFM showed a globular and flattened shape of EVs, although several samples were characterized by a lot of contaminants and EVs lost their prototypal spherical shape; EVs comprehensively maintained their morphology at high altitude. Raman spectroscopy revealed some typical phospholipidic-like pattern, often masked by contaminants of spectra that most often refer to high-altitude samples. Collectively, short-term exposure to altitude hypoxia increased renal concentrating ability, produced non-pathological impairment or renal function, and triggered an oxyinflammatory burden with heterogeneous response of NO system. The combination of AFM and Raman spectroscopy revealed that EVs collected at high altitude more likely are fused together and incorporated into a sediment matrix, and contain contaminants peaks that make the purification process less efficient. The combination of analytical procedures as in the present study offers novel possibilities to detect the biological and clinical effects of high altitude on renal system.
暴露于高原缺氧环境中的人类会出现泌尿系统功能障碍。尿液作为一种非侵入性、易于管理且信息丰富的生物样本,是检测高海拔探险期间泌尿系统临床损伤以及其他系统和细胞外囊泡(EVs)生物学变化的相关基质。然而,在全面评估高海拔急性暴露导致的功能障碍、分子负担和EVs生物学方面还存在差距。本研究旨在寻找尿液EVs的生物物理和生物化学特征,以了解缺氧引起的泌尿系统功能变化(可能伴有氧化炎症负担)。在一次阿尔卑斯项目中,在低海拔和高海拔地区采集了 15 名参与者的尿样(7 名女性和 8 名男性,年龄在 24 至 63 岁之间,体重指数在 17.93 至 30.76 公斤/平方米之间),并分析了肌酐和白蛋白、脂质过氧化、IL6、NO 衍生物;通过蔗糖梯度超速离心法分离尿液 EVs 后,进行了原子力显微镜和拉曼光谱分析。在高海拔地区,白蛋白与肌酐的比率增加,IL6和8-异前列腺素也增加了。原子力显微镜(AFM)显示,EVs呈球状和扁平状,但有几个样本含有大量杂质,EVs失去了球状的原型;EVs在高海拔地区全面保持了其形态。拉曼光谱显示了一些典型的磷脂样模式,这些模式往往被高海拔样本的光谱污染物所掩盖。总之,短期暴露于高海拔缺氧环境中会增加肾脏的浓缩能力,产生非病理性的肾功能损害,并引发氧化性炎症负担和氮氧化物系统的异质性反应。结合原子力显微镜和拉曼光谱分析发现,在高海拔地区采集的肠病毒更有可能融合在一起并融入沉积物基质中,而且含有杂质峰,从而降低了纯化过程的效率。本研究结合了多种分析方法,为检测高海拔对肾脏系统的生物和临床影响提供了新的可能性。
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引用次数: 0
Kinetics of i-motif folding within the duplex context i-motif 在双链中折叠的动力学。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-12 DOI: 10.1016/j.bpc.2024.107350
Rugiya Alieva , Anna Keshek , Timofei Zatsepin , Victor Orlov , Andrey Aralov , Elena Zavyalova
Non-canonical nucleic acid structures possess an ability to interact selectively with proteins, thereby exerting influence over various intracellular processes. Numerous studies indicate that genomic G-quadruplexes and i-motifs are involved in the regulation of transcription. These structures are formed temporarily during the unwinding of the DNA double helix; and their direct determination is a rather difficult task. In addition, i-motif folding is pH-dependent, with most i-motifs having low stability at neutral pH. However, some genomic i-motifs with long cytosine repeats were shown to be stable at pH 7.3, suggesting their functionality within the nucleus. Here we studied pH-dependent behavior of a model i-motif with flanking sequences that forms a duplex motif. Kinetic studies on bimodular structures with cytosine residues replaced with an environment-sensitive fluorescent label reveal the stabilization of the i-motif structure near the i-motif-duplex junction. These results highlight the importance of the natural environment of i-motifs for the correct assessment of their stability.
非典型核酸结构具有选择性地与蛋白质相互作用的能力,从而对各种细胞内过程产生影响。大量研究表明,基因组 G-四链体和 i-位点参与了转录的调控。这些结构是在 DNA 双螺旋解旋过程中临时形成的,直接测定它们是一项相当困难的任务。此外,i-motif 的折叠与 pH 值有关,大多数 i-motif 在中性 pH 值下稳定性较低。然而,一些具有长胞嘧啶重复序列的基因组 i-motif 在 pH 值为 7.3 时是稳定的,这表明它们在细胞核内具有功能性。在这里,我们研究了一个带有侧翼序列的模型 i-motif的pH依赖性行为,它形成了一个双链图案。对胞嘧啶残基被环境敏感性荧光标签取代的双模结构进行的动力学研究表明,i-motif 结构在 i-motif 双链交界处附近趋于稳定。这些结果凸显了 i-motifs 的自然环境对于正确评估其稳定性的重要性。
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引用次数: 0
Supramolecular arrangements in human amyloid tissues using SAXS 利用 SAXS 分析人体淀粉样组织中的超分子排列。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-07 DOI: 10.1016/j.bpc.2024.107349
N.S. Mohd Nor Ihsan , S.F. Abdul Sani , L.M. Looi , Dharini Pathmanathan , P.L. Cheah , S.F. Chiew , Sirinart Chio-Srichan , Siriwat Soontaranon , D.A. Bradley
Amyloid diseases are characterized by the accumulation of misfolded protein aggregates in human tissues, pose significant challenges for both diagnosis and treatment. Protein aggregations known as amyloids are linked to several neurodegenerative conditions including Alzheimer's disease, Parkinson's disease, and systemic amyloidosis. The key goal of this research is to employ Small-Angle X-ray Scattering (SAXS) to examine the supramolecular structures of amyloid aggregates in human tissues. We present the structural analysis of amyloid using SAXS, which is employed directly to analyze thin tissue samples without damaging the tissues. This technique provides size and shape information of fibrils, which can be used to generate low-resolution 2D models. The present study investigates the structural changes in amyloid fibril axial d-spacing and scattering intensity in different human tissues, including kidney, heart, thyroid, and others, while also accounting for the presence of triglycerides in these tissues. Tissue structural components were examined at momentum transfer values between q = 0.2 nm−1 and 1.5 nm−1. The d-spacing is a critical parameter in SAXS that provides information about the periodic distances between structures within a sample. From the supramolecular SAXS patterns, the axial d-spacing of fibrils in amyloid tissues is prominent and exists within the 3rd to 10th order, compared to that of healthy tissues which do not have notable peak orders. The axial period of fibrils in amyloid tissues is within the scattering vector range 57.40–64.64 nm−1 while in normal tissues the range is between 60.68 and 61.41 nm−1, which is 3.0 nm−1 smaller than amyloid-containing tissues. Differences in d-spacing are often correlate with distinct pathological mechanisms or stages of disease progression. The application of SAXS to investigate amyloid structures in human tissues has enormous potential to further knowledge of amyloid disorders. This work will open the path for novel diagnostic instruments and therapeutic strategies meant to reduce the burden of amyloid-related diseases by offering a thorough structural examination of amyloid aggregates.
淀粉样蛋白疾病的特征是折叠错误的蛋白质聚集在人体组织中,给诊断和治疗带来了巨大挑战。被称为淀粉样蛋白的蛋白质聚集与多种神经退行性疾病有关,包括阿尔茨海默病、帕金森病和全身性淀粉样变性。这项研究的主要目标是利用小角 X 射线散射(SAXS)来研究人体组织中淀粉样蛋白聚集体的超分子结构。我们介绍了利用 SAXS 对淀粉样蛋白进行结构分析的方法,该方法可直接用于分析薄组织样本,而无需破坏组织。该技术提供了纤维的尺寸和形状信息,可用于生成低分辨率的二维模型。本研究调查了不同人体组织(包括肾脏、心脏、甲状腺等)中淀粉样蛋白纤维轴向 d-间距和散射强度的结构变化,同时还考虑了这些组织中甘油三酯的存在。在 q = 0.2 nm-1 和 1.5 nm-1 之间的动量传递值下对组织结构成分进行了研究。d 间距是 SAXS 中的一个关键参数,它提供了样品内结构间周期性距离的信息。从超分子 SAXS 图谱来看,淀粉样变组织中纤维的轴向 d-间距很明显,在 3 至 10 阶范围内,而健康组织中的纤维则没有明显的峰值阶数。淀粉样组织中纤维的轴向周期在散射矢量范围 57.40-64.64 nm-1 之间,而正常组织的范围在 60.68-61.41 nm-1 之间,比含淀粉样组织小 3.0 nm-1。d-间距的差异通常与不同的病理机制或疾病进展阶段相关。应用 SAXS 研究人体组织中的淀粉样蛋白结构对于进一步了解淀粉样蛋白疾病具有巨大的潜力。这项工作将为新型诊断仪器和治疗策略开辟道路,通过对淀粉样蛋白聚集体进行彻底的结构检查,减轻淀粉样蛋白相关疾病的负担。
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引用次数: 0
Characterization of a novel salt- and solvent-tolerant esterase Dhs82 from soil metagenome capable of hydrolyzing estrogenic phthalate esters 土壤元基因组中能水解雌激素邻苯二甲酸酯的新型耐盐和耐溶剂酯酶 Dhs82 的特征。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-11-05 DOI: 10.1016/j.bpc.2024.107348
Yuanyan Wang , Chunmei Deng , Xin Wang
Esterases that can function under extreme conditions are important for industrial processing and environmental remediation. Here, we report the identification of a salt- and solvent-tolerant esterase, Dhs82, from a soil metagenomic library. Dhs82 prefers short-chain p-nitrophenyl (p-NP) esters and exhibits enzymatic activity up to 1460 ± 61 U/mg towards p-NP butyrate. Meanwhile, Dhs82 can catalyze the hydrolysis of dialkyl phthalate esters, especially the widely-used diethyl phthalate (DEP), dipropyl phthalate (DPP) and di-n-butyl phthalate (DBP). Importantly, as an acidic protein with negative charges dominating its surface, Dhs82 is highly active and extraordinarily stable at high salinity. This property is quite rare among previously reported esterases/hydrolases capable of degrading phthalate esters (PAEs). In addition, Dhs82 activity can be significantly enhanced in the presence of solvents over a concentration range of 10–30 % (v/v). Notably, Dhs82 also showed high stability towards these solvents and solvent concentrations as high as 50–60 % (v/v) are required to inactivate Dhs82. Furthermore, molecular docking revealed the key residues, including the catalytic triad (Ser156, His281, and Asp251) and the surrounding Gly84 and Gly85, involved in the interaction of Dhs82 with DBP, depicting how Dhs82 degrades PAEs as a family IV esterase. Together, these diverse properties make Dhs82 a valuable candidate for both basic research and biotechnological applications.
能在极端条件下发挥作用的酯酶对工业加工和环境修复非常重要。在此,我们报告了从土壤元基因组文库中鉴定出的耐盐和耐溶剂酯酶 Dhs82。Dhs82 偏爱短链对硝基苯(p-NP)酯,对对硝基苯丁酸酯的酶活性高达 1460 ± 61 U/mg 。同时,Dhs82 还能催化水解邻苯二甲酸二烷基酯,尤其是广泛使用的邻苯二甲酸二乙酯(DEP)、邻苯二甲酸二丙酯(DPP)和邻苯二甲酸二正丁酯(DBP)。重要的是,作为一种表面带负电荷的酸性蛋白质,Dhs82 在高盐度条件下具有高度活性和超常稳定性。这一特性在之前报道的能够降解邻苯二甲酸酯(PAEs)的酯酶/水解酶中非常罕见。此外,在溶剂存在的情况下,Dhs82 的活性可在 10-30 %(v/v)的浓度范围内显著增强。值得注意的是,Dhs82 对这些溶剂也表现出很高的稳定性,溶剂浓度高达 50-60 % (v/v) 时才能使 Dhs82 失活。此外,分子对接揭示了参与 Dhs82 与 DBP 相互作用的关键残基,包括催化三元组(Ser156、His281 和 Asp251)以及周围的 Gly84 和 Gly85,描绘了 Dhs82 如何作为 IV 族酯酶降解 PAE。Dhs82 的这些不同特性使其成为基础研究和生物技术应用的重要候选物。
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引用次数: 0
The Drosophila RNA binding protein Hrp48 binds a specific RNA sequence of the msl-2 mRNA 3’ UTR to regulate translation 果蝇 RNA 结合蛋白 Hrp48 与 msl-2 mRNA 3' UTR 的特定 RNA 序列结合,从而调节翻译。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-29 DOI: 10.1016/j.bpc.2024.107346
Andrea Lomoschitz , Julia Meyer , Tanit Guitart , Miroslav Krepl , Karine Lapouge , Clara Hayn , Kristian Schweimer , Bernd Simon , Jiří Šponer , Fátima Gebauer , Janosch Hennig
Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3’ untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3’ UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.
抑制msl-2 mRNA的翻译对黑腹果蝇雌体的存活至关重要,以防止两条X染色体的过度转录。这种翻译控制事件由雌性特异性蛋白Sex-lethal(Sxl)协调,Sxl将RNA结合蛋白Unr和Hrp48招募到msl-2转录本的3'非翻译区(UTR),并抑制翻译启动。Hrp48在翻译抑制过程中的作用机制及其与msl-2的相互作用尚不十分清楚。在这里,我们研究了 Hrp48 的串联 RNA 识别基团的 RNA 结合特异性和亲和性。利用核磁共振光谱、分子动力学模拟和等温滴定量热法,我们确定了 Hrp48 识别的 msl-2 3' UTR 的确切区域。其他生物物理实验和翻译测定进一步揭示了 Hrp48、Unr、Sxl 和 RNA 形成复合物的情况。我们的结果表明,Hrp48与msl-2的Sxl和Unr的E和F结合位点下游结合,而不依赖于Sxl和Unr。
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引用次数: 0
Understanding Cu+2 binding with DNA: A molecular dynamics study comparing Cu2+ and Mg2+ binding to the Dickerson DNA 了解 Cu+2 与 DNA 的结合:比较 Cu2+ 和 Mg2+ 与 Dickerson DNA 结合的分子动力学研究。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-28 DOI: 10.1016/j.bpc.2024.107347
Angad Sharma, Hari O.S. Yadav, Pradipta Bandyopadhyay
Cu2+ ions led DNA damage by reactive oxygen species (ROS) is widely known biological phenomena. The ionic radii of Cu2+ and Mg2+ being similar, the binding of Cu2+ ions to DNA is expected to be similar to that of the Mg2+ ions. However, little is known how Cu2+ ions bind in different parts (phosphate, major and minor grooves) of a double-strand (ds) DNA, especially at atomic level. In the present study, we employ molecular dynamic (MD) simulations to investigate the binding of Cu2+ ions with the Dickerson DNA, a B-type dodecamer double stranded (ds) DNA. The binding characteristics of Cu2+ and Mg2+ ions with this dsDNA are compared to get an insight into the differences and similarities in binding behavior of both ions. Unlike Mg2+ ions, the first hydration shell of Cu2+ is found to be labile, thus it shows both direct and indirect binding with the dsDNA, i.e., binding through displacement of water from the hydration shell or through the hydration shell. Though the binding propensity of Cu2+ ions with dsDNA is observed relatively stronger, the binding order to phosphates, major groove, and minor groove is found qualitatively similar (phosphates > major groove > minor groove) for both ions. The study gives a deep understanding of Cu2+ binding to DNA, which could be helpful in rationalizing the Cu2+ led ROS-mediated DNA damage.
Cu2+ 离子导致活性氧(ROS)损伤 DNA 是广为人知的生物现象。Cu2+ 和 Mg2+ 的离子半径相似,因此 Cu2+ 离子与 DNA 的结合情况预计与 Mg2+ 离子相似。然而,人们对 Cu2+ 离子如何与双链(ds)DNA 的不同部分(磷酸、主要沟槽和次要沟槽)结合,尤其是在原子水平上结合知之甚少。在本研究中,我们采用分子动力学(MD)模拟来研究 Cu2+ 离子与 Dickerson DNA(一种 B 型十二聚体双链(ds)DNA)的结合。通过比较 Cu2+ 离子和 Mg2+ 离子与该 dsDNA 的结合特性,可以深入了解两种离子结合行为的异同。与 Mg2+ 离子不同,Cu2+ 离子的第一个水合壳是易变的,因此它与 dsDNA 的结合既有直接结合,也有间接结合,即通过水合壳中的水置换结合或通过水合壳结合。虽然观察到 Cu2+ 离子与 dsDNA 的结合倾向相对较强,但发现这两种离子与磷酸盐、主沟和小沟的结合顺序在性质上相似(磷酸盐 > 主沟 > 小沟)。该研究深入揭示了 Cu2+ 与 DNA 的结合,有助于合理解释 Cu2+ 导致的 ROS 介导的 DNA 损伤。
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引用次数: 0
Biophysical significance of fluorescence spectroscopy in deciphering nucleic acid dynamics: From fundamental to recent advancements 荧光光谱在解读核酸动力学方面的生物物理意义:从基础到最新进展
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-22 DOI: 10.1016/j.bpc.2024.107345
Vivek Pandey , Tejasvi Pandey
Fluorescence spectroscopy has revolutionized the study of nucleic acids, providing invaluable insights into the dynamic processes that underpin gene expression, replication, and repair. This review explores the application of fluorescence probes in monitoring the conformational changes, interactions, and regulatory mechanisms of DNA and RNA. We discuss the utility of intercalating and non-intercalating fluorescent probes in real-time tracking of nucleic acid dynamics, highlighting their role in elucidating the molecular mechanisms of DNA replication, transcriptional regulation, and DNA repair. By offering a detailed analysis of recent advancements, this review underscores the significance of fluorescence-based techniques in enhancing our understanding of nucleic acid behavior and their implications for genomic stability and therapeutic interventions.
荧光光谱学彻底改变了核酸研究,为了解基因表达、复制和修复的动态过程提供了宝贵的见解。本综述探讨了荧光探针在监测 DNA 和 RNA 的构象变化、相互作用和调控机制方面的应用。我们讨论了插层和非插层荧光探针在实时跟踪核酸动态中的应用,强调了它们在阐明 DNA 复制、转录调控和 DNA 修复的分子机制中的作用。通过对最新进展的详细分析,这篇综述强调了基于荧光的技术在加深我们对核酸行为及其对基因组稳定性和治疗干预的影响的理解方面所具有的重要意义。
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引用次数: 0
How fingers affect folding of a thumb: Inter-subdomain cooperation in the folding of SARS-CoV-2 RdRp protein 手指如何影响拇指的折叠:SARS-CoV-2 RdRp 蛋白折叠过程中的亚域间合作
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.bpc.2024.107342
Anushree Sinha , Angel Mary Chiramel Tony , Susmita Roy
The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a critical enzyme essential for the virus's replication and transcription, making it a key therapeutic target. The RdRp protein exhibits a characteristic cupped right-hand shaped structure with two vital subdomains: the fingers and the thumb. Despite being distinct, biophysical experiments suggest that these subdomains cooperate to facilitate RNA accommodation, ensuring RdRp functionality. To investigate the structure-based mechanisms underlying the fingers-thumb interaction in both apo and RNA-bound RdRp, we constructed a coarse-grained structure-based model based on recent cryo-electron microscopy data. The simulations reveal frequent open-to-closed conformational transitions in apo RdRp, akin to a breathing-like motion. These conformational changes are regulated by the fingers-thumb association and the folding dynamics of the thumb subdomain. The thumb adopts a stable fold only when tethered by the fingers-thumb interface; when these subdomains are disconnected, the thumb transitions into an open state. A significant number of open-to-closed transition events were analyzed to generate a transition contact probability map, which highlights a few specific residues at the thumb-fingers interface, distant from the RNA accommodation sites, as essential for inducing the thumb's folding process. Given that thumb subdomain folding is critical for RNA binding and viral replication, the study proposes that these interfacial residues may function as remote regulatory switches and could be targeted for the development of allosteric drugs against SARS-CoV-2 and similar RNA viruses.
SARS-CoV-2 的 RNA 依赖性 RNA 聚合酶(RdRp)是病毒复制和转录所必需的关键酶,因此成为关键的治疗靶点。RdRp 蛋白具有独特的右手杯状结构,有两个重要的子域:手指和拇指。尽管各不相同,但生物物理实验表明,这两个亚域相互合作,促进 RNA 的容纳,确保了 RdRp 的功能。为了探究apo和RNA结合的RdRp中手指与拇指相互作用的结构机制,我们根据最近的冷冻电镜数据构建了一个粗粒度的结构模型。模拟结果表明,在 apo RdRp 中,从打开到关闭的构象转变非常频繁,类似于呼吸运动。这些构象变化受手指与拇指的关联以及拇指亚域的折叠动力学的调控。拇指只有在被手指-拇指界面拴住时才会采用稳定的折叠;当这些子域断开时,拇指就会过渡到开放状态。通过分析大量从打开到关闭的转换事件,生成了转换接触概率图,该图突出显示了拇指-手指界面上远离 RNA 容纳位点的几个特定残基,它们对于诱导拇指的折叠过程至关重要。鉴于拇指亚域的折叠对 RNA 结合和病毒复制至关重要,该研究认为这些界面残基可能具有远程调控开关的功能,可以作为开发针对 SARS-CoV-2 和类似 RNA 病毒的异构药物的目标。
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引用次数: 0
In vitro and in silico effect of meldrum's acid-derived compounds on Staphylococcus aureus strains as NorA efflux pump inhibitors 作为 NorA 外排泵抑制剂,梅尔德隆酸衍生化合物对金黄色葡萄球菌菌株的体外和硅学效应。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.bpc.2024.107344
Isaac Moura Araújo , Raimundo Luiz Silva Pereira , Ana Carolina Justino de Araújo , Sheila Alves Gonçalves , Saulo Relison Tintino , Cícera Datiane de Morais Oliveira-Tintino , Irwin Rose Alencar de Menezes , Renata Salamoni , Iêda Maria Begnini , Ricardo Andrade Rebelo , Luiz Everson da Silva , Carolina Bandeira Domiciano , Henrique Douglas Melo Coutinho
The misuse of antibiotics has led to an alarming increase in bacterial strains resistant to these drugs. Efflux pumps, which expel antibiotics from bacterial cells, have emerged as one of the key mechanisms of bacterial resistance. In the quest to combat and mitigate bacterial resistance, researchers have turned their attention to efflux pump inhibitors as a potential solution. Meldrum's acid, a synthetic molecule widely utilized in the synthesis of bioactive compounds, has garnered significant interest in this regard. Hence, this study aims to investigate the antibacterial activity and evaluate the efficacy of three derivatives of meldrum's acid in inhibiting efflux mechanisms, employing both in silico and in vitro approaches. The antibacterial activity of the derivatives was assessed through rigorous broth microdilution testing. While the derivatives themselves did not exhibit direct antibacterial activity, they demonstrated remarkable potential in potentiating the effects of antibiotics. Additionally, fluorescence emission assays using ethidium bromide (EtBr) revealed fluorescence levels comparable to the positive control, indicating a possible blockade of efflux pumps. Molecular docking studies conducted in silico further supported these findings by revealing binding interactions similar to norfloxacin and CCCP, known efflux pump inhibitors. These results underscore the potential of meldrum's acid derivatives as effective inhibitors of efflux pumps. By inhibiting these mechanisms, the derivatives hold promise in enhancing the effectiveness of antibiotics and combatting bacterial resistance. This study contributes valuable insights into the development of novel strategies to address the pressing issue of bacterial resistance and paves the way for further research and exploration in this field.
抗生素的滥用导致对这些药物产生抗药性的细菌菌株数量惊人地增加。将抗生素排出细菌细胞的外排泵已成为细菌产生耐药性的关键机制之一。为了消除和减轻细菌的耐药性,研究人员将目光转向了外排泵抑制剂,将其作为一种潜在的解决方案。梅尔杜姆酸是一种广泛用于合成生物活性化合物的合成分子,在这方面引起了极大的兴趣。因此,本研究采用了硅学和体外方法,旨在研究美杜莎酸的三种衍生物的抗菌活性,并评估其在抑制外流机制方面的功效。衍生物的抗菌活性是通过严格的肉汤微稀释试验进行评估的。虽然这些衍生物本身没有表现出直接的抗菌活性,但它们在增强抗生素效果方面表现出了显著的潜力。此外,使用溴化乙锭(EtBr)进行的荧光发射测定显示,其荧光水平与阳性对照相当,这表明它们可能阻断了外排泵。在硅学中进行的分子对接研究进一步证实了这些发现,研究显示了与诺氟沙星和CCCP(已知的外排泵抑制剂)类似的结合相互作用。这些结果凸显了美杜莎酸衍生物作为有效外排泵抑制剂的潜力。通过抑制这些机制,这些衍生物有望提高抗生素的疗效并对抗细菌耐药性。这项研究为开发解决细菌耐药性这一紧迫问题的新策略提供了宝贵的见解,并为这一领域的进一步研究和探索铺平了道路。
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引用次数: 0
Solubilisation & purification of membrane proteins using benzylamine-modified SMA polymers 使用苄胺改性 SMA 聚合物增溶和纯化膜蛋白。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.bpc.2024.107343
Aneel Akram , Waled Hadasha , Gestél C. Kuyler , Michael-Phillip Smith , Shauna Bailey-Dallaway , Aiden Preedy , Caolan Browne , Luke Broadbent , Adam Hill , Tahreem Javaid , Haroon Nazar , Nikita Samra , Anadil Naveed , Holly Tregunna , Hetal Joshi , Nusheen Akhtar , Aneesa Javed , Jessica Bowater , Joel Ravenhill , Patrik Hajdu , Alice J. Rothnie
Extraction of proteins from the membrane using styrene maleic acid co-polymers (SMA), forming SMA lipid particles (SMALPs), has allowed for the first time the purification of membrane proteins with their lipid bilayer environment. To date, SMA2000 has been the most effective polymer used for this purpose, with a 2:1 ratio of styrene:maleic acid, and styrene and maleic acid moieties spread statistically throughout the chain. However, SMA2000 is a highly polydisperse polymer that contains an array of different polymer lengths and sequences. RAFT polymerisation offers much better control over the polymer length; however, homogeneous distribution of styrene and maleic acid throughout the polymer is difficult to achieve. Instead, here RAFT polymerisation was used to produce a 1:1 styrene:maleic anhydride polymer, which was then modified with benzylamine. This mimics the 2:1 hydrophobic:hydrophilic nature of SMA2000, while controlling the length and obtaining a homogeneous distribution of the hydrophobic moieties (styrene and N-benzylmaleimide). SMA-benzylamine (SMA-BA) polymers of three different lengths (2, 4, and 7 kDa) were all able to solubilise purified lipids, cellular membranes, and a range of specific proteins. However, the larger 7 kDa polymer solubilised membranes more slowly and less efficiently than the shorter polymers. This also affected the yield of purified protein obtained by affinity purification with this polymer. The smallest 2 kDa polymer solubilised membranes the fastest but appeared to offer less stability to the extracted proteins. The SMA-BA polymers were more sensitive to Mg2+ ions than SMA2000. SMA-BA 4 kDa was otherwise comparable to SMA2000 and even gave a higher degree of purity.
使用苯乙烯-马来酸共聚物(SMA)从膜中提取蛋白质,形成 SMA 脂质颗粒(SMALPs),首次实现了在脂质双分子层环境中纯化膜蛋白质。迄今为止,SMA2000 是用于此目的最有效的聚合物,其苯乙烯和马来酸的比例为 2:1,苯乙烯和马来酸分子分布在整个链中。不过,SMA2000 是一种高度分散的聚合物,含有一系列不同长度和序列的聚合物。RAFT 聚合可以更好地控制聚合物的长度;但是,很难实现苯乙烯和马来酸在整个聚合物中的均匀分布。因此,这里采用了 RAFT 聚合法来生产 1:1 苯乙烯:马来酸酐聚合物,然后用苄胺对其进行改性。这就模仿了 SMA2000 2:1 的疏水:亲水性质,同时控制了长度并获得了疏水分子(苯乙烯和 N-苄基马来酰亚胺)的均匀分布。三种不同长度(2、4 和 7 kDa)的 SMA-苄胺(SMA-BA)聚合物都能溶解纯化的脂质、细胞膜和一系列特定蛋白质。不过,与较短的聚合物相比,较大的 7 kDa 聚合物溶解膜的速度更慢,效率更低。这也影响了使用这种聚合物进行亲和纯化所获得的纯化蛋白质的产量。最小的 2 kDa 聚合物溶解膜的速度最快,但提取蛋白质的稳定性似乎较差。与 SMA2000 相比,SMA-BA 聚合物对 Mg2+ 离子更敏感。SMA-BA 4 kDa 在其他方面与 SMA2000 相当,甚至纯度更高。
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Biophysical chemistry
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