Biochimica et biophysica acta. Biomembranes最新文献_第10页

Growth of Staphylococcus aureus in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides 金黄色葡萄球菌在油酸存在下的生长会改变糖脂脂肪酸谱，并增加对抗菌肽的耐药性。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-11-03 DOI: 10.1016/j.bbamem.2024.184395

Djuro Raskovic , Gloria Alvarado , Kelly M. Hines , Libin Xu , Craig Gatto , Brian J. Wilkinson , Antje Pokorny

Staphylococcus aureus readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, S. aureus presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When S. aureus is grown conventionally, its membrane lipids contain a mix of branched-chain and straight-chain saturated fatty acids. However, when unsaturated fatty acids are present in the growth medium, they become a major part of the total fatty acid composition. This study explores the biophysical effects of incorporating straight-chain unsaturated fatty acids into S. aureus membrane lipids. Membrane preparations from cultures supplemented with oleic acid showed more complex differential scanning calorimetry scans than those grown in tryptic soy broth alone. When grown in the presence of oleic acid, the cultures exhibited a transition significantly above the growth temperature, attributed to the presence of glycolipids with long-chain fatty acids causing acyl chain packing frustration within the bilayer. Functional aspects of the membrane were assessed by studying the kinetics of dye release from unilamellar vesicles induced by the antimicrobial peptide mastoparan X. Dye release was slower from liposomes prepared from cells grown in oleic acid-supplemented cultures, suggesting that changes in membrane lipid composition and biophysics protect the cell membrane against peptide-induced lysis. These findings underscore the intricate relationship between the growth environment, membrane lipid composition, and the physical properties of the bacterial membrane, which should be considered when developing new strategies against S. aureus infections.

金黄色葡萄球菌很容易适应各种环境，并迅速产生抗生素耐药性，导致耐多药感染增加。因此，金黄色葡萄球菌是一个重大的全球健康问题，它对宿主环境的适应性对于了解发病机理和抗生素敏感性至关重要。当金黄色葡萄球菌以传统方式生长时，其膜脂质含有支链和直链饱和脂肪酸的混合物。然而，当生长培养基中含有不饱和脂肪酸时，它们就会成为总脂肪酸组成的主要部分。本研究探讨了在金黄色葡萄球菌膜脂中加入直链不饱和脂肪酸的生物物理效应。与仅在胰蛋白酶大豆肉汤中生长的培养物相比，添加了油酸的培养物膜制备物显示出更复杂的差示扫描量热扫描。在有油酸存在的情况下生长时，培养物会出现明显高于生长温度的转变，这归因于含有长链脂肪酸的糖脂的存在导致了双分子层内酰基链堆积失调。通过研究抗菌肽马斯托帕兰 X 诱导的单纤毛膜囊泡释放染料的动力学，评估了膜的功能方面。这些发现强调了生长环境、膜脂成分和细菌膜的物理特性之间错综复杂的关系，在开发抗金葡菌感染的新策略时应考虑到这一点。

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引用次数: 0

Voltage- and Ca2+-inducible PLC activity for analyzing PI(4,5)P2 sensitivity of ion channels in Xenopus oocytes 电压和 Ca2+ 诱导的 PLC 活性，用于分析爪蟾卵母细胞中离子通道对 PI(4,5)P2 的敏感性。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-30 DOI: 10.1016/j.bbamem.2024.184396

Takafumi Kawai , Natsuki Mizutani , Yasushi Okamura

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP₂ levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP₂ levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP₂. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca²⁺ influx driven by the increased driving force of Ca²⁺. High Ca²⁺ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca²⁺-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP₂-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca²⁺-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP₂ levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP₂ levels, (2) PIP₂ levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP₂ levels were reduced by Ca²⁺ administration. Therefore, PLCζ effectively supports understanding how PIP₂ regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP₂ and the PLC pathway in Xenopus oocytes.

磷脂酰肌醇 4,5-二磷酸（PIP2）是调节各种离子通道活动的关键膜脂。目前，有多种分子工具可用于调节 PIP2 的水平，但每种工具都有各自的优缺点。在本研究中，我们提出了一种利用异源爪蟾卵母细胞的新方法，即利用能水解 PIP2 的磷脂酶 C（PLC）-ζ 来精确操纵 PIP2 水平。注射了 PLCζ 的爪蟾卵母细胞在 Ca2+ 驱动力增加的驱动下表现出明显的超极化诱导 Ca2+ 流入。PLCζ 对 Ca2+ 的高敏感性促进了超极化诱导的 PLC 在章鱼卵母细胞中的活性，这种活性是电压和 Ca2+ 依赖性的。这项研究证明了 PLCζ 以电压和 Ca2+ 依赖性方式调节 PIP2 敏感离子通道（如 KCNQ2/3 和 GIRK 通道）的调节能力。此外，PLCζ的激活途径只需要一个双电极电压钳装置，这使其成为一种方便的分子工具，可与电压感应磷酸酶（VSP）相结合来操纵 PIP2 水平。与 VSP 相比，PLCζ 具有明显的特点和优势：(1) 超极化（而非去极化）会降低 PIP2 水平；(2) PIP2 水平降低的同时，磷脂酰肌醇 4-单磷酸（PIP）水平不会增加；(3) 施加 Ca2+ 会降低 PIP2 水平。因此，PLCζ 有效地支持了对 PIP2 如何与 VSP 一起调节离子通道的理解。总之，本研究强调了 PLCζ 的独特性及其在分析异种卵母细胞中 PIP2 和 PLC 通路对离子通道调控的独特优势。

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引用次数: 0

Lipid bilayer permeabilities and antibiotic effects of tetramethylguanidinium and choline fatty acid ionic liquids 四甲基胍和胆碱脂肪酸离子液体的脂质双分子层渗透性和抗生素效应。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-22 DOI: 10.1016/j.bbamem.2024.184393

Achismita Dutta , Brandon Burrell , Esha Prajapati , Sierra Cottle , Hailey Y. Maurer , Matthew J. Urban , Samuel R. Pennock , Arwa M. Muhamed , Janiyah Harris , Yesenia Flores , Lauren Staman , Benjamin R. Carone , Gregory A. Caputo , Timothy D. Vaden

Ionic liquids (ILs) have been studied as potential components in antibiotic formulations based on their abilities to permeabilize and penetrate lipid bilayer, which correlate with their antibacterial effects. Fatty acid-based ILs (FAILs), in which the anion is a long-chain fatty acid, can permeabilize lipid membranes and have been used in biomedical applications since they have low human cell cytotoxicity. In this work we investigated the abilities of several different FAILs to permeabilize lipid bilayers and how that permeabilization correlates with antibacterial activity, cell membrane permeability, and cytotoxicity. The FAILs consisted of the cations tetramethylguanidinium (TMG) or choline combined with octanoate or decanoate. These FAILs were tested on model bilayer vesicles with three different lipid compositions for membrane permeabilization using a leakage assay. They were then tested for antibiotic and membrane permeabilization on bacterial and mammalian cells. The results show that while the octanoate-based FAILs do not form micelles and have low activities on vesicles and biological cells, the decanoate-based FAILs can permeabilize bilayers and have biological activities that correlate with the model vesicle results. The ILs with both cation and fatty-acid anion have strong activities while the decanoate alone has only minimal permeabilization and antibiotic activity. Membrane permeabilization occurs at FAIL concentrations below their CMC values which suggests that their mechanism of action may not involve micelle formation.

离子液体（ILs）具有渗透和穿透脂质双分子层的能力，与抗菌效果相关，因此被研究用作抗生素制剂的潜在成分。脂肪酸基 IL（FAILs）中的阴离子是长链脂肪酸，可以渗透脂膜，由于对人体细胞的细胞毒性较低，因此已被用于生物医学领域。在这项工作中，我们研究了几种不同的 FAILs 对脂质双分子层的渗透能力，以及这种渗透能力与抗菌活性、细胞膜渗透性和细胞毒性之间的关系。FAIL 由阳离子四甲基胍（TMG）或胆碱与辛酸酯或癸酸酯组合而成。在具有三种不同脂质成分的模型双层囊泡上对这些 FAIL 进行了测试，使用渗漏测定法检测其膜渗透性。然后在细菌和哺乳动物细胞上进行了抗生素和膜渗透测试。结果表明，辛酸酯类 FAIL 不能形成胶束，对囊泡和生物细胞的活性较低，而癸酸酯类 FAIL 则能渗透双层膜，并具有与模型囊泡结果相关的生物活性。同时具有阳离子和脂肪酸阴离子的IL具有很强的活性，而单独的癸酸酯只有极低的渗透性和抗生素活性。当 FAIL 的浓度低于其 CMC 值时就会发生膜渗透，这表明它们的作用机制可能不涉及胶束的形成。

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引用次数: 0

TNF receptors: Structure-function relationships and therapeutic targeting strategies TNF 受体：结构-功能关系和靶向治疗策略

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-22 DOI: 10.1016/j.bbamem.2024.184394

Chih Hung Lo

Tumor necrosis factor receptors (TNFR1 and TNFR2) play key roles in mediating inflammatory response and cell death signaling, which are associated with autoimmune disorders, neurodegenerative diseases, and cancers. The structure-function relationships of TNF receptors and their ligands determine the activation or inhibition of downstream signaling pathways. Available crystal structures have provided critical insights into the therapeutic targeting strategies of TNF receptors and their signaling networks. In this review, we discuss the potential of targeting receptor-ligand and receptor-receptor interactions in a competitive manner as well as perturbing receptor conformational dynamics through an allosteric mechanism to modulate TNF receptor signaling. We propose that conformational states of TNF receptors can act as a molecular switch in determining their functions and are important therapeutic targets. The knowledge of the structure-function relationships of TNF receptors can be applied to translational high-throughput drug screening and design of novel receptor-specific modulators with enhanced pharmacological properties.

肿瘤坏死因子受体（TNFR1 和 TNFR2）在介导炎症反应和细胞死亡信号传导方面发挥着关键作用，而炎症反应和细胞死亡信号传导与自身免疫性疾病、神经退行性疾病和癌症有关。TNF 受体及其配体的结构-功能关系决定了下游信号通路的激活或抑制。现有的晶体结构为 TNF 受体及其信号网络的靶向治疗策略提供了重要见解。在这篇综述中，我们讨论了以竞争方式靶向受体-配体和受体-受体相互作用的潜力，以及通过异构机制扰乱受体构象动力学以调节 TNF 受体信号传导的潜力。我们提出，TNF受体的构象状态可作为决定其功能的分子开关，是重要的治疗靶点。有关 TNF 受体结构-功能关系的知识可应用于转化型高通量药物筛选，并设计出药理特性更强的新型受体特异性调节剂。

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引用次数: 0

Conformational heterogeneity and structural features for function of the prototype viroporin influenza AM2 原型病毒蛋白流感 AM2 功能的构象异质性和结构特征。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-17 DOI: 10.1016/j.bbamem.2024.184387

Kyriakos Georgiou, Antonios Kolocouris

The 97-residue influenza A matrix 2 (ΑM2) protein, a prototype for viroporins, transports protons through water molecules and His37. We discuss structural biology and molecular biophysics experiments and some functional assays that have transformed over 40 years our understanding of the structure and function of AM2. The structural studies on ΑM2 have been performed with different conditions (pH, temperature, lipid, constructs) and using various protein constructs, e.g., AM2 transmembrane (AM2TM) domain, AM2 conductance domain (AM2CD), ectodomain-containing or ectodomain-truncated, AM2 full length (AM2FL) and aimed to describe the different conformations and structural details that are necessary for the stability and function of AM2. However, the conclusions from these experiments appeared sometimes ambiguous and caused exciting debates. This was not due to inaccurate measurements, but instead because of the different membrane mimetic environment used, e.g., detergent, micelles or phospholipid bilayer, the method (e.g., X-ray crystallography, solid state NMR, solution NMR, native mass spectrometry), the used protein construct (e.g., AM2TM or AM2CD), or the amino acids residues to follow observables (e.g., NMR chemical shifts). We present these results according to the different used biophysical methods, the research groups and often by keeping a chronological order for presenting the progress in the research. We discuss ideas for additional research on structural details of AM2 and how the present findings can be useful to explore new routes of influenza A inhibition. The AM2 research can provide inspiration to study other viroporins as drug targets.

97个残基的甲型流感基质2（ΑM2）蛋白是病毒蛋白的原型，它通过水分子和His37转运质子。我们讨论了结构生物学和分子生物物理学实验以及一些功能测定，这些实验和测定在过去 40 年中改变了我们对 AM2 结构和功能的理解。对ΑM2的结构研究是在不同条件（pH值、温度、脂质、构建体）下进行的，并使用了不同的蛋白质构建体，如AM2跨膜（AM2TM）结构域、AM2传导结构域（AM2CD）、含外结构域或外结构域截断、AM2全长（AM2FL），旨在描述AM2稳定性和功能所必需的不同构象和结构细节。然而，这些实验的结论有时显得模棱两可，引起了激烈的争论。这并不是因为测量结果不准确，而是因为使用了不同的膜模拟环境，如去垢剂、胶束或磷脂双分子层，使用了不同的方法（如 X 射线晶体学、固态核磁共振、溶液核磁共振、原生质谱），构建了不同的蛋白质（如 AM2TM 或 AM2CD），或跟踪了不同的氨基酸残基。我们根据所使用的不同生物物理方法、研究小组来介绍这些成果，并通常按照时间顺序介绍研究进展。我们讨论了对 AM2 结构细节进行更多研究的想法，以及目前的发现如何有助于探索抑制甲型流感的新途径。AM2的研究可以为研究其他作为药物靶点的病毒蛋白提供灵感。

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引用次数: 0

Polymer nanodiscs support the functional extraction of an artificial transmembrane cytochrome 聚合物纳米盘支持人工跨膜细胞色素的功能提取

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-15 DOI: 10.1016/j.bbamem.2024.184392

Benjamin J. Hardy, Holly C. Ford, May Rudin, J.L. Ross Anderson, Paul Curnow

Polymer nanodiscs are an attractive alternative to surfactants for studying integral membrane proteins within their native lipid environment. Here, we investigate the use of such polymers to isolate a computationally-designed de novo membrane cytochrome named CytbX. We show that the block copolymers known as CyclAPols can efficiently extract CytbX directly from biomembranes and are compatible with the downstream purification and biophysical characterisation of this artificial protein. CyclAPol-solubilised CytbX is well-folded and highly robust with properties that are essentially identical to those observed for the same protein in a detergent micelle. However, electron transfer to CytbX from a diffusive flavoprotein is substantially faster in micelles than in the nanodisc system. Our results confirm that polymer nanodiscs will be a useful tool for the ongoing study and application of de novo membrane proteins.

在研究原生脂质环境中的整体膜蛋白时，聚合物纳米盘是一种极具吸引力的表面活性剂替代品。在这里，我们研究了如何利用这种聚合物来分离一种通过计算重新设计的膜细胞色素 CytbX。我们的研究表明，被称为 CyclAPols 的嵌段共聚物能直接从生物膜中有效地提取 CytbX，并能与这种人工蛋白的下游纯化和生物物理特性分析相兼容。CyclAPol溶解后的CytbX具有良好的折叠性和高度的稳健性，其特性与在洗涤剂胶束中观察到的相同蛋白质的特性基本相同。然而，在胶束中，扩散黄素蛋白向 CytbX 的电子传递速度远远快于纳米盘系统。我们的研究结果证实，聚合物纳米盘将成为研究和应用新膜蛋白的有用工具。

引用次数: 0

A comparison of lipid diffusive dynamics in monolayers and bilayers in the context of interleaflet coupling 在小叶间耦合的背景下比较单层和双层中的脂质扩散动力学。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-12 DOI: 10.1016/j.bbamem.2024.184388

Titas Mandal , Nadine Brandt , Carmelo Tempra , Matti Javanainen , Balázs Fábián , Salvatore Chiantia

Cellular membranes are composed of lipids typically organized in a double-leaflet structure. Interactions between these two leaflets – often referred to as interleaflet coupling – play a crucial role in various cellular processes. Despite extensive study, the mechanisms governing such interactions remain incompletely understood. Here, we investigate the effects of interleaflet coupling from a specific point of view, i.e. by comparing diffusive dynamics in bilayers and monolayers, focusing on potential lipid-specific interactions between opposing leaflets. Through quantitative fluorescence microscopy techniques, we characterize lipid diffusion and mean molecular area in monolayers and bilayers composed of different lipids. Our results suggest that the observed decrease in bilayer lipid diffusion compared to monolayers depends on lipid identity. Furthermore, our analysis suggests that lipid acyl chain structure and spatial configuration at the bilayer may strongly influence interleaflet interactions and dynamics in bilayers. These findings provide insights into the role of lipid structure in mediating interleaflet coupling and underscore the need for further experimental investigations to elucidate the underlying mechanisms.

细胞膜由脂质组成，通常呈双叶结构。这两个小叶之间的相互作用（通常称为小叶间耦合）在各种细胞过程中起着至关重要的作用。尽管进行了广泛的研究，但人们对这种相互作用的机制仍不甚了解。在这里，我们从一个特定的角度研究了小叶间耦合的影响，即通过比较双层膜和单层膜中的扩散动力学，重点研究对立小叶间潜在的脂质特异性相互作用。通过定量荧光显微镜技术，我们描述了由不同脂质组成的单层和双层中的脂质扩散和平均分子面积。我们的结果表明，与单层膜相比，观察到的双层膜脂质扩散的减少取决于脂质的特性。此外，我们的分析表明，脂质酰基链结构和双分子层的空间构型可能会强烈影响双分子层中小叶间的相互作用和动力学。这些发现让我们深入了解了脂质结构在介导小叶间耦合中的作用，并强调了进一步开展实验研究以阐明内在机制的必要性。

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引用次数: 0

Evidence for phospholipid self-organisation in concentrated ammonia-water environments 浓氨水环境中磷脂自组织的证据。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-09 DOI: 10.1016/j.bbamem.2024.184391

Sean M. Mackay , Ben Sutherland , Richard A. Easingwood , Andrew Hopkins , Mihnea Bostina , Eng Wui Tan

Titan, the largest moon of Saturn is thought to have the potential to support primordial life. The surface of Titan contains bodies of liquid hydrocarbons, and modelling suggests that an ammonia-water ocean resides deep beneath the surface, both of which have been speculated to support primordial chemistry. Here we present the first evidence that both preformed and self-organised phospholipid vesicles remain stable and can maintain concentration gradients in ammonia-water environments; a fundamental requirement for primordial chemistry and biology to originate. We further reveal the remarkable stability of a diether phospholipid, such as those found in extremophilic bacteria, under these conditions and demonstrate that electron microscopy and tomography are useful tools to investigate macromolecular structure under diverse physico-chemical environments.

土卫六是土星最大的卫星，被认为有可能孕育原始生命。土卫六表面含有液态碳氢化合物体，模型显示表面下深处存在氨水海洋，这两种物质都被推测为支持原始化学。在这里，我们首次证明了预形成和自组织磷脂囊泡都能保持稳定，并能在氨水环境中维持浓度梯度；这是原始化学和生物学起源的基本要求。我们进一步揭示了二元磷脂（如嗜极细菌中发现的磷脂）在这些条件下的显著稳定性，并证明电子显微镜和层析成像技术是研究不同物理化学环境下大分子结构的有用工具。

引用次数: 0

Nanodisc-associated acetylcholinesterase as a novel model system of physiological relevant membrane-bound cholinesterases. Inhibition by phenolic compounds 纳米盘相关乙酰胆碱酯酶作为生理相关膜结合胆碱酯酶的新型模型系统。酚类化合物的抑制作用。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-08 DOI: 10.1016/j.bbamem.2024.184389

Paula Belén Salazar , Fernando Gabriel Dupuy , Mariana C. Fiori , Samantha M. Stanfield , Jon McCord , Guillermo A. Altenberg , Carlos Javier Minahk

Acetylcholinesterase (AChE) plays a pivotal role in the cholinergic system, and its inhibition is sought after in a wide range of applications, from insect control to Alzheimer's disease treatment. While the primary physiological isoforms of AChE are membrane-bound proteins, most assays for discovering new, safer, and potent inhibitors are conducted using commercially available soluble isoforms, such as the electric eel AChE (eeAChE). In this study, we conducted a comparative analysis of the activity and selectivity to phenolic inhibitors of recombinant human AChE, eeAChE and a mutant variant of human AChE known as dAChE4. Despite numerous mutations, dAChE4 closely resembles its parental protein and serves as a suitable model for monomeric human AChE. We also established an in vitro system of membrane-bound AChE to create a model that closely mimics the physiological isoforms. This system ensures the proper work of the enzyme and allowed us to control the exact concentration of enzyme and lipids per assay.

乙酰胆碱酯酶（AChE）在胆碱能系统中起着举足轻重的作用，从昆虫防治到阿尔茨海默病治疗，人们都在寻求抑制乙酰胆碱酯酶的方法。虽然 AChE 的主要生理异构体是膜结合蛋白，但大多数发现新的、更安全、更有效的抑制剂的试验都是使用市售的可溶性异构体（如电鳗 AChE（eeAChE））进行的。在这项研究中，我们对重组人 AChE、eeAChE 和一种称为 dAChE4 的人 AChE 突变变体的酚类抑制剂的活性和选择性进行了比较分析。尽管存在许多突变，但 dAChE4 与其亲本蛋白非常相似，是人类 AChE 单体的合适模型。我们还建立了一个膜结合 AChE 体外系统，以创建一个近似生理异构体的模型。该系统确保了酶的正常工作，并使我们能够精确控制每次测定的酶和脂质浓度。

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引用次数: 0

The dimer of human SVCT1 is key for transport function 人类 SVCT1 的二聚体是运输功能的关键。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-10-04 DOI: 10.1016/j.bbamem.2024.184390

Menebere Woubshete , Lok I. Chan , George Diallinas , Bernadette Byrne

Humans and other primates lack the ability to synthesize the essential nutrient, Vitamin C, which is derived exclusively from the diet. Crucial for effective vitamin C uptake are the Na⁺ dependent Vitamin C transporters, SVCT1 and SVCT2, members of the nucleobase ascorbate transporter (NAT) family. SVCT1 and 2 actively transport the reduced form of Vitamin C, ascorbic acid, into key tissues. The recent structure of the mouse SVCT1 revealed the molecular basis of substrate binding and that, like the other structurally characterised members of the NAT family, it exists as a closely associated dimer. SVCT1 is likely to function via the elevator mechanism with the core domain of each protomer able to bind substrate and move through the membrane carrying the substrate across the membrane. Here we explored the function of a range of variants of the human SVCT1, revealing a range of residues involved in substrate selection and binding, and confirming the importance of the C-terminus in membrane localisation. Furthermore, using a dominant negative mutant we show that the dimer is essential for transport function, as previously seen in the fungal homologue, UapA. In addition, we show that a localisation deficient C-terminal truncation of SVCT1 blocks correct localisation of co-expressed, associated wildtype SVCT1. These results clearly show the importance of the dimer in both correct SVCT1 trafficking and transport activity.

人类和其他灵长类动物缺乏合成必需营养素维生素 C 的能力，而维生素 C 完全来自饮食。依赖 Na+ 的维生素 C 转运体 SVCT1 和 SVCT2 是有效吸收维生素 C 的关键，它们是核碱基抗坏血酸转运体（NAT）家族的成员。SVCT1 和 2 能积极地将还原型维生素 C（抗坏血酸）转运到关键组织中。小鼠 SVCT1 的最新结构揭示了其与底物结合的分子基础，而且与 NAT 家族中其他具有结构特征的成员一样，SVCT1 也是以紧密结合的二聚体形式存在的。SVCT1 可能通过升降机机制发挥作用，每个原体的核心结构域都能结合底物，并携带底物穿过膜。在这里，我们探索了人类 SVCT1 的一系列变体的功能，发现了一系列参与底物选择和结合的残基，并证实了 C 端在膜定位中的重要性。此外，我们利用显性负突变体表明，二聚体对于运输功能至关重要，这与之前在真菌同源物 UapA 中看到的情况相同。此外，我们还发现，SVCT1 的 C 端截短定位缺陷会阻碍与野生型 SVCT1 共同表达的正确定位。这些结果清楚地表明了二聚体在 SVCT1 的正确运输和运输活性中的重要性。

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引用次数: 0