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

Elucidating the structure and function of a membrane-active plant protein domain using in silico mutagenesis 阐明膜活性植物蛋白结构域在硅诱变中的结构和功能。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-01-07 DOI: 10.1016/j.bbamem.2025.184409

Lennie K.Y. Cheung , Sebastian Thallmair , Rickey Y. Yada

The Solanum tuberosum (common potato) plant specific insert (StPSI) is an antimicrobial protein domain that exhibits membrane-disrupting and membrane-fusing activity upon dimerization at acidic pH, activity proposed to involve electrostatic attraction and membrane anchoring mediated by specific positively-charged and conserved tryptophan residues, respectively. This study is the first to employ an in silico mutagenesis approach to clarify the structure-function relationship of a plant specific insert (PSI), where ten rationally-mutated StPSI variants were investigated using all-atom and coarse-grained molecular dynamics. The tryptophan (W) residue at position 18 (W18) of wild-type StPSI was predicted to confer structural flexibility to the dimer and mediate a partial separation of the assembled monomers upon bilayer contact, while residues including W77 and the lysine (K) residue at position 83 (K83) were predicted to stabilize secondary structure and influence association with the model membrane. Mechanisms predicted to influence StPSI-membrane association included the partial separation of assembled monomers on the bilayer surface, formation of a specific salt bridge, and membrane anchoring of hinge 2 residues. The findings suggested that the structure-function relationship of StPSI involved several mechanisms that may each be modulated by specific key residues, insights that may support efforts to develop PSI with tailored membrane association for novel applications in plant biotechnology and crop protection.

Solanum tuberosum（普通马铃薯）植物特异性插入体（StPSI）是一种抗菌蛋白结构域，在酸性pH值下表现出膜破坏和膜融合活性，活性可能涉及静电吸引和膜锚定，分别由特定的带正电的色氨酸残基和保守的色氨酸残基介导。本研究首次采用硅诱变方法来阐明植物特异性插入体（PSI）的结构-功能关系，其中使用全原子和粗粒度分子动力学研究了10个合理突变的StPSI变体。野生型StPSI的18位色氨酸(W)残基被预测赋予二聚体结构灵活性，并在双层接触时介导组装单体的部分分离，而包括W77和83位赖氨酸(K)残基（K83）在内的残基被预测稳定二级结构并影响与模型膜的结合。预测影响stpsi -膜结合的机制包括双层表面组装单体的部分分离，特定盐桥的形成以及铰链2残基的膜锚定。研究结果表明，StPSI的结构-功能关系涉及多种机制，每种机制都可能由特定的关键残基调节，这些见解可能支持开发具有定制膜关联的PSI，以用于植物生物技术和作物保护的新应用。

{"title":"Elucidating the structure and function of a membrane-active plant protein domain using in silico mutagenesis","authors":"Lennie K.Y. Cheung , Sebastian Thallmair , Rickey Y. Yada","doi":"10.1016/j.bbamem.2025.184409","DOIUrl":"10.1016/j.bbamem.2025.184409","url":null,"abstract":"<div><div>The <em>Solanum tuberosum</em> (common potato) plant specific insert (<em>St</em>PSI) is an antimicrobial protein domain that exhibits membrane-disrupting and membrane-fusing activity upon dimerization at acidic pH, activity proposed to involve electrostatic attraction and membrane anchoring mediated by specific positively-charged and conserved tryptophan residues, respectively. This study is the first to employ an in silico mutagenesis approach to clarify the structure-function relationship of a plant specific insert (PSI), where ten rationally-mutated <em>St</em>PSI variants were investigated using all-atom and coarse-grained molecular dynamics. The tryptophan (W) residue at position 18 (W18) of wild-type <em>St</em>PSI was predicted to confer structural flexibility to the dimer and mediate a partial separation of the assembled monomers upon bilayer contact, while residues including W77 and the lysine (K) residue at position 83 (K83) were predicted to stabilize secondary structure and influence association with the model membrane. Mechanisms predicted to influence <em>St</em>PSI-membrane association included the partial separation of assembled monomers on the bilayer surface, formation of a specific salt bridge, and membrane anchoring of hinge 2 residues. The findings suggested that the structure-function relationship of <em>St</em>PSI involved several mechanisms that may each be modulated by specific key residues, insights that may support efforts to develop PSI with tailored membrane association for novel applications in plant biotechnology and crop protection.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 3","pages":"Article 184409"},"PeriodicalIF":2.8,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142943676","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Improving the antimicrobial potential of the peptide CIDEM-501 through acylation: A computational approach 通过酰化提高肽CIDEM-501的抗菌潜力：一种计算方法。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-01-07 DOI: 10.1016/j.bbamem.2025.184407

Daniel Alpízar-Pedraza , Adrian Romero-Rivero , Rolando Perdomo-Morales , Niurys Mantilla-García , Claudia Pérez-Martínez , Hilda Garay-Pérez , Frank Rosenau , Ludger Ständker , Vivian Montero-Alejo

Acylation is a common method used to modify antimicrobial peptides to enhance their effectiveness. It increases the interactions between the peptide and the bacterial cell membranes. However, acylation can also reduce the selectivity of the peptides by making them more active on eukaryotic membranes, which can lead to unintended toxicity. This study examines the potential of using in silico tools to evaluate the interaction and selectivity of the antimicrobial peptide CIDEM-501 when acylated with decanoic acid at the N-terminus, compared to the non-acylated counterpart. Circular dichroism, microdilution, and hemolysis assays were used to determine the peptide's secondary structure, antimicrobial activity, and selectivity to validate the theoretical predictions. The acylated peptide showed a more stable interaction with the bacterial membrane by inserting the acyl chain into the membrane's hydrophobic core, which led to tighter adsorption and a greater buried surface area. Additionally, it significantly altered membrane order more than the non-acylated counterpart, suggesting superior antimicrobial potential. Finally, in vitro activity assays confirmed theoretical predictions, showing that the acylated peptide had lower Minimum Inhibitory Concentration (MIC) values than the non-acylated peptide. Neither peptide showed significant hemolytic activity at their MIC. The computational techniques used in this study displayed strong predictive capability and helped to elucidate the interaction between the peptide and the membranes.

酰化是一种常用的方法，用于修饰抗菌肽，以提高其有效性。它增加了肽和细菌细胞膜之间的相互作用。然而，酰化也可以降低肽的选择性，使它们在真核生物膜上更活跃，这可能导致意想不到的毒性。本研究考察了使用硅工具评估抗菌肽CIDEM-501在n端与癸酸酰化时的相互作用和选择性的潜力，与未酰化的对应物相比。圆二色性、微量稀释和溶血试验用于确定肽的二级结构、抗菌活性和选择性，以验证理论预测。通过将酰基链插入细菌膜的疏水核心，酰基化肽与细菌膜的相互作用更加稳定，从而导致更紧密的吸附和更大的埋藏表面积。此外，它比非酰化对应物更能显著改变膜序，表明其具有更强的抗菌潜力。最后，体外活性测定证实了理论预测，表明酰化肽具有比非酰化肽更低的最低抑制浓度（MIC）值。两种肽在MIC处均未显示出明显的溶血活性。本研究中使用的计算技术显示出很强的预测能力，并有助于阐明肽与膜之间的相互作用。

{"title":"Improving the antimicrobial potential of the peptide CIDEM-501 through acylation: A computational approach","authors":"Daniel Alpízar-Pedraza , Adrian Romero-Rivero , Rolando Perdomo-Morales , Niurys Mantilla-García , Claudia Pérez-Martínez , Hilda Garay-Pérez , Frank Rosenau , Ludger Ständker , Vivian Montero-Alejo","doi":"10.1016/j.bbamem.2025.184407","DOIUrl":"10.1016/j.bbamem.2025.184407","url":null,"abstract":"<div><div>Acylation is a common method used to modify antimicrobial peptides to enhance their effectiveness. It increases the interactions between the peptide and the bacterial cell membranes. However, acylation can also reduce the selectivity of the peptides by making them more active on eukaryotic membranes, which can lead to unintended toxicity. This study examines the potential of using <em>in silico</em> tools to evaluate the interaction and selectivity of the antimicrobial peptide CIDEM-501 when acylated with decanoic acid at the N-terminus, compared to the non-acylated counterpart. Circular dichroism, microdilution, and hemolysis assays were used to determine the peptide's secondary structure, antimicrobial activity, and selectivity to validate the theoretical predictions. The acylated peptide showed a more stable interaction with the bacterial membrane by inserting the acyl chain into the membrane's hydrophobic core, which led to tighter adsorption and a greater buried surface area. Additionally, it significantly altered membrane order more than the non-acylated counterpart, suggesting superior antimicrobial potential. Finally, <em>in vitro</em> activity assays confirmed theoretical predictions, showing that the acylated peptide had lower Minimum Inhibitory Concentration (MIC) values than the non-acylated peptide. Neither peptide showed significant hemolytic activity at their MIC. The computational techniques used in this study displayed strong predictive capability and helped to elucidate the interaction between the peptide and the membranes.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 3","pages":"Article 184407"},"PeriodicalIF":2.8,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142943679","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

Differential insertion of arginine in saturated and unsaturated lipid vesicles 精氨酸在饱和和不饱和脂质囊泡中的不同插入。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-12-18 DOI: 10.1016/j.bbamem.2024.184405

Hugo A. Perez, María A. Brandan, Aníbal Disalvo, María de los A. Frías

In this work, the effects of L- Arginine (L-Arg) insertion on saturated and unsaturated lipid membranes were assessed by fluorescence spectroscopy, dynamic light scattering (DLS) and monolayer measurements. The studied systems were composed by DPPC, 16:0 DietherPC, 16:1 Δ9-CisPC, DPPC:Chol, 16:1 Δ9-CisPC:Chol, and 16:1 Δ9-CisPC:DPPC in the presence of increasing concentrations of L-Arg.

The information obtained using fluorescence spectral Laurdan properties indicates that L- Arg produces a decrease in the polarizability of saturated lipids congruent with the increase in vesicle size and area per lipid. However, in unsaturated lipids, the polarizability increases without significant changes in size and area per lipid denoting a different mechanism of insertion. The two opposite effects can be modulated by the saturated and unsaturated ratio and are independent of carbonyl groups. This modulation is damped by cholesterol. The differences in the L-Arg insertion can be explained considering that in the presence of the double bonds, L-Arg decreases the organized water in the lipid matrix without expanding the bilayer. Instead, in saturated lipid membranes, L-Arg inserts into the acyl chains dragging water and expanding the membrane area.

本研究通过荧光光谱、动态光散射（DLS）和单层测量来评估L-精氨酸（L- arg）插入对饱和和不饱和脂质膜的影响。在l -精氨酸浓度增加的情况下，该体系由DPPC、16:0双醚pc、16:1 Δ9-CisPC、DPPC:Chol、16:1 Δ9-CisPC:Chol和16:1 Δ9-CisPC:DPPC组成。利用荧光光谱Laurdan性质获得的信息表明，L- Arg使饱和脂质的极化率随着囊泡大小和每个脂质的面积的增加而降低。然而，在不饱和脂质中，极化率增加而每个脂质的大小和面积没有显著变化，这表明插入机制不同。这两种相反的效应可以通过饱和和不饱和的比例来调节，并且与羰基无关。这种调节受到胆固醇的抑制。l -精氨酸插入的差异可以解释为，在双键存在的情况下，l -精氨酸减少了脂质基质中的组织水，而没有扩大双分子层。相反，在饱和脂质膜中，l -精氨酸插入到酰基链中，拖动水并扩大膜面积。

{"title":"Differential insertion of arginine in saturated and unsaturated lipid vesicles","authors":"Hugo A. Perez, María A. Brandan, Aníbal Disalvo, María de los A. Frías","doi":"10.1016/j.bbamem.2024.184405","DOIUrl":"10.1016/j.bbamem.2024.184405","url":null,"abstract":"<div><div>In this work, the effects of L- Arginine (L-Arg) insertion on saturated and unsaturated lipid membranes were assessed by fluorescence spectroscopy, dynamic light scattering (DLS) and monolayer measurements. The studied systems were composed by DPPC, 16:0 DietherPC, 16:1 Δ9-CisPC, DPPC:Chol, 16:1 Δ9-CisPC:Chol, and 16:1 Δ9-CisPC:DPPC in the presence of increasing concentrations of L-Arg.</div><div>The information obtained using fluorescence spectral Laurdan properties indicates that L- Arg produces a decrease in the polarizability of saturated lipids congruent with the increase in vesicle size and area per lipid. However, in unsaturated lipids, the polarizability increases without significant changes in size and area per lipid denoting a different mechanism of insertion. The two opposite effects can be modulated by the saturated and unsaturated ratio and are independent of carbonyl groups. This modulation is damped by cholesterol. The differences in the L-Arg insertion can be explained considering that in the presence of the double bonds, L-Arg decreases the organized water in the lipid matrix without expanding the bilayer. Instead, in saturated lipid membranes, L-Arg inserts into the acyl chains dragging water and expanding the membrane area.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 3","pages":"Article 184405"},"PeriodicalIF":2.8,"publicationDate":"2024-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871178","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

Nanodomains enriched in arachidonic acid promote P2Y12 receptor oligomerization in the platelet plasma membrane 富含花生四烯酸的纳米域可促进血小板质膜中 P2Y12 受体的寡聚化。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-11-16 DOI: 10.1016/j.bbamem.2024.184402

Florentin Allemand , Semen Yesylevskyy , Jennifer Lagoutte-Renosi , Siamak Davani , Christophe Ramseyer

P2Y12 receptors on the platelet plasma membrane are targeted by several antiplatelets drugs. Although oligomerization and functioning of P2Y12 receptors depend on the membrane environment, little is known about their preferred membrane localization and the role of surrounding lipid composition, especially the arachidonic acids (ARA), which are abundant in platelets. Coarse-grained molecular dynamics simulations of platelet plasma membrane based on the lipidomics data were used to investigate the P2Y12 lipid environment and the involvement of ARA in its oligomerization in platelet plasma membranes. The platelet plasma membrane contains two types of lipids nanodomains: ordered, enriched in SM and cholesterol, and disordered, enriched in ARA-containing lipids. P2Y12 receptors prefer to localize in these ARA-rich domains and induce the sorting of the ARA-containing lipids in their vicinity. This ARA-rich environment promotes the oligomerization of P2Y12 receptors and stabilizes the protein-protein interfaces of oligomers. As summary, oligomerization of P2Y12 receptors is promoted in ARA-rich nano-domains of the platelet plasma membrane.

血小板浆膜上的 P2Y12 受体是多种抗血小板药物的靶点。虽然 P2Y12 受体的寡聚化和功能取决于膜环境，但人们对其首选膜定位以及周围脂质成分，尤其是血小板中含量丰富的花生四烯酸（ARA）的作用知之甚少。基于脂质组学数据的血小板质膜粗粒度分子动力学模拟被用来研究血小板质膜中 P2Y12 的脂质环境以及 ARA 在其低聚作用中的参与。血小板浆膜包含两种类型的脂质纳米域：有序的，富含SM和胆固醇；无序的，富含ARA脂质。P2Y12 受体喜欢定位在这些富含 ARA 的结构域中，并诱导其附近的含 ARA 脂质分选。这种富含 ARA 的环境促进了 P2Y12 受体的寡聚化，并稳定了寡聚体的蛋白质-蛋白质界面。综上所述，血小板质膜上富含 ARA 的纳米区促进了 P2Y12 受体的寡聚化。

{"title":"Nanodomains enriched in arachidonic acid promote P2Y12 receptor oligomerization in the platelet plasma membrane","authors":"Florentin Allemand , Semen Yesylevskyy , Jennifer Lagoutte-Renosi , Siamak Davani , Christophe Ramseyer","doi":"10.1016/j.bbamem.2024.184402","DOIUrl":"10.1016/j.bbamem.2024.184402","url":null,"abstract":"<div><div>P2Y12 receptors on the platelet plasma membrane are targeted by several antiplatelets drugs. Although oligomerization and functioning of P2Y12 receptors depend on the membrane environment, little is known about their preferred membrane localization and the role of surrounding lipid composition, especially the arachidonic acids (ARA), which are abundant in platelets. Coarse-grained molecular dynamics simulations of platelet plasma membrane based on the lipidomics data were used to investigate the P2Y12 lipid environment and the involvement of ARA in its oligomerization in platelet plasma membranes. The platelet plasma membrane contains two types of lipids nanodomains: ordered, enriched in SM and cholesterol, and disordered, enriched in ARA-containing lipids. P2Y12 receptors prefer to localize in these ARA-rich domains and induce the sorting of the ARA-containing lipids in their vicinity. This ARA-rich environment promotes the oligomerization of P2Y12 receptors and stabilizes the protein-protein interfaces of oligomers. As summary, oligomerization of P2Y12 receptors is promoted in ARA-rich nano-domains of the platelet plasma membrane.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 1","pages":"Article 184402"},"PeriodicalIF":2.8,"publicationDate":"2024-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142667214","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

Successful strategies for expression and purification of ABC transporters 表达和纯化 ABC 转运体的成功策略。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-11-12 DOI: 10.1016/j.bbamem.2024.184401

Bea Berner , Georgia Daoutsali , Emilia Melén , Natália Remper , Emma Weszelovszká , Alice Rothnie , Kristina Hedfalk

ATP-binding cassette (ABC) transporters are proteins responsible for active transport of various compounds, from small ions to macromolecules, across membranes. Proteins from this superfamily also pump drugs out of the cell resulting in multidrug resistance. Based on the cellular functions of ABC-transporters they are commonly associated with diseases like cancer and cystic fibrosis. To understand the molecular mechanism of this critical family of integral membrane proteins, structural characterization is a powerful tool which in turn requires successful recombinant production of stable and functional protein in good yields. In this review we have used high resolution structures of ABC transporters as a measure of successful protein production and summarized strategies for prokaryotic and eukaryotic proteins, respectively. In general, Escherichia coli is the most frequently used host for production of prokaryotic ABC transporters while human embryonic kidney 293 (HEK293) cells are the preferred host system for eukaryotic proteins. Independent of origin, at least two-steps of purification were required after solubilization in the most used detergent DDM. The purification tag was frequently cleaved off before structural characterization using cryogenic electron microscopy, or crystallization and X-ray analysis for prokaryotic proteins.

ATP结合盒（ABC）转运体是一种蛋白质，负责各种化合物（从小肠离子到大分子）的跨膜主动转运。该超家族的蛋白质还能将药物泵出细胞，从而产生多药耐药性。根据 ABC 转运体的细胞功能，它们通常与癌症和囊性纤维化等疾病有关。要了解这一重要的整体膜蛋白家族的分子机理，结构表征是一个强有力的工具，而这反过来又需要成功地重组生产稳定的功能性蛋白。在这篇综述中，我们将 ABC 转运体的高分辨率结构作为衡量成功生产蛋白质的标准，并分别总结了原核和真核蛋白质的生产策略。一般来说，大肠杆菌是生产原核 ABC 转运体最常用的宿主，而人胚肾 293（HEK293）细胞则是生产真核蛋白质的首选宿主系统。无论来源如何，在最常用的去垢剂 DDM 中溶解后至少需要两步纯化。在使用低温电子显微镜或结晶和 X 射线分析原核蛋白质的结构特征之前，纯化标签通常会被切掉。

{"title":"Successful strategies for expression and purification of ABC transporters","authors":"Bea Berner , Georgia Daoutsali , Emilia Melén , Natália Remper , Emma Weszelovszká , Alice Rothnie , Kristina Hedfalk","doi":"10.1016/j.bbamem.2024.184401","DOIUrl":"10.1016/j.bbamem.2024.184401","url":null,"abstract":"<div><div>ATP-binding cassette (ABC) transporters are proteins responsible for active transport of various compounds, from small ions to macromolecules, across membranes. Proteins from this superfamily also pump drugs out of the cell resulting in multidrug resistance. Based on the cellular functions of ABC-transporters they are commonly associated with diseases like cancer and cystic fibrosis. To understand the molecular mechanism of this critical family of integral membrane proteins, structural characterization is a powerful tool which in turn requires successful recombinant production of stable and functional protein in good yields. In this review we have used high resolution structures of ABC transporters as a measure of successful protein production and summarized strategies for prokaryotic and eukaryotic proteins, respectively. In general, <em>Escherichia coli</em> is the most frequently used host for production of prokaryotic ABC transporters while human embryonic kidney 293 (HEK293) cells are the preferred host system for eukaryotic proteins. Independent of origin, at least two-steps of purification were required after solubilization in the most used detergent DDM. The purification tag was frequently cleaved off before structural characterization using cryogenic electron microscopy, or crystallization and X-ray analysis for prokaryotic proteins.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 2","pages":"Article 184401"},"PeriodicalIF":2.8,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614113","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Bacterial flotillins as destabilizers of phospholipid membranes 作为磷脂膜脱稳剂的细菌絮凝物。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-11-08 DOI: 10.1016/j.bbamem.2024.184399

Ana Álvarez-Mena , Estelle Morvan , Denis Martinez , Melanie Berbon , Abigail Savietto Scholz , Axelle Grélard , Sarah Turpin , Erick J. Dufourc , Marc Bramkamp , Birgit Habenstein

From archaea to mammals evolutionary conserved flotillins are scaffolding proteins, recognized for their nandomain-segregating activity. Flotillins form basket-like oligomeric architectures on the membrane, based on a conserved secondary structure composition of the monomeric subunits: a membrane-targeting region, an SPFH domain and a coiled-coil “flotillin” domain. In B. subtilis, the two flotillins FloT and FloA are present, localizing mainly in distinct nanodomains and executing multiple cellular functions. We here use deuterium and phosphorus solid-state NMR to monitor the effect of the different flotillins FloT and FloA and their structural components on model membranes. We find a clear disordering effect of FloT and FloA on the membranes reaching the carbon positions in the centre of the membrane. This effect is imposed by the hydrophobic region and the adjacent SPFH domain and, surprisingly, further supported by the membrane-distant flotillin domain. Biological implications of this disordering action are discussed.

从古生菌到哺乳动物，在进化过程中得到保护的绒毛膜蛋白是一种支架蛋白，因其具有核苷酸聚集活性而得到认可。基于单体亚基保守的二级结构组成：一个膜靶区、一个 SPFH 结构域和一个线圈 "flotillin "结构域，磷脂酰蛋白在膜上形成篮状寡聚体结构。在枯草芽孢杆菌中，存在 FloT 和 FloA 两种菌素，它们主要定位于不同的纳米域中，执行多种细胞功能。在此，我们利用氘和磷固态核磁共振来监测不同的FloT和FloA及其结构成分对模型膜的影响。我们发现 FloT 和 FloA 对到达膜中心碳位置的膜有明显的失调效应。这种作用是由疏水区域和邻近的 SPFH 结构域造成的，而且令人惊讶的是，这种作用还得到了膜远端 flotillin 结构域的进一步支持。本文讨论了这种紊乱作用的生物学意义。

{"title":"Bacterial flotillins as destabilizers of phospholipid membranes","authors":"Ana Álvarez-Mena , Estelle Morvan , Denis Martinez , Melanie Berbon , Abigail Savietto Scholz , Axelle Grélard , Sarah Turpin , Erick J. Dufourc , Marc Bramkamp , Birgit Habenstein","doi":"10.1016/j.bbamem.2024.184399","DOIUrl":"10.1016/j.bbamem.2024.184399","url":null,"abstract":"<div><div>From archaea to mammals evolutionary conserved flotillins are scaffolding proteins, recognized for their nandomain-segregating activity. Flotillins form basket-like oligomeric architectures on the membrane, based on a conserved secondary structure composition of the monomeric subunits: a membrane-targeting region, an SPFH domain and a coiled-coil “flotillin” domain. In <em>B. subtilis</em>, the two flotillins FloT and FloA are present, localizing mainly in distinct nanodomains and executing multiple cellular functions. We here use deuterium and phosphorus solid-state NMR to monitor the effect of the different flotillins FloT and FloA and their structural components on model membranes. We find a clear disordering effect of FloT and FloA on the membranes reaching the carbon positions in the centre of the membrane. This effect is imposed by the hydrophobic region and the adjacent SPFH domain and, surprisingly, further supported by the membrane-distant flotillin domain. Biological implications of this disordering action are discussed.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 1","pages":"Article 184399"},"PeriodicalIF":2.8,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614074","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Membrane fusion by dengue virus: The first step 登革热病毒的膜融合：第一步

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-11-08 DOI: 10.1016/j.bbamem.2024.184400

José Villalaín

Flaviviruses include important human pathogens such as Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as some emerging viruses that affect millions of people worldwide. They fuse their membrane with the late endosomal one in a pH-dependent way and therefore the merging of the membranes is one of the main goals for obtaining new antivirals. The envelope E protein, a membrane fusion protein, is accountable for fusion and encompasses different domains involved in the fusion mechanism, including the fusion peptide segment. In this work we have used molecular dynamics to study the interaction of the distal end of domain II of the DENV envelope E protein with a membrane like the late endosomal membrane in order to observe the initiation of membrane fusion carried out by a number of trimers of the DENV envelope E protein interacting with a complex biomembrane and demonstrate its feasibility. Our results demonstrate the likelihood of membrane disorganization and pore formation by trimer complex organization, the amino acids responsible for such condition and the secondary structure arrangements needed for such fundamental process. At the same time, we define new targets of the envelope E protein sequence which could permit designing potent antiviral bioactive molecules.

黄病毒包括登革热、寨卡、西尼罗河病毒、黄热病、日本脑炎、蜱传脑炎病毒等重要的人类病原体，以及一些影响全球数百万人的新出现的病毒。它们的膜与晚期内体膜融合的方式取决于 pH 值，因此膜的融合是获得新抗病毒药物的主要目标之一。包膜 E 蛋白是一种膜融合蛋白，它负责融合，并包含参与融合机制的不同结构域，包括融合肽段。在这项工作中，我们利用分子动力学研究了 DENV 包膜 E 蛋白结构域 II 的远端与类似晚期内体膜的膜的相互作用，以观察 DENV 包膜 E 蛋白的多个三聚体与复杂生物膜相互作用时膜融合的启动过程，并证明其可行性。我们的研究结果证明了三聚体复合物组织膜解构和孔形成的可能性、造成这种情况的氨基酸以及这种基本过程所需的二级结构排列。同时，我们还确定了包膜 E 蛋白序列的新目标，从而可以设计出强效的抗病毒生物活性分子。

{"title":"Membrane fusion by dengue virus: The first step","authors":"José Villalaín","doi":"10.1016/j.bbamem.2024.184400","DOIUrl":"10.1016/j.bbamem.2024.184400","url":null,"abstract":"<div><div>Flaviviruses include important human pathogens such as Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as some emerging viruses that affect millions of people worldwide. They fuse their membrane with the late endosomal one in a pH-dependent way and therefore the merging of the membranes is one of the main goals for obtaining new antivirals. The envelope E protein, a membrane fusion protein, is accountable for fusion and encompasses different domains involved in the fusion mechanism, including the fusion peptide segment. In this work we have used molecular dynamics to study the interaction of the distal end of domain II of the DENV envelope E protein with a membrane like the late endosomal membrane in order to observe the initiation of membrane fusion carried out by a number of trimers of the DENV envelope E protein interacting with a complex biomembrane and demonstrate its feasibility. Our results demonstrate the likelihood of membrane disorganization and pore formation by trimer complex organization, the amino acids responsible for such condition and the secondary structure arrangements needed for such fundamental process. At the same time, we define new targets of the envelope E protein sequence which could permit designing potent antiviral bioactive molecules.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 1","pages":"Article 184400"},"PeriodicalIF":2.8,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Antimicrobial and antibiofilm potential of α-MSH derived cationic and hydrophobic peptides against Escherichia coli: Mechanistic insight through peptide-lipopolysaccharide interactions α-MSH衍生的阳离子肽和疏水肽对大肠杆菌的抗菌和抗生物膜潜力：通过肽与脂多糖相互作用的机理研究。

IF 2.8 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2024-11-07 DOI: 10.1016/j.bbamem.2024.184398

Priya Patel , Swaleeha Jaan Abdullah , Kanchan Tiwari , Surajit Bhattacharjya , Kasturi Mukhopadhyay

The prevalence of infections caused by various Gram-negative pathogens specifically Escherichia coli continuously poses a significant challenge in health care as well as community settings owing to their ability to form biofilm and escalating tolerance towards available antibiotics. While most treatment regimes are targeted at eliminating the E. coli cells, the pathogenicity factors called endotoxin (lipopolysaccharides), associated with the sepsis initiation and the leading cause of death in intensive care units globally, are often ignored. In this study, the potency of alpha-melanocyte stimulating hormone based-peptides, particularly Ana-9 and Ana-10 against E. coli was investigated through microbiological, biophysical, and microscopic assays. Both Ana-9 and Ana-10 demonstrated enhanced activity against planktonic E. coli cells, and retained their activity against biofilm, which was supported by confocal microscopy. From the mechanistic perspective, spectroscopic studies indicated that the binding of peptides with LPS led to structural alteration of peptides due to their insertion into the hydrophobic environment of LPS. The electrostatic interaction of the peptide with LPS leads to outer membrane disorganization, allowing the peptide to access the inner membrane, depolarize it and ultimately inhibit the bacterial cells within the biofilm. These observations were further confirmed by atomic force and scanning electron microscopy. Thus, this study deepens our understanding of the structural characteristics of peptides attached to LPS, which could lead to the gradual improvement in developing more potent, broad-spectrum endotoxin neutralizers.

由于各种革兰氏阴性病原体，特别是大肠埃希氏菌，具有形成生物膜的能力，而且对现有抗生素的耐受性不断提高，因此它们引起的感染在医疗保健和社区环境中持续流行，构成了一项重大挑战。虽然大多数治疗方案都以消灭大肠杆菌细胞为目标，但被称为内毒素（脂多糖）的致病因子却常常被忽视，而内毒素与败血症的发生有关，是全球重症监护室的主要死因。在这项研究中，我们通过微生物学、生物物理学和显微镜实验研究了基于α-黑色素细胞刺激素的肽，特别是 Ana-9 和 Ana-10 对大肠杆菌的作用。Ana-9 和 Ana-10 对浮游大肠杆菌细胞的活性增强，对生物膜的活性保持不变，共聚焦显微镜证实了这一点。从机理角度来看，光谱研究表明，肽与 LPS 结合后，由于肽插入到 LPS 的疏水环境中，导致肽的结构发生了改变。多肽与 LPS 的静电作用导致外膜紊乱，使多肽进入内膜，使内膜去极化，最终抑制生物膜内的细菌细胞。原子力和扫描电子显微镜进一步证实了这些观察结果。因此，这项研究加深了我们对附着在 LPS 上的肽的结构特征的了解，这将有助于逐步开发出更强效、更广谱的内毒素中和剂。

{"title":"Antimicrobial and antibiofilm potential of α-MSH derived cationic and hydrophobic peptides against Escherichia coli: Mechanistic insight through peptide-lipopolysaccharide interactions","authors":"Priya Patel , Swaleeha Jaan Abdullah , Kanchan Tiwari , Surajit Bhattacharjya , Kasturi Mukhopadhyay","doi":"10.1016/j.bbamem.2024.184398","DOIUrl":"10.1016/j.bbamem.2024.184398","url":null,"abstract":"<div><div>The prevalence of infections caused by various Gram-negative pathogens specifically <em>Escherichia coli</em> continuously poses a significant challenge in health care as well as community settings owing to their ability to form biofilm and escalating tolerance towards available antibiotics. While most treatment regimes are targeted at eliminating the <em>E. coli</em> cells, the pathogenicity factors called endotoxin (lipopolysaccharides), associated with the sepsis initiation and the leading cause of death in intensive care units globally, are often ignored. In this study, the potency of alpha-melanocyte stimulating hormone based-peptides, particularly Ana-9 and Ana-10 against <em>E. coli</em> was investigated through microbiological, biophysical, and microscopic assays. Both Ana-9 and Ana-10 demonstrated enhanced activity against planktonic <em>E. coli</em> cells, and retained their activity against biofilm, which was supported by confocal microscopy. From the mechanistic perspective, spectroscopic studies indicated that the binding of peptides with LPS led to structural alteration of peptides due to their insertion into the hydrophobic environment of LPS. The electrostatic interaction of the peptide with LPS leads to outer membrane disorganization, allowing the peptide to access the inner membrane, depolarize it and ultimately inhibit the bacterial cells within the biofilm. These observations were further confirmed by atomic force and scanning electron microscopy. Thus, this study deepens our understanding of the structural characteristics of peptides attached to LPS, which could lead to the gradual improvement in developing more potent, broad-spectrum endotoxin neutralizers.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 1","pages":"Article 184398"},"PeriodicalIF":2.8,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614066","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

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。

{"title":"Growth of Staphylococcus aureus in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides","authors":"Djuro Raskovic , Gloria Alvarado , Kelly M. Hines , Libin Xu , Craig Gatto , Brian J. Wilkinson , Antje Pokorny","doi":"10.1016/j.bbamem.2024.184395","DOIUrl":"10.1016/j.bbamem.2024.184395","url":null,"abstract":"<div><div><em>Staphylococcus aureus</em> readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, <em>S. aureus</em> presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When <em>S. aureus</em> 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 <em>S. aureus</em> 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 <em>S. aureus</em> infections.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 1","pages":"Article 184395"},"PeriodicalIF":2.8,"publicationDate":"2024-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142582032","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

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。

{"title":"Voltage- and Ca2+-inducible PLC activity for analyzing PI(4,5)P2 sensitivity of ion channels in Xenopus oocytes","authors":"Takafumi Kawai , Natsuki Mizutani , Yasushi Okamura","doi":"10.1016/j.bbamem.2024.184396","DOIUrl":"10.1016/j.bbamem.2024.184396","url":null,"abstract":"<div><div>Phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP<sub>2</sub> levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous <em>Xenopus</em> oocytes to precisely manipulate PIP<sub>2</sub> levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP<sub>2</sub>. <em>Xenopus</em> oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca<sup>2+</sup> influx driven by the increased driving force of Ca<sup>2+</sup>. High Ca<sup>2+</sup> sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in <em>Xenopus</em> oocytes that was voltage- and Ca<sup>2+</sup>-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP<sub>2</sub>-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca<sup>2+</sup>-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP<sub>2</sub> 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 PIP<sub>2</sub> levels, (2) PIP<sub>2</sub> levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP<sub>2</sub> levels were reduced by Ca<sup>2+</sup> administration. Therefore, PLCζ effectively supports understanding how PIP<sub>2</sub> regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP<sub>2</sub> and the PLC pathway in <em>Xenopus</em> oocytes.</div></div>","PeriodicalId":8831,"journal":{"name":"Biochimica et biophysica acta. Biomembranes","volume":"1867 1","pages":"Article 184396"},"PeriodicalIF":2.8,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142557053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0