Methods in enzymology最新文献_第3页

Regulation of phospholipid scramblases by cholesterol. 胆固醇对磷脂重组酶的调节。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-21 DOI: 10.1016/bs.mie.2026.01.020

Sabita Chourasia, Haodong Wang, Anant K Menon

{"title":"Regulation of phospholipid scramblases by cholesterol.","authors":"Sabita Chourasia, Haodong Wang, Anant K Menon","doi":"10.1016/bs.mie.2026.01.020","DOIUrl":"10.1016/bs.mie.2026.01.020","url":null,"abstract":"The activity of membrane proteins is often regulated by their lipid environment, either via physical properties of the membrane or by direct interaction with specific lipids. Testing these effects with purified proteins reconstituted into lipid vesicles is challenging because of the compositional heterogeneity of the vesicle sample. Cholesterol influences membrane properties and binds to a variety of membrane proteins, thereby regulating their activity. To study the effect of cholesterol on the phospholipid scramblase activity of G protein-coupled receptors (GPCRs), we developed a protocol to introduce cholesterol into vesicles after reconstituting the protein. This approach sidesteps the problem of having to account for the possible effect of cholesterol on the reconstitution process itself, enabling direct evaluation of the effect of cholesterol on activity. Here we describe the cholesterol loading protocol and how to quantify the amount loaded by colorimetric assays and membrane fluidity measurements. We provide sample data on the effect of cholesterol on GPCRs. Our protocol is broadly applicable and can be used in any study of the effect of cholesterol on a reconstituted membrane protein.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"1-16"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Metabolic labeling of glycerophospholipids. 甘油磷脂的代谢标记。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-01 DOI: 10.1016/bs.mie.2025.11.010

Robert J Maraski, Christelle F Ancajas, Jinchao Lou, Michael D Best

引用次数: 0

Assembling Retromer-coated membrane tubules for biochemical and structural studies. 组装用于生化和结构研究的反转录物涂层膜管。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-23 DOI: 10.1016/bs.mie.2026.01.012

Kai-En Chen, Vikas A Tillu, Nicholas Ariotti, Brett M Collins

{"title":"Assembling Retromer-coated membrane tubules for biochemical and structural studies.","authors":"Kai-En Chen, Vikas A Tillu, Nicholas Ariotti, Brett M Collins","doi":"10.1016/bs.mie.2026.01.012","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.01.012","url":null,"abstract":"The evolutionarily conserved Retromer complex, composed of Vps29, Vps26 and Vps35, is an essential regulator of endosomal retrieval of transmembrane cargo proteins. For cargo sorting and trafficking to take place, Retromer assembles into coated tubulovesicular carriers together with various sorting nexin (SNX) adaptor proteins including SNX3 and SNX27 in metazoans, and Snx3 or the dimeric Vps5-Vps17 SNX-BAR proteins in yeast. Although Retromer-coated tubulovesicular carriers are vital for its function, the in vitro reconstitution of these membrane assemblies for structural and functional studies can be technically challenging. Approaches include the use of giant unilamellar vesicles and supported membrane tubules for fluorescence imaging, or smaller multilamellar vesicles (MLVs) to generate uniform tubules for imaging by cryoelectron tomography (CryoET). This chapter describes protocols for producing MLVs for membrane binding studies of Retromer and assembling the yeast Retromer-Vps5-Vps17 heteropentameric complex for reconstituting membrane tubulation for CryoET studies. We also discuss our observations of both poorly ordered and well-ordered Retromer coats observed in this experimental setup.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"101-116"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Detecting ZDHHC acyltransferase inhibition using acylation-coupled lipophilic induction of polarization (Acyl-cLIP) or LC-MS. 利用酰基偶联亲脂诱导极化（酰基- clip）或LC-MS检测ZDHHC酰基转移酶抑制。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-05 DOI: 10.1016/bs.mie.2026.01.015

Wenzhe Chen, Jiaqi Zhao, Zhenquan Sun, Kewen Peng, Wendy K Greentree, Maurine E Linder, Hening Lin

{"title":"Detecting ZDHHC acyltransferase inhibition using acylation-coupled lipophilic induction of polarization (Acyl-cLIP) or LC-MS.","authors":"Wenzhe Chen, Jiaqi Zhao, Zhenquan Sun, Kewen Peng, Wendy K Greentree, Maurine E Linder, Hening Lin","doi":"10.1016/bs.mie.2026.01.015","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.01.015","url":null,"abstract":"Acylation of protein substrates by ZDHHC (zinc finger Asp-His-His-Cys) acyltransferases is a key regulator of cell signaling. Potent and selective small molecule inhibitors for ZDHHCs have been long desired for the field but so far still do not exist. Robust activity assay methods are needed to discover selective ZDHHC inhibitors. Here we present two complementary assays for in vitro ZDHHC activity assay and inhibitor profiling. The Acylation-Coupled Lipophilic Induction of Polarization (Acyl-cLIP) assay is a homogeneous fluorescence polarization (FP) assay that uses a fluorophore-tagged peptide substrate; upon palmitoylation by ZDHHC enzymes, the substrate partitions into detergent micelles, increasing FP signal. This assay enables real-time kinetic measurements in 384-well plates suitable for high-throughput screening. As an orthogonal validation method, we use liquid chromatography-mass spectrometry (LC-MS) to directly detect palmitoylation through a +238 Da mass shift on model peptide substrates, allowing more reliable quantification and confirmation. Together, these methods offer a platform for identifying and validating ZDHHC inhibitors.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"129-141"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

One-pot biosynthesis of genetically encoded amphiphiles via protein prenylation. 基因编码的两亲植物通过蛋白质酰化的一锅生物合成。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-11 DOI: 10.1016/bs.mie.2026.01.038

Md Mahbubul Alam, Md Shahadat Hossain, James L Hougland, Davoud Mozhdehi

引用次数: 0

Reconstitution strategies for exploring interactions of Ebola virus matrix protein with host membrane mimetics. 探索埃博拉病毒基质蛋白与宿主膜模拟物相互作用的重组策略。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-16 DOI: 10.1016/bs.mie.2026.01.006

Sreetama Pal, Shalini T Low-Nam, Robert V Stahelin

{"title":"Reconstitution strategies for exploring interactions of Ebola virus matrix protein with host membrane mimetics.","authors":"Sreetama Pal, Shalini T Low-Nam, Robert V Stahelin","doi":"10.1016/bs.mie.2026.01.006","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.01.006","url":null,"abstract":"Ebola virus (EBOV), an enveloped virus of the Filoviridae family, is an excellent biophysical model for studying enveloped virus assembly, despite its status as a BSL-4 pathogen. The expression of the EBOV matrix protein VP40 (viral protein 40 kDa) is necessary and sufficient for virus assembly/budding in human cells. VP40, in its dimeric form, interacts with the host plasma membrane (PM) inner leaflet and forms higher-order hexamers critical for EBOV assembly. The dependence of viral maturation on VP40 dynamics provides the opportunity to quantify biophysical requirements for the EBOV life cycle using a reductionist approach to measure VP40-membrane interactions. There are significant gaps in our understanding of the regulation underlying VP40 recruitment at the host cell PM, and how it culminates in the assembly/budding of mature, infectious virions. Here, we describe (i) reconstitution strategies for mimicking the VP40-host cell membrane interface in a controlled, biologically relevant manner and (ii) total internal reflection fluorescence microscopy-based detection of endogenous VP40 association with membranes under regulation of anionic lipid content. These methods are applicable for analyzing peripheral proteins and their interactions with lipid membranes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"289-303"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Real-time measurement of the ATG8 lipidation reaction by fluorescence spectroscopy. 荧光光谱法实时测量ATG8脂化反应。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-03 DOI: 10.1016/bs.mie.2026.01.011

Wenxin Zhang, Sharon A Tooze, Taki Nishimura

{"title":"Real-time measurement of the ATG8 lipidation reaction by fluorescence spectroscopy.","authors":"Wenxin Zhang, Sharon A Tooze, Taki Nishimura","doi":"10.1016/bs.mie.2026.01.011","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.01.011","url":null,"abstract":"Autophagy is a highly conserved intracellular degradation pathway, in which damaged organelles and/or dysfunctional cytosolic components are enveloped via double-membraned autophagosomes and subsequently delivered to lysosomes for degradation. The ubiquitin-like ATG8 family proteins (LC3s and GABARAPs) are covalently conjugated to phosphatidylethanolamine (PE) on autophagic membranes via ubiquitin-like conjugation systems, a process known as ATG8 lipidation. Lipidated ATG8 is the most widely used membrane marker for autophagosomes, and its flux is commonly used as a readout for autophagy activity. In vitro reconstitution of the ATG8 lipidation reaction is well-established, and the end-point reaction is typically resolved by SDS-PAGE. This endpoint readout is not suitable to monitor the kinetics of this reaction, and tools to study this process have been lacking. Here, we describe a real-time assay to measure the ATG8 lipidation reaction. This approach not only reveals the subsequential formation of covalently bound intermediates of ATG8 with the E1 (ATG7) and E2 (ATG3) enzymes, as well as ATG8-PE itself, but also provides insights into the interaction interface of ATG8 with proteins and membranes during the conjugation reaction.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"327-345"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530745","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

High-resolution sphingolipid analysis in vitro using LC-qToF MS. LC-qToF质谱体外高分辨率鞘脂分析。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-04 DOI: 10.1016/bs.mie.2025.11.008

Shweta Chitkara, G Ekin Atilla-Gokcumen

{"title":"High-resolution sphingolipid analysis in vitro using LC-qToF MS.","authors":"Shweta Chitkara, G Ekin Atilla-Gokcumen","doi":"10.1016/bs.mie.2025.11.008","DOIUrl":"10.1016/bs.mie.2025.11.008","url":null,"abstract":"The complexity of the sphingolipidome, characterized by variations in chain length, saturation, and headgroup composition, makes it essential to develop analytical strategies capable of high sensitivity and structural precision. Traditional biochemical methods lack the resolution to discriminate among closely related species, underscoring the transformative role of liquid chromatography-mass spectrometry (LC-MS) in sphingolipidomics. LC-MS provides unparalleled capabilities for sphingolipid analysis, combining chromatographic separation with high-resolution mass detection to achieve both qualitative and quantitative accuracy. In particular, liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-qToF MS) has emerged as a powerful platform, offering mass accuracy, broad dynamic range, and rapid acquisition rates. These features enable confident identification of isobaric and structurally related sphingolipids, which is essential for understanding their roles in cellular physiology and pathology. This chapter focuses on an optimized LC-qToF MS method tailored for sphingolipid profiling in cultured mammalian cells. By focusing on the analytical strengths of LC-MS, the approach provides a robust foundation for dissecting sphingolipid metabolism and its dysregulation in cellular processes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"726 ","pages":"157-180"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13006889/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146258011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Use of small unilamellar vesicles to study the biochemical properties of the GRAM domain of GRAMD1. 利用小的单层囊泡研究了GRAMD1的GRAM结构域的生化特性。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-05 DOI: 10.1016/bs.mie.2025.11.014

Dylan Hong Zheng Koh, Yasunori Saheki

{"title":"Use of small unilamellar vesicles to study the biochemical properties of the GRAM domain of GRAMD1.","authors":"Dylan Hong Zheng Koh, Yasunori Saheki","doi":"10.1016/bs.mie.2025.11.014","DOIUrl":"10.1016/bs.mie.2025.11.014","url":null,"abstract":"Cellular membranes are composed of a diverse array of lipids, which are essential for maintaining membrane integrity and facilitating cellular signaling. While proteins often exhibit selective affinities for specific lipid species, the precise thresholds and determinants of these interactions need to be carefully studied to elucidate protein functions. Here, we employ an in vitro liposome-based assay to assess lipid-protein interactions using small unilamellar vesicles of defined lipid compositions. Specifically, we outline the methodology for purifying the GRAM domain of GRAMD1b/Aster-B protein, which has been previously characterized for its ability to detect accessible cholesterol and anionic lipids, including phosphatidylserine, at cellular membranes. We further detail the preparation of liposomes and demonstrate that the purified GRAM domain proteins bind preferentially to liposomes containing both cholesterol and anionic lipids, rather than to those containing either lipid alone, thereby confirming their role as co-incidence detectors. This finding highlights the utility of small unilamellar vesicles as a robust and versatile system to assess the lipid-binding preference of proteins, providing a complementary approach to cell-based assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"429-451"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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

Quantitative analysis of H-Ras localization using confocal microscopy. 用共聚焦显微镜定量分析H-Ras定位。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-11 DOI: 10.1016/bs.mie.2025.11.009

Anjana P Sundaresan, Mary E Brown, Mark D Distefano

引用次数: 0