Methods in enzymology最新文献_第2页

Reconstitution approaches for understanding cellular lipid dynamics. 理解细胞脂质动力学的重构方法。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-10 DOI: 10.1016/bs.mie.2025.11.022

Dazhi Li, Justin L Korfhage, Karin M Reinisch

引用次数: 0

Non-invasive imaging of phosphoinositides with genetically encoded lipid biosensors. 用基因编码脂质生物传感器对磷酸肌苷进行无创成像。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-09 DOI: 10.1016/bs.mie.2025.11.017

Michael Worcester, Magdalene Motter, Gerald R V Hammond

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Expanding the multiplexing capability of HIDE probes via fluorescence lifetime imaging microscopy. 通过荧光寿命成像显微镜扩展HIDE探针的多路复用能力。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-16 DOI: 10.1016/bs.mie.2025.11.011

Justin H Kwon, Alanna Schepartz

引用次数: 0

Mass spectrometry-based lipid analysis in NPC1 disease: Methods for phosphoinositide quantification, lipid imaging, and myelin lipid profiling. NPC1疾病中基于质谱的脂质分析：磷酸肌肽定量、脂质成像和髓磷脂脂质谱分析方法。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-01 DOI: 10.1016/bs.mie.2025.11.003

Koralege C Pathmasiri, Stephanie M Cologna

{"title":"Mass spectrometry-based lipid analysis in NPC1 disease: Methods for phosphoinositide quantification, lipid imaging, and myelin lipid profiling.","authors":"Koralege C Pathmasiri, Stephanie M Cologna","doi":"10.1016/bs.mie.2025.11.003","DOIUrl":"10.1016/bs.mie.2025.11.003","url":null,"abstract":"Loss of NPC cholesterol transporter 1 protein function results in severe lipid dysregulation in multiple vital organs, including the brain, in Niemann-Pick Type C1 (NPC1) disease. Investigation of lipid changes and lipid metabolism disruptions in NPC1 is critical to elucidating the disease mechanisms driving the pathophysiology, identifying potential biomarkers, and guiding therapeutic strategies. One such example is phosphoinositides, which are key lipids involved in multiple signaling pathways relevant to NPC1 that are challenging to study due to their low abundance and detection difficulty. In this chapter, we present a detailed phosphoinositide analysis protocol using mass spectrometry. When studying lipids, spatial information is also important because it reveals distribution within the tissue, which can provide insights into functional roles and disease-related alterations. MALDI-MS lipid imaging is a powerful tool for investigating the spatial distribution of lipids. Herein, we also discuss a protocol for lipid imaging using MALDI-MSI, along with key precautions and troubleshooting tips. Finally, we present a myelin isolation protocol integrated with LC-MS lipidomics to investigate the myelin lipidome in tissues such as the brain, as myelin lipid composition is crucial for maintaining neuronal function and is often disrupted in neurodegenerative diseases like NPC1, including the investigation of phosphoinositides.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"726 ","pages":"333-355"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146258328","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

Photoaffinity strategies to profile cholesterol metabolite interactomes and binding sites. 光亲和策略分析胆固醇代谢物相互作用组和结合位点。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-03 DOI: 10.1016/bs.mie.2026.01.002

Yu-Shiuan Cheng, Alison E Ondrus

引用次数: 0

A fluorescence and HPLC-based activity assay system for lipid-modifying enzymes: Lipin/Pah phosphatidic acid phosphatase as an optimized example. 一种基于荧光和高效液相色谱的脂质修饰酶活性测定系统：脂质/多环芳烃磷脂酸磷酸酶为优化示例。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-23 DOI: 10.1016/bs.mie.2026.01.003

Franceine S Welcome, Lingshuang Wu, Tereza Vitkovska, Jiyao Chai, Shujuan Gao, Michael V Airola

{"title":"A fluorescence and HPLC-based activity assay system for lipid-modifying enzymes: Lipin/Pah phosphatidic acid phosphatase as an optimized example.","authors":"Franceine S Welcome, Lingshuang Wu, Tereza Vitkovska, Jiyao Chai, Shujuan Gao, Michael V Airola","doi":"10.1016/bs.mie.2026.01.003","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.01.003","url":null,"abstract":"Quantitating the enzymatic activity of lipid-modifying enzymes is a critical aspect of their study. Here, we describe a general approach to measure the activity of lipid-modifying enzymes using fluorescently labeled lipid substrates. This approach uses high-performance liquid chromatography (HPLC) to separate the resulting fluorescent lipid product(s) of enzymatic catalysis from the fluorescent lipid substrates, and to concurrently quantify the products using a fluorescence detector. Here, we focus on a version of this assay that has been optimized for lipin/Pah phosphatidic acid phosphatases, a class of enzymes that catalyze the magnesium-dependent hydrolysis of phosphatidic acid into diacylglycerol. Details for delivery of a fluorescent lipid substrate to an enzyme in detergent mixed-micelles and liposomes are provided. We also describe methods to purify recombinant lipin/Pah phosphatidic acid phosphatases using E. coli as an overexpression system, which can present challenges given the role of phosphatidic acid as a central precursor in bacterial phospholipid synthesis. Overall, the fluorescence and HPLC-based activity assay described here is generally applicable to any lipid modifying enzyme and additionally allows for the detection of unsuspected products that would remain undetected by conventional methods.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"235-251"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530954","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

Application of EPR spectroscopy in the structural studies of Aβ oligomers and fibrils. EPR光谱在Aβ低聚物和原纤维结构研究中的应用。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-03 DOI: 10.1016/bs.mie.2026.01.030

Diana Portugal Barron, Zhefeng Guo

引用次数: 0

Cryo-EM methods to study binding between amyloid fibrils and chemical compounds. 冷冻电镜方法研究淀粉样蛋白原纤维与化合物之间的结合。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-28 DOI: 10.1016/bs.mie.2026.01.029

Qinyue Zhao, Kaien Liu, Dan Li, Cong Liu

{"title":"Cryo-EM methods to study binding between amyloid fibrils and chemical compounds.","authors":"Qinyue Zhao, Kaien Liu, Dan Li, Cong Liu","doi":"10.1016/bs.mie.2026.01.029","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.01.029","url":null,"abstract":"Amyloid fibrils formed by amyloid proteins such as α-synuclein (α-syn), Amyloid-β (Aβ) and Tau are central to the pathology of neurodegenerative diseases like Parkinson's disease (PD) and Alzheimer's disease (AD). Structural elucidation of fibril-ligand interactions is essential for the rational design of imaging probes and therapeutic inhibitors targeting these pathological aggregates. Here, we present a comprehensive cryo-electron microscopy (cryo-EM)-based workflow for modeling small-molecule binding to amyloid fibrils, with a focus on α-syn-ligand complexes. The protocol integrates optimized fibril sample preparation, helical reconstruction, and iterative 2D/3D classification to yield high-resolution density maps suitable for atomic modeling. Ligands are incorporated by generating coordinates from SMILES strings and restraint files from Phenix eLBOW, followed by manual docking and real-space refinement. Using CCA-α-syn complex as a case study, we demonstrate precise ligand placement into specific fibril binding sites (the C-pocket, N-pocket, and a back-surface groove of the fibril core distinct from typical globular protein pockets). Subsequent structural refinement preserved key interaction features, including π-π stacking and side-chain hydrogen bonding. Validation metrics confirm the stereochemical integrity and good model-to-map fit of the final fibril-ligand complex structures. Overall, this workflow enables accurate modeling of ligand engagement with amyloids even at ∼3-4 Å resolution and provides a scalable framework for structure-guided ligand discovery in neurodegenerative disease research.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"729 ","pages":"107-144"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147776277","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

All-atom MD simulations of amyloid-membrane interactions: Setup, execution, and analysis. 淀粉样蛋白-膜相互作用的全原子MD模拟：设置，执行和分析。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-03-09 DOI: 10.1016/bs.mie.2026.01.031

Bastian F Bundschuh, Franziska Kley, Hebah Fatafta, Birgit Strodel

引用次数: 0

Lipid imaging using bifunctional lipid probes. 脂质成像使用双功能脂质探针。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-21 DOI: 10.1016/bs.mie.2026.01.019

Martin Buitrago-Arango, Chia-Yi Chou, André Nadler

引用次数: 0