Methods in enzymology最新文献_第3页

A dual-color FRET assay for detection and quantitative analysis of O-glycopeptidases. 双色FRET检测和定量分析的o糖肽酶。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-04-15 DOI: 10.1016/bs.mie.2026.03.007

Olivia Canil, Liam Mihalynuk, Nicole Thompson, Warren Wakarchuk, Alisdair Boraston

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Tracing sphingolipid metabolism kinetics using radioactive and non-radioactive labels. 使用放射性和非放射性标记追踪鞘脂代谢动力学。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-21 DOI: 10.1016/bs.mie.2026.01.017

Alec Griffith, Helene Jahn, Fikadu G Tafesse

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Imaging intracellular lipid heterogeneity with fluorogen-activating coincidence encounter sensing. 用氟激活偶遇传感成像细胞内脂质异质性。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-04 DOI: 10.1016/bs.mie.2025.11.015

William M Moore, Itay Budin

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Imaging interorganelle membrane contact sites using dimerization-dependent fluorescent proteins. 利用二聚化依赖的荧光蛋白成像细胞器间膜接触位点。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-27 DOI: 10.1016/bs.mie.2026.01.024

Victoria H Williams, Chih-Hsuan Hsu, Gregory E Miner, Sarah Cohen

{"title":"Imaging interorganelle membrane contact sites using dimerization-dependent fluorescent proteins.","authors":"Victoria H Williams, Chih-Hsuan Hsu, Gregory E Miner, Sarah Cohen","doi":"10.1016/bs.mie.2026.01.024","DOIUrl":"10.1016/bs.mie.2026.01.024","url":null,"abstract":"Membrane contact sites (MCSs), sites of close apposition between membrane-bound organelles, mediate key intracellular processes to coordinate organelle function and are implicated in a wide range of human diseases. Because MCSs can span as little as 10 nm of distance, the limited resolution of light microscopy can hamper the ability to study these structures in live cells. Dimerization-dependent fluorescent proteins consist of a weakly fluorescent and non-fluorescent monomer that produce greater signal when the two monomers interact, thereby allowing the user to identify sites of proximity through protein-protein interaction. Here, we describe a protocol using Contact-FP - our suite of organelle-targeted dimerization-dependent fluorescent proteins - to study MCSs using confocal or Airyscan microscopy in live cells. The protocol includes guidance for transfection, imaging, and analysis of Contact-FP biosensors. It includes instructions on how to leverage this tool to study a single MCS type, identify two MCSs involving the same organelle, or induce MCSs using high levels of transfection of Contact-FP probes. We also suggest troubleshooting steps for transfection, imaging, and analysis. This protocol provides a specific example for using this tool in U-2 OS osteosarcoma cells but is amenable to adjustment for other cell types.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"321-354"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326601","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}

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

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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

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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。

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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

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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。

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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":"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。

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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

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MS2DECIDE: Harnessing the complementarity of mass spectrometry annotation tools for prioritizing novel natural products. MS2DECIDE：利用质谱注释工具的互补性优先考虑新的天然产物。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-03-25 DOI: 10.1016/bs.mie.2026.03.002

Yassine Mejri, Olivier Cailloux, Pierre Le Pogam, Meltem Öztürk-Escoffier, Mehdi A Beniddir

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Mass spectrometry-based mapping of the ubiquitin chaperone code. 基于质谱的泛素伴侣代码映射。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-01 DOI: 10.1016/bs.mie.2026.05.047

Ayush Nigam, Prasun Kumar Bhunia, Trayambak Basak, Prasad Kasturi

{"title":"Mass spectrometry-based mapping of the ubiquitin chaperone code.","authors":"Ayush Nigam, Prasun Kumar Bhunia, Trayambak Basak, Prasad Kasturi","doi":"10.1016/bs.mie.2026.05.047","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.047","url":null,"abstract":"Maintenance of proteome integrity is essential for cellular homeostasis and organismal health. This integrity depends on proteostasis, a coordinated network of protein quality control systems that regulate protein folding, stabilization, and degradation. Molecular chaperones, together with proteolytic pathways such as the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway, prevent the accumulation of misfolded and aggregation-prone proteins. Perturbations, including genetic mutations, environmental stress, and aging challenge protein folding fidelity, leading to proteotoxic stress and contributing to the pathogenesis of neurodegenerative disorders. Among the chaperone machinery, the HSP70 and HSP90 families play central roles in maintaining protein conformational homeostasis and directing damaged or misfolded substrates toward refolding or degradation pathways. Recent studies show that chaperone activity is dynamically regulated by diverse post-translational modifications (PTMs), including phosphorylation, acetylation, and ubiquitination, collectively termed the \"chaperone code.\" These modifications modulate chaperone-client interactions, enzymatic activity, localization, and coordination with protein degradation systems. Mass spectrometry (MS)-based proteomics has emerged as a powerful approach for mapping ubiquitination sites and quantifying ubiquitin signaling dynamics. This chapter outlines experimental and computational strategies for MS-based analysis of the ubiquitin chaperone code, including di-glycine peptide enrichment, site identification, quantitative analysis, and validation.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"734 ","pages":"199-216"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148888022","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