Methods in enzymology最新文献

High-resolution phosphoinositide analysis. 高分辨率磷酸肌苷分析。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-09 DOI: 10.1016/bs.mie.2025.11.016

Helene Jahn, Berit Blume, Alexis E Traynor-Kaplan, Fikadu G Tafesse, Carsten Schultz

引用次数: 0

Quantitative analysis of S-acylation. s -酰化的定量分析。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-17 DOI: 10.1016/bs.mie.2025.12.001

Carla Busquets Hernández, Alexandra Tsiotsia, Gemma Triola

{"title":"Quantitative analysis of S-acylation.","authors":"Carla Busquets Hernández, Alexandra Tsiotsia, Gemma Triola","doi":"10.1016/bs.mie.2025.12.001","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.12.001","url":null,"abstract":"S-acylation is a protein post-translational modification that relies on the attachment of a hydrophobic fatty acid chain to a cysteine residue through the formation of a thioester bond. This reversible modification is controlled by the counteraction of protein acyltransferases and acyl protein thioesterases. The bonded lipid moiety can modulate the physicochemical properties of the substrate proteins, their membrane-binding affinity, subcellular localization, protein stability, or interactions with other proteins or cell components, thereby playing a key role in several cell processes such as protein trafficking, signal transduction, or cell proliferation. Moreover, increasing evidence has associated S-acylation malfunction with several pathological processes, including various types of cancers or neurodegenerative disorders, making S-acylation and its controlling enzymes an ideal target for therapeutic strategies. S-acylation has been commonly known as S-palmitoylation because palmitic acid was considered the predominant fatty acid attached to proteins. However, recent advances, especially in the mass spectrometry field, have suggested a diversity in the identity of the attached lipids greater than that previously considered. Moreover, since variations in the length and saturation degree of the fatty acyl chains may alter the intracellular localization and biological function of proteins, its potential role in the regulation of protein function is just being explored. However, this heterogeneous lipid composition could not be systematically studied, mainly due to the lack of suitable methods. In order to address these challenges, this protocol reports a method to identify and quantify the fatty acids attached to S-acylated proteins.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"117-128"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530764","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

Inhibition of sterol transport protein function. 抑制甾醇转运蛋白的功能。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-30 DOI: 10.1016/bs.mie.2026.01.009

Hogan P Bryce-Rogers, Laura Depta, Thomas Whitmarsh-Everiss, Luca Laraia

引用次数: 0

Pharmacological modulation of lipid signaling. 脂质信号的药理调节。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-22 DOI: 10.1016/bs.mie.2026.01.025

Ashutosh Sharma, Wonhwa Cho

{"title":"Pharmacological modulation of lipid signaling.","authors":"Ashutosh Sharma, Wonhwa Cho","doi":"10.1016/bs.mie.2026.01.025","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.01.025","url":null,"abstract":"Lipids regulate a broad spectrum of cellular functions through spatiotemporally controlled lipid-protein interactions. Dysregulation of lipid metabolism and lipid-mediated signaling is associated with diverse human diseases, including cancer, metabolic disorders, and neurodegenerative conditions. Consequently, selective inhibition of site-specific lipid-protein interactions has emerged as a promising therapeutic strategy to modulate aberrant cell signaling at the membrane interface. Here, we present a streamlined, quantitative workflow for the discovery, characterization, and evaluation of small-molecule inhibitors that disrupt lipid-dependent membrane association and activation of cytosolic signaling proteins. The protocol integrates a high-throughput fluorescence-quenching assay for inhibitor screening, detailed biochemical and cellular target-validation methods, and standardized procedures for in vitro and in vivo assessment of inhibitor potency, specificity, and safety. These general protocols provide a versatile and reproducible platform for developing potent, specific, and mechanistically defined inhibitors targeting a wide range of lipid-binding proteins implicated in disease.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"369-387"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530638","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

Single-molecule fluorescence spectroscopy and imaging of heterogeneous amyloid β aggregation. 非均相淀粉样蛋白β聚集的单分子荧光光谱与成像。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-28 DOI: 10.1016/bs.mie.2026.01.033

Jae-Yeol Kim, Eunho Song, Annie Aniana, John M Louis, Hoi Sung Chung

引用次数: 0

Two-dimensional FTIR methods on amyloid aggregation and folding pathways. 淀粉样蛋白聚集和折叠途径的二维FTIR方法。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-28 DOI: 10.1016/bs.mie.2026.01.027

Michał Maj

引用次数: 0

Prediction and characterization of lipid-interacting proteins. 脂质相互作用蛋白的预测和表征。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-09 DOI: 10.1016/bs.mie.2025.11.013

Sebastian Alfonso, Cassandra M Decosto, Poulami Chatterjee, Timothy Precord, Laura M K Dassama

{"title":"Prediction and characterization of lipid-interacting proteins.","authors":"Sebastian Alfonso, Cassandra M Decosto, Poulami Chatterjee, Timothy Precord, Laura M K Dassama","doi":"10.1016/bs.mie.2025.11.013","DOIUrl":"10.1016/bs.mie.2025.11.013","url":null,"abstract":"Lipids are essential to all life forms. These molecules serve diverse purposes that range from cell membrane formation to energy storage and inter-cellular signaling. Lipids can be natively synthesized or sourced from the environment, often through the action of proteins engaging with specific lipid molecules. Characterizing lipid-interacting proteins is a key frontier in therapeutic science, as dysfunction in lipid metabolism is implicated in a range of human diseases. A substantial bottleneck that precludes the identification and characterization of lipid-interacting proteins pertains to the nature of the lipid substrates: they are not genetically encoded, their hydrophobic nature results in non-specific interactions, they exist in complex cellular environments, and they are structurally diverse. Regardless, the identification, characterization, and specific targeting of proteins that maintain proper lipid homeostasis is important for efforts to restore dysregulated metabolism. In this chapter, we outline bioinformatic and experimental approaches employed by our research group and others to study lipids and the proteins that directly bind them. The chapter covers methods for proteome-wide computational screening to reveal lipid binding proteins, characterization of total lipid composition in mammalian and bacterial cells, and the use of analytical and biophysical methods to study target protein-lipid interactions.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"253-289"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326650","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

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。

4区生物学

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

William M Moore, Itay Budin

引用次数: 0

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。

4区生物学

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}

引用次数: 0

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。

4区生物学

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

Alec Griffith, Helene Jahn, Fikadu G Tafesse

引用次数: 0