Methods in enzymology最新文献

Methods for studying mucin-microbe interactions. 黏液-微生物相互作用的研究方法。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-13 DOI: 10.1016/bs.mie.2026.05.024

Olga Sokolovskaya, Reuben Allen, Bradley Turner, Katharina Ribbeck

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":"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

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":"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

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

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

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

ChIPmentation-based mapping of histone modifications in aging: Experimental design and analytical considerations. 基于芯片的组蛋白修饰在衰老中的映射：实验设计和分析考虑。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-16 DOI: 10.1016/bs.mie.2026.05.032

Amalanandan Johna, Sreedarsanam Sreya, Sengupta Tiyas, Pavithra Elumalai, Avik Sengupta, Rahul Kumar, Mayilaadumveettil Nishana

{"title":"ChIPmentation-based mapping of histone modifications in aging: Experimental design and analytical considerations.","authors":"Amalanandan Johna, Sreedarsanam Sreya, Sengupta Tiyas, Pavithra Elumalai, Avik Sengupta, Rahul Kumar, Mayilaadumveettil Nishana","doi":"10.1016/bs.mie.2026.05.032","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.032","url":null,"abstract":"Aging is accompanied by progressive alterations in chromatin organization that contribute to transcriptional dysregulation, genomic instability, and loss of cellular identity. Among the epigenetic mechanisms implicated, histone modifications play a central role in regulating chromatin accessibility and gene expression during aging. Changes in key histone marks, including H3K9me3, H3K27me3, H4K20me3, and histone acetylation signatures, are associated with heterochromatin loss, increased transcriptional noise, and altered cellular function across diverse tissues and organisms. These modifications are dynamically regulated by histone-modifying enzymes and are responsive to metabolic and stress signaling pathways, linking environmental cues to chromatin state and longevity. Genome-wide profiling of histone modifications is essential to understand these age-associated chromatin changes. ChIPmentation, a streamlined approach that integrates chromatin immunoprecipitation with transposase-mediated tagmentation, enables rapid and sensitive mapping of histone marks with reduced input requirements and simplified library preparation. This method is particularly advantageous for aging studies, where sample availability is often limiting. In this chapter, we outline the principles and experimental workflow of ChIPmentation, including crosslinking, chromatin fragmentation, immunoprecipitation, tagmentation, library preparation, sequencing, and data analysis. Additionally, we discuss computational strategies for processing and analyzing ChIPmentation data, including alignment, normalization, peak calling, and reproducibility assessment. Together, this chapter provides a comprehensive methodological framework for studying histone modification landscapes in aging systems, enabling robust investigation of epigenetic mechanisms underlying age-associated phenotypes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"367-384"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795478","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

Integrated SDH, COX histochemistry, and MHC fibre typing in skeletal muscles: Methods and interpretation. 骨骼肌中SDH、COX组织化学和MHC纤维分型：方法和解释。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-09 DOI: 10.1016/bs.mie.2026.05.046

Aastha Munjal, Ridhi Singh, Ashwini Bhelave, Shwetha Sekar, Ninitha Asirvatham-Jeyaraj, Nagalingam R Sundaresan

{"title":"Integrated SDH, COX histochemistry, and MHC fibre typing in skeletal muscles: Methods and interpretation.","authors":"Aastha Munjal, Ridhi Singh, Ashwini Bhelave, Shwetha Sekar, Ninitha Asirvatham-Jeyaraj, Nagalingam R Sundaresan","doi":"10.1016/bs.mie.2026.05.046","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.046","url":null,"abstract":"Skeletal muscle plays a vital role in metabolic homeostasis, accounting for the majority of glucose uptake, lipid oxidation, and adaptive thermogenesis. Its plasticity enables rapid, controlled remodelling in response to exercise, nutrients, hormonal changes, ageing, and disease. This metabolic plasticity is due to fibre-type heterogeneity. Each muscle fibre has distinct contractile and bioenergetic properties. Sirtuins are known critical regulators of skeletal muscle mitochondrial content and oxidative metabolism. Sirtuins, are NAD+-dependent acetylases and deacetylases that regulate mitochondrial biogenesis, redox balance, and cellular response to stress. Thus, studying the role of sirtuins in muscle physiology requires assays that can identify metabolic and contractile phenotypes. In this chapter, we provide a comprehensive histochemistry protocol for succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) to assess mitochondrial oxidative capacity, and Myosin Heavy Chain (MHC) immunohistochemistry to assess fibre-type classification. Additionally, we have discussed detailed guidance for troubleshooting the critical steps of the protocol, including cryoinjury, tissue sectioning, staining optimisation, and imaging.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"223-251"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795530","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