Methods in enzymology最新文献_第6页

Chromatin immunoprecipitation assay for analyzing histone H3-mediated regulation of copper homeostasis. 染色质免疫沉淀法分析组蛋白h3介导的铜稳态调节。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-03 DOI: 10.1016/bs.mie.2026.05.030

Sakshi Singh, Rakesh Kumar Sahu, Raghuvir Singh Tomar

{"title":"Chromatin immunoprecipitation assay for analyzing histone H3-mediated regulation of copper homeostasis.","authors":"Sakshi Singh, Rakesh Kumar Sahu, Raghuvir Singh Tomar","doi":"10.1016/bs.mie.2026.05.030","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.030","url":null,"abstract":"Histone tails undergo various post-translational modifications that aid in recruiting effector proteins involved in diverse transcriptional responses. Our previous work explored the role of histone H3 N-terminal residues and their modifications on the transcriptional regulation of the CUP1 metallothionein gene during cellular copper stress. To pinpoint critical histone residues, we used complementary methods, including growth assays and gene expression kinetics. We screened a library of synthetic histone H3 and H4 mutants for copper response defects by assessing their sensitivity to copper stress and quantifying CUP1 transcript levels via real-time PCR. We further investigated the mechanisms of impaired CUP1 transcription by assessing the recruitment of Ace1, a copper-sensing transcription factor that activates CUP1 in response to high intracellular copper, and of TATA-binding protein (TBP), using chromatin immunoprecipitation (ChIP) assays. Our results showed that mutations in the H3 N-terminal tail impair the recruitment of Ace1 and TBP to the CUP1 promoter, causing defects in its induction. Collectively, these findings highlight the significance of histone residues and their modifications in regulating metal homeostasis. This chapter outlines the methods used to study histone-mediated regulation of copper homeostasis, with a focus on the chromatin immunoprecipitation assay.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"55-79"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795525","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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Preface. 前言。

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Methods in enzymology Pub Date : 2026-01-01 DOI: 10.1016/S0076-6879(26)00363-0

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Evolving thiazole synthases for expression in plants. 进化噻唑合成酶在植物中的表达。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-20 DOI: 10.1016/bs.mie.2026.06.013

Edmar Ramos de Oliveira Filho, Anuran K Gayen, J Donato García-García, Ulschan Bathe, Kristen Van Gelder

{"title":"Evolving thiazole synthases for expression in plants.","authors":"Edmar Ramos de Oliveira Filho, Anuran K Gayen, J Donato García-García, Ulschan Bathe, Kristen Van Gelder","doi":"10.1016/bs.mie.2026.06.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.013","url":null,"abstract":"Thiazole synthases (THI4s) catalyze the synthesis of the thiazole moiety of thiamin (vitamin B1) from glycine, NAD, and a sulfur atom. The sulfur atom can originate from an active site cysteine residue, as is the case for cysteine-dependent THI4s (cys-THI4s), or from an external sulfide source, as it is for sulfide-dependent THI4s (noncys-THI4s). Cys-THI4s are also known as suicidal enzymes, as catalysis leads to enzyme inactivation, followed by protein degradation. These enzymes can be considered energy intensive and catalytically inefficient enzymes. Replacement of plant cys-THI4s with noncys-THI4s which are not self-inactivating though still slow catalysts, could lead to energy conservation through decreased protein turnover. However, noncys-THI4s are anaerobic or microoxic enzymes and thus would be poor replacements for plant cys-THI4s that are aerobic enzymes. Engineering a plant cys-THI4 to be a true catalyst or a noncys-THI4 to function in plant-like conditions would be necessary to achieve safe respiratory energy. In this chapter, we will provide methods outlining: (1) how to use comparative genomics to select the best candidate THI4s to be engineered via rational design or continuous directed evolution (CDE); (2) how to use rational design to engineer THI4s; and (3) how to use OrthoRep, a yeast-based CDE tool, to engineering noncys-THI4s to function under plant-like conditions.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"3-42"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030815","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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A general method for the assay of thiamine pyrophosphokinase activity. 硫胺素焦磷酸激酶活性测定的一般方法。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-07-10 DOI: 10.1016/bs.mie.2026.06.026

Lucien Bettendorff

{"title":"A general method for the assay of thiamine pyrophosphokinase activity.","authors":"Lucien Bettendorff","doi":"10.1016/bs.mie.2026.06.026","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.026","url":null,"abstract":"Thiamine diphosphate (ThDP) is a coenzyme for several essential enzymes in cell metabolism (mainly transketolase, pyruvate and 2-oxoglutarate dehydrogenases). In eukaryotic cells ThDP is synthesized by pyrophosphorylation of thiamine (vitamin B1) by ATP or UTP and catalyzed by thiamine pyrophosphokinase isoform 1 (TPK1). Complete inactivation of TPK1 is lethal, but recently several hereditary mutations in the corresponding gene were described in human families. The phenotype is generally severe with acute encephalopathy and fatal outcome when not treated with excess thiamine. This suggests that the mutated enzyme still retains a fraction of its native activity. In order to estimate the effect of the mutations on enzyme activity it is essential to benefit from a reliable assay of TPK1 activity. This enzyme has a low KM for thiamine (KM < 0.1 µM) but a low turnover (kcat = 0.03 s-1) requiring very sensitive methods for the detection of the product ThDP by HPLC followed by fluorometric (after derivatization into fluorescent thiochrome derivatives) or mass spectrometric detection. Moreover, ThDP is a noncompetitive inhibitor of TPK1 with a KI = 0.4 µM. Hence, when measuring TPK1 activity care must be taken that the reaction is stopped before inhibition becomes significant. Here, we present a powerful HPLC method coupled to fluorometric detection allowing TPK1 assay either in the forward or the reverse direction. The importance of such a method is further highlighted by recent data showing decreased expression of TPK1 in blood and brain of Alzheimer patients making TPK1 assay an interesting alternative to the estimation of TPK1 expression.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"239-256"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030832","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

Scaffold engineering enables high-resolution cryo-EM analysis of thiamine pyrophosphate riboswitch architecture and dynamics. 支架工程使高分辨率低温电镜分析硫胺素焦磷酸核糖开关的结构和动力学。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-07-02 DOI: 10.1016/bs.mie.2026.06.024

Madison M Maille, Boris Rudolfs, Daniel B Haack, Navtej Toor

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

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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}

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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

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Strain-promoted bioorthogonal cycloadditions to investigate sialyltransferases promiscuity towards unnatural sialic acids. 菌株促进的生物正交环添加研究唾液基转移酶对非天然唾液酸的混杂性。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-29 DOI: 10.1016/bs.mie.2026.05.008

Zoeisha S Chinoy, Frédéric Friscourt

{"title":"Strain-promoted bioorthogonal cycloadditions to investigate sialyltransferases promiscuity towards unnatural sialic acids.","authors":"Zoeisha S Chinoy, Frédéric Friscourt","doi":"10.1016/bs.mie.2026.05.008","DOIUrl":"10.1016/bs.mie.2026.05.008","url":null,"abstract":"Bioorthogonal chemical reporters have revolutionized the study of glycosylation by enabling the selective labeling, visualization, and enrichment of glycans in living systems. Monosaccharide analogs bearing small, inert bioorthogonal tags such as azides, alkynes, cyclopropenes or sydnones, are metabolically incorporated into glycoconjugates by the cell's endogenous glycosyltransferases (GTs). Subsequent chemoselective ligation (e.g., strain-promoted alkyne-azide cycloaddition or inverse electron-demand Diels-Alder reactions) permits downstream detection or pull-down with minimal perturbation to cellular physiology. This chapter demonstrates the impact of a sydnone bioorthogonal chemical reporter on sialyltransferases (STs) substrate tolerance and catalytic efficiency. While many STs display surprising promiscuity toward modified CMP-sialic acid donors, incorporation efficiencies vary widely depending on the position and steric demand of the bioorthogonal handle, and can alter kinetic parameters, and influence donor/acceptor specificity. Herein, we present detailed protocols for assessing the impact of bioorthogonal chemical reporters, with a focus on 1,3-dipoles, on the activity of recombinant human sialyltransferases. The methods include qualitative evaluation by in-gel fluorescence imaging and quantitative analysis through determination of enzyme-specific kinetic parameters. These protocols can be extended to other glycosyltransferases collectively expanding the enzymatic toolkit for glycobiology, enabling additional mechanistic studies in the interrogation of glycosylation dynamics and GT function.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"31-44"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148330974","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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Click and release through bioorthogonal hydroamination. 点击并通过生物正交氢胺化释放。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-28 DOI: 10.1016/bs.mie.2026.05.021

Yiming Guo, Tyler J Brown, Justin Kim

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Molecular imaging enabled by bioorthogonal click labeling. 通过生物正交点击标记实现分子成像。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-30 DOI: 10.1016/bs.mie.2026.05.013

Katharina Götz, Marcel Streit, Patrick Eiring, Gerti Beliu

{"title":"Molecular imaging enabled by bioorthogonal click labeling.","authors":"Katharina Götz, Marcel Streit, Patrick Eiring, Gerti Beliu","doi":"10.1016/bs.mie.2026.05.013","DOIUrl":"10.1016/bs.mie.2026.05.013","url":null,"abstract":"Efficient protein labeling with minimal linkage error is a key requirement for super-resolution fluorescence microscopy. Many commonly used labeling strategies, including antibodies, fluorescent proteins, and self-labeling enzymes are limited by steric hindrance, and therefore they limit labeling density or constrain labeling to protein termini, thereby restricting achievable resolution at the molecular scale. Here, we describe a practical and broadly applicable protocol for site-specific protein labeling based on genetic code expansion and bioorthogonal inverse electron-demand Diels-Alder click chemistry. The method relies on the incorporation of a strained alkene-modified noncanonical amino acid at a defined position within a protein of interest, followed by rapid and selective covalent labeling with tetrazine-conjugated organic fluorophores. This approach enables the attachment of small, bright dyes with minimal linkage error and is compatible with both live-cell and fixed-cell imaging. The protocol provides detailed guidance on the design of suitable click sites, expression of amber mutants in mammalian cells, selection of appropriate tetrazine dyes, and optimization of labeling conditions for super-resolution microscopy, including single-molecule localization microscopy. Critical parameters, common pitfalls, and limitations are discussed to facilitate robust implementation across different protein classes and experimental systems. This workflow supports high-density, stoichiometric labeling and enables molecular-scale imaging of proteins in their native cellular context.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"353-386"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148331009","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