Methods in enzymology最新文献

{"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":"<p><p>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.</p>","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":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13220709/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":"10.1016/bs.mie.2026.01.003","url":null,"abstract":"<p><p>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.</p>","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":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13264702/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of thiamine status in humans.","authors":"Kerry S Jones, Lucien Bettendorff, Elizabeth L Frank, Kyly C Whitfield","doi":"10.1016/bs.mie.2026.06.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.018","url":null,"abstract":"<p><p>Thiamine (vitamin B1) is an essential nutrient with established adverse health consequences associated with acute deficiency. Emerging evidence also suggests long-term neurocognitive deficits due to sub-clinical deficiency in early life, and there is a growing appreciation of a potential role for thiamine in Alzheimer's disease. Therefore, the assessment of thiamine biomarkers, particularly in large, population-based studies, is critical to grasp the true scope of this public health problem. The most reliable biomarkers of thiamine status are whole blood concentrations of thiamine diphosphate (ThDP) measured by high performance liquid chromatography (HPLC) with fluorescence detection or liquid chromatography tandem mass spectrometry (LC-MS/MS), and the functional assay of transketolase activity (erythrocyte transketolase activity coefficient, ETKAC). Plasma thiamine reflects recent intake, and is not a useful biomarker of thiamine status. This manuscript describes the different biomarkers and methods of assessment, their advantages and disadvantages, and related cutoffs for deficiency. A table of 'performance characteristics' is provided to guide users on the quality and performance characteristics of each assay, and practicalities related to their cost, resource requirement, and maintenance. Provision of these insights will help inform decisions about inclusion of thiamine analysis in future work to better understand the extent of global thiamine deficiency.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"189-216"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030811","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}

{"title":"DNA methylation and histone modifications in tumor hypoxia.","authors":"Shruti Ganesh Dhamdhere, Parik Kakani, Sanjeev Shukla","doi":"10.1016/bs.mie.2026.05.035","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.035","url":null,"abstract":"<p><p>Hypoxia, an inherent condition to tumors, contributes to cancer development, metastasis, and therapy resistance. Cancer cells employ epigenetic remodelling for survival under hypoxia. DNA methylation and various histone modifications are two key mechanisms of epigenetic regulation of gene expression. Histone modifications in collaboration with DNA methylation play a vital role in controlling gene expression and modulating chromatin structure. Recently it has become evident that hypoxia plays key roles in the EMT, angiogenesis, autophagy, and ECM remodeling via (i) modulating DNA methylation at the promoter of the gene or at loci undergoing alternative splicing; and (ii) locus-specific histone modifications such as H3K27Ac, H3K9Ac, H3K4me3, H4R3me2s, and H3R8me2s. Thus, it is important to mechanistically assess how hypoxia-mediated DNA methylation changes and histone modification affect tumorigenesis using suitable methods. In this chapter, we describe the methods used in our laboratory to assess the locus-specific DNA methylation and histone modifications profiling. The goals of this chapter are twofold. First, we discuss immunoprecipitation-based approaches applied to assess DNA methylation or hydroxymethylation, histone modification, and transcription factor binding profile at specific loci. Second, we discuss methods used to modify the epigenetic landscape, such as DNA methylation, binding of transcription factors such as HIF1α and CTCF, and histone marks on a given locus using dCas9 fused with epigenetic modifiers such as DNMT3A, TET1, PRMT5, etc. Collectively, these approaches provide a template that can be applied to evaluate roles of any epigenetic mark involved in development and/or progression of cancer.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"143-162"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795436","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}

{"title":"Histone deacetylase as a potential therapeutic target for memory dysfunction during aging and neurodegenerative disorders.","authors":"Padmanabh Singh, Mahendra Kumar Thakur","doi":"10.1016/bs.mie.2026.05.037","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.037","url":null,"abstract":"<p><p>Brain aging is a natural biological process characterized by gradual structural, molecular, and functional changes in the brain over time. Aging is the major risk factor for neurodegenerative diseases characterized by progressive neuronal loss, cognitive decline, and impaired motor function. In recent years, growing evidence has highlighted the importance of epigenetic regulation in the development and progression of these disorders. Among these epigenetic regulators, histone deacetylases (HDACs) have emerged as critical modulators of gene expression and neuronal function. Dysregulation of HDAC activity has been increasingly implicated in the pathogenesis of several neurodegenerative diseases. Abnormal HDAC expression or activity can alter transcription of genes involved in neuronal survival and synaptic integrity, which leads to neuronal dysfunction and degeneration. Pharmacological inhibition of HDAC activity has been shown to restore histone acetylation levels, enhance the expression of neuroprotective genes, and improve synaptic plasticity in various experimental models.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"279-295"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795532","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}

{"title":"ArtiSt: An organocatalyst inspired by TPP installed in a steroid-carrying protein creates an artificial Stetterase.","authors":"Alice MacAulay, Süleyman Mert Ünal, Evangelia Notari, Christopher W Wood, Amanda G Jarvis, Dominic J Campopiano","doi":"10.1016/bs.mie.2026.07.032","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.07.032","url":null,"abstract":"<p><p>The various methods (protein expression, mass spectrometry, chemical synthesis, protein design, engineering, product analysis) presented in this chapter were used to create the first artificial Stetterase constructed from an otherwise inactive protein scaffold and a synthetic thiamine pyrophosphate-inspired organocatalyst. The N-heterocyclic carbene (NHC)-derived organocatalyst was covalently incorporated into engineered protein scaffolds, creating an artificial \"Stetterase\" (ArtiSt). Cysteine residues were strategically introduced into selected scaffolds to enable site-specific bioconjugation of a synthetic NHC bearing a maleimide handle. Scaffold selection and mutagenesis were guided by structural analysis and computational modelling. Recombinant scaffold expression, purification, and optimization of functionalization conditions, generated hybrid catalysts that were evaluated using an intramolecular Stetter reaction as a model transformation. The NHC-functionalized ArtiSt catalyzed the reaction under ambient, aqueous conditions with low catalyst loading, affording modest yields and enantioselectivities. Activity was enhanced more than 20-fold by adopting a more stable protein scaffold, highlighting the critical role of the protein environment in controlling the catalytic behavior of the embedded organocatalyst. The modular platform described here establishes a foundation for optimizing activity and enantioselectivity and provides a general strategy for expanding NHC-mediated transformations within protein environments.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"736 ","pages":"369-397"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149075273","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}

{"title":"Plant and fungal acetohydroxyacid synthases (AHAS): Methods for heterologous expression, kinetic characterization, and structural analysis.","authors":"Mario Daniel Garcia, Liliana Cerda-Mejía, Vivian Gavilanes-Flores, Thierry Lonhienne, Luke Guddat","doi":"10.1016/bs.mie.2026.07.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.07.018","url":null,"abstract":"<p><p>Acetohydroxyacid synthase (AHAS; EC 2.2.1.6), also known as acetolactate synthase, catalyzes the first common step in the biosynthesis of the branched-chain amino acids valine, leucine, and isoleucine. AHAS is present in plants and microorganisms but absent in animals. For this reason, it has become an important target for the development of herbicides and antimicrobial agents. AHAS is the target of five major families of commercial herbicides, which collectively represent one of the most successful groups of herbicides used in modern agriculture. Biochemical, kinetic, and structural studies have revealed that AHAS exhibits a complex catalytic mechanism involving the cofactors thiamine diphosphate (ThDP), flavin adenine dinucleotide (FAD), and Mg²⁺, as well adopting unique inhibition processes associated with oxidative side reactions that occur under turnover conditions and in the presence of AHAS-inhibiting compounds. Consequently, robust experimental methodologies have been required to investigate AHAS structure, function, and inhibition. Here, we describe a comprehensive set of protocols for the heterologous expression and purification of plant and fungal AHASs, determination of steady-state kinetic parameters, characterization of reversible accumulative inhibition by commercial herbicides, and analysis of herbicide-induced cofactor degradation. Detailed procedures are also provided for monitoring ThDP degradation, FAD redox transitions, and obtaining diffraction-quality crystals suitable for X-ray crystallographic studies. These protocols address several experimental factors that critically influence enzyme activity, including cofactor requirements, lag-phase kinetics, oxygen-dependent side reactions, and inhibitor-induced oxidative processes. Although these methods have been optimized for plant and fungal AHAS enzymes, several of the biochemical and structural approaches described here may also facilitate similar studies of bacterial AHAS enzymes.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"736 ","pages":"157-215"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149075279","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}

{"title":"Comparative methods for recombinant production and characterization of 1-deoxy-D-xylulose-5-phosphate synthase from bacterial and apicomplexan sources.","authors":"Tyler Holets, Imani S McCalla, Nathaniel O Johnson, David J Merkler","doi":"10.1016/bs.mie.2026.07.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.07.002","url":null,"abstract":"<p><p>1-Deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyzes the first step of the methylerythritol phosphate (MEP) pathway, which is required for isoprenoid biosynthesis in bacteria and apicomplexan parasites but is absent in humans. This pathway represents an important target for drug development, necessitating methods for recombinant production and biochemical characterization of DXPS from diverse organisms. This chapter describes a comparative framework for the cloning, expression, purification, and characterization of DXPS from the bacterium Deinococcus radiodurans and the apicomplexan Plasmodium vivax and Plasmodium falciparum. Strategies for construct design are presented, including systematic truncation of N-terminal targeting sequences required to obtain active enzymes. Expression optimization is detailed, along with chromatography workflows that preserve cofactor-dependent stability. Biochemical characterization was performed using a continuous spectrophotometric assay based on a coupled DXPS-DXR system. In addition, computational methodologies are presented, including molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) approaches for modeling thiamine diphosphate (ThDP)-dependent catalysis and active site electrostatics. Collectively, these methods provide a comprehensive and generalizable platform for the production, analysis, and mechanistic investigation of DXPS and related ThDP-dependent enzymes.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"736 ","pages":"97-125"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149075321","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}

{"title":"Expanding the multiplexing capability of HIDE probes via fluorescence lifetime imaging microscopy.","authors":"Justin H Kwon, Alanna Schepartz","doi":"10.1016/bs.mie.2025.11.011","DOIUrl":"10.1016/bs.mie.2025.11.011","url":null,"abstract":"<p><p>Fluorescence lifetime imaging microscopy (FLIM) measures the average time that a fluorophore spends in the excited state. FLIM is exceptionally useful as an imaging tool because the lifetime of a fluorophore is independent of its excitation and emission profiles. As such, even spectrally identical fluorophores can often be distinguished on the basis of their fluorescence lifetimes. Furthermore, given the dependence of fluorescence lifetime on the relative magnitude of radiative and nonradiative decay rates, it is possible to both predict and modulate the lifetime of a single fluorophore for applications in FLIM multiplexing. Herein, we provide an overview of the protocols necessary for transfection-free FLIM multiplexing in live cells. Protocols are provided for the in vitro reactions required to initially screen bioorthogonal handles and dyes, incubation of the small molecule probes required for FLIM multiplexing with live mammalian cells, and the setup of FLIM experiments. We also provide a discussion of methods available to analyze FLIM data to extract lifetime data.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"726 ","pages":"1-18"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13182769/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146257934","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}

{"title":"Cell-based mucin arrays.","authors":"Felix Goerdeler, Yoshiki Narimatsu, Christian Büll","doi":"10.1016/bs.mie.2026.03.006","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.03.006","url":null,"abstract":"<p><p>The cell-based mucin array is a versatile platform for expressing and interrogating recombinant mucin reporter proteins with representative patterning and customizable O-glycan structures. The platform is based on glycoengineered mammalian cell lines (HEK293/CHO), in which the glycosylation machinery is genetically rewritten to enable controlled display of specific O-glycan core structures and terminal epitopes with defined sialylation, fucosylation, and sulfation. Uniquely, the platform presents glycans in their native protein context, enabling investigation of how O-glycan density, clustering, and multivalency influence interactions with mucins. A conceptual framework for recognition of such glycan-context cues is provided by the patterned arrangement of O-glycans within mucin O-glycodomains, often composed of tandem repeat (TR) sequences with distinct O-glycosites resembling molecular barcodes. Mucin reporters mimic the serine-, threonine-, and proline-rich domains of natural mucins and mucin-like proteins or they can be designed as artificial Glycocarriers with model O-glycan cluster motifs. Reporters expressed as membrane-bound forms for cell display or as secreted fusion proteins for production can be applied in diverse bioassays. They have been used to probe glycan/mucin binding by viral and microbial adhesins, as well as human Siglec immune receptors. Moreover, the platform provides defined substrates for functional analyses of mucin and O-glycodomain degradation by microbial O-glycopeptidases or mucinases, revealing information of substrate specificities, cleavage points, and catalytic mechanisms. This chapter describes how the cell-based mucin array can be used to dissect interactions with mucins by glycan- and mucin-binding receptors as well as mucinases. The platform overcomes limitations of contemporary technologies by enabling studies with mimics of natural mucins and O-glycodomains that preserve clustered and patterned O-glycan contexts.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"732 ","pages":"177-218"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148382298","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}