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Identification of 2,4-dihydroxy-azaflavanone as an allosteric activator of SIRT1 and its potential role in cellular models of Parkinson's disease. 2,4-二羟基-氮黄酮作为SIRT1变构激活剂的鉴定及其在帕金森病细胞模型中的潜在作用
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-07-02 DOI: 10.1016/bs.mie.2026.06.011
Chitrakshi Pant, Vidya Jyothi Alli, Genji Sukumar, Abhijit Mohapatra, Surender Singh Jadav, Srihari Pabbaraja, Shasi V Kalivendi
{"title":"Identification of 2,4-dihydroxy-azaflavanone as an allosteric activator of SIRT1 and its potential role in cellular models of Parkinson's disease.","authors":"Chitrakshi Pant, Vidya Jyothi Alli, Genji Sukumar, Abhijit Mohapatra, Surender Singh Jadav, Srihari Pabbaraja, Shasi V Kalivendi","doi":"10.1016/bs.mie.2026.06.011","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.011","url":null,"abstract":"<p><p>Mitochondrial dysfunction is one of the significant aspects of Parkinson's disease (PD) pathophysiology, marked by a gradual decline in oxidative phosphorylation, an abnormal increase in free radical species, dysfunctional mitochondrial quality control, and faulty mitochondrial biogenesis. Sirtuin 1 (SIRT1), a NAD<sup>+</sup>-dependent class-III deacetylase, acts as a crucial metabolic sensor that orchestrates transcriptional programs related to mitochondrial biogenesis, respiratory chain assembly, and stress resilience, mainly by way of deacetylation and activation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). While screening for the small molecule activators, we have identified that 2,4-dihydroxy-Azaflavanone allosterically activates SIRT1. This chapter outlines a detailed, multi-layered methodological framework for assessing the allosteric activation of SIRT1 by 2,4-dihydroxy-azaflavanone and its downstream effects in a cellular models of PD. The validation process involves synthesis of small molecules, molecular docking studies utilizing crystallographic SIRT1 coordinates (PDB: 5BTR), in vitro fluorometric deacetylase assays with recombinant enzyme, and cellular thermal shift assays (CETSA) to confirm direct, isoform-selective target engagement. The activation of downstream pathways is evaluated by immunoblotting and quantitative PCR for PGC-1α, TFAM, and quantification of mitochondrial DNA (mtDNA) copy number. Functional restoration of mitochondria in cells is analyzed by assessing the overall mitochondrial bioenergetics parameters using Seahorse extracellular flux analyzer. Overall, this integrated approach offers robust, reproducible results for exploring SIRT1-activators in the mechanisms mediating neurodegenerative disease models.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"734 ","pages":"121-149"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148888077","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
1,2-Aminothiol-specific conjugation for dual-color fluorescent labeling via ultrafast TAMM conjugates. 1,2-氨基巯基特异性偶联超快TAMM偶联双色荧光标记。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-22 DOI: 10.1016/bs.mie.2026.05.011
Linlin Wang, Gaocan Dai, Yu-Hsuan Tsai, Sicong Yao
{"title":"1,2-Aminothiol-specific conjugation for dual-color fluorescent labeling via ultrafast TAMM conjugates.","authors":"Linlin Wang, Gaocan Dai, Yu-Hsuan Tsai, Sicong Yao","doi":"10.1016/bs.mie.2026.05.011","DOIUrl":"10.1016/bs.mie.2026.05.011","url":null,"abstract":"<p><p>This chapter describes a practical protocol for dual-color fluorescent labeling of two different cell-surface proteins using ultrafast 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) conjugates. Each target protein is engineered with an N-terminal protease-recognition sequence that, upon cleavage by a specific protease, exposes an N-terminal cysteine and thus a 1,2-aminothiol handle for bioorthogonal TAMM conjugation. Accordingly, sequential protease treatment followed by labeling with distinct TAMM-fluorophore conjugates enables robust dual-color visualization of cell-surface proteins on mammalian cells.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"99-118"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148331144","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
Non-invasive imaging of phosphoinositides with genetically encoded lipid biosensors. 用基因编码脂质生物传感器对磷酸肌苷进行无创成像。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-09 DOI: 10.1016/bs.mie.2025.11.017
Michael Worcester, Magdalene Motter, Gerald R V Hammond
{"title":"Non-invasive imaging of phosphoinositides with genetically encoded lipid biosensors.","authors":"Michael Worcester, Magdalene Motter, Gerald R V Hammond","doi":"10.1016/bs.mie.2025.11.017","DOIUrl":"10.1016/bs.mie.2025.11.017","url":null,"abstract":"<p><p>The localization and metabolism of lipids are difficult to dynamically resolve. Whereas immunofluorescence techniques rely on fixing cells and subsequent postmortem examination, and the employment of fluorescent lipid analogs in turn depends on approximative probes, genetically encoded lipid biosensors are not invasive and report on endogenous targets in living cells. Here we outline a protocol for imaging PI(4,5)P<sub>2</sub> localization in live HEK293A cells by expression of the genetically encoded lipid biosensor PH-PLCδ1. The protocol is presented as having two branching paths, with the first detailing how to overexpress the biosensor for confocal microscopy. The second path describes expression of the biosensor at single molecule levels for subsequent TIRF microscopy.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"726 ","pages":"181-191"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146258352","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
Photoaffinity strategies to profile cholesterol metabolite interactomes and binding sites. 光亲和策略分析胆固醇代谢物相互作用组和结合位点。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-02-03 DOI: 10.1016/bs.mie.2026.01.002
Yu-Shiuan Cheng, Alison E Ondrus
{"title":"Photoaffinity strategies to profile cholesterol metabolite interactomes and binding sites.","authors":"Yu-Shiuan Cheng, Alison E Ondrus","doi":"10.1016/bs.mie.2026.01.002","DOIUrl":"10.1016/bs.mie.2026.01.002","url":null,"abstract":"<p><p>Cholesterol and its metabolites regulate diverse and essential roles in biological systems and feature prominently in human health and disease. Key to the activity of these molecules are the proteins that they interact with in a given cell type and condition. In this Chapter, we describe the use of chemical photoaffinity probes to map the interactome of cholesterol metabolites throughout the proteome and to pinpoint the site of binding within a protein of interest. We pay close attention to variables that influence the study of cholesterol-based molecules, describing experimental and data analysis parameters to optimize proteome/binding site coverage and robustness of results.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"728 ","pages":"23-63"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147530689","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
Molecular engineering of activatable cyclopropenes for bioorthogonal labeling. 用于生物正交标记的可活化环丙烯分子工程。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-29 DOI: 10.1016/bs.mie.2026.05.019
Evans Kusi, Kangqiao Wen, Scott Laughlin
{"title":"Molecular engineering of activatable cyclopropenes for bioorthogonal labeling.","authors":"Evans Kusi, Kangqiao Wen, Scott Laughlin","doi":"10.1016/bs.mie.2026.05.019","DOIUrl":"10.1016/bs.mie.2026.05.019","url":null,"abstract":"<p><p>Bioorthogonal reactions enable selective biomolecular labeling in complex biological environments with minimal perturbation, and activatable (caged) reagents further provide spatial and temporal control of labeling. In this chapter, we apply a molecular engineering framework to cyclopropene-tetrazine ligation by treating cyclopropene reactivity as a tunable property that can be programmed through substituent electronics, scaffold architecture, and caging-group installation. Building on established activatable cyclopropene designs, we focus on strategies aimed at increasing ligation kinetics through structural constraint of an azaspirocyclopropene framework. Although the targeted ring-contracted scaffold was not sufficiently stable for isolation and direct evaluation in tetrazine ligation, these studies enabled access to functionalized 4-azaspiro[2.3]hexanes from dibromo intermediates. This scaffold offers a practical entry point to piperidine bioisosteres and expands the synthetic utility of the platform beyond molecular labeling. Overall, this work highlights how molecular-level design decisions govern stability and reactivity in activatable cyclopropenes and outlines actionable principles for engineering next-generation tetrazine-ligation partners.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"387-426"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148331052","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
Metabolic labeling of glycerophospholipids. 甘油磷脂的代谢标记。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-01 DOI: 10.1016/bs.mie.2025.11.010
Robert J Maraski, Christelle F Ancajas, Jinchao Lou, Michael D Best
{"title":"Metabolic labeling of glycerophospholipids.","authors":"Robert J Maraski, Christelle F Ancajas, Jinchao Lou, Michael D Best","doi":"10.1016/bs.mie.2025.11.010","DOIUrl":"10.1016/bs.mie.2025.11.010","url":null,"abstract":"<p><p>The approach of metabolic labeling provides an invaluable tool for elucidating previously unknown and poorly understood metabolic processes within cells. By introducing clickable versions of substrates into cells, the products of these biomolecule mimics can be conveniently tracked via post-derivatization of the clickable tag with a variety of reporter groups. Here, we will describe lipid metabolic labeling as an invaluable approach for interrogating lipid metabolic pathways, which can yield crucial information regarding complex lipid biosynthesis and trafficking networks that can open new therapeutic targets involving downstream natural products. In this chapter, we present detailed experimental procedures for the development of clickable serine probes for the labeling of phosphatidylserine (PS) and other lipids, including probe design and synthesis as well as analysis of biological incorporation via confocal microscopy, thin-layer chromatography (TLC), and liquid chromatography mass spectrometry (LCMS). This strategy provides a powerful approach for interrogating lipid biosynthetic pathways centered around PS.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"726 ","pages":"289-319"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146258349","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
Exploration of mucin-protein interactions using liquid glycan array. 利用液体聚糖阵列探索黏液-蛋白相互作用。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-27 DOI: 10.1016/bs.mie.2026.05.003
Shreyas Gupta, Guilherme Meira Lima, Chuanhao Peng, Ratmir Derda
{"title":"Exploration of mucin-protein interactions using liquid glycan array.","authors":"Shreyas Gupta, Guilherme Meira Lima, Chuanhao Peng, Ratmir Derda","doi":"10.1016/bs.mie.2026.05.003","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.003","url":null,"abstract":"<p><p>M13 phage makes it possible to produce DNA-encoded display of any molecules stable in water by prospective DNA-barcoding. For example, chemical conjugation of a glycan to an M13 virion that contains a prospectively introduced DNA barcode in the M13 genome creates a DNA-barcoded glycophage. In this glycophage, there is a 1:1 correspondence between the DNA sequence inside the phage and the structure of the glycan displayed on the surface of the phage. The same construct simultaneously illustrates the concept of DNA-encoded multivalency because there is a one-to-one correspondence between the DNA barcode and the displayed density. In this chapter, we show that a collection of these DNA-barcoded multivalent glycophages, known as Liquid Glycan Array (LiGA), can measure the glycan-recognition profile of mucin-binding proteins. Using DNA-encoded multivalency, we make an intriguing observation that a simple ultra-dense display of D-galactose on M13 \"looks like mucin\" because it binds to the proteolytically inactive E447D mutant of mucinase StcE (StcE<sup>E447D</sup>). The binding is specific because StcE<sup>E447D</sup> does not bind to the ultra-high-density display of the enantiomer control, L-galactose, nor to the diastereomer controls (D-glucose and D-mannose).</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"732 ","pages":"391-453"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148382307","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
Evolutionary genetic approaches to analyze mucins. 用进化遗传学方法分析粘蛋白。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-04-07 DOI: 10.1016/bs.mie.2026.03.009
Petar Pajic, Omer Gokcumen
{"title":"Evolutionary genetic approaches to analyze mucins.","authors":"Petar Pajic, Omer Gokcumen","doi":"10.1016/bs.mie.2026.03.009","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.03.009","url":null,"abstract":"<p><p>Mucins are heavily glycosylated proteins that form protective mucus barriers at host-environment interfaces. Mucin genes frequently contain exonic variable number tandem repeat (exVNTR) domains that encode peptides enriched in proline, threonine, and serine. These repeat domains create substantial challenges for comparative and population genetic analyses because short-read sequencing often collapses repeat arrays and obscures haplotype structure. Recent advances in long-read sequencing, pangenome resources, and specialized VNTR analysis tools now enable systematic investigation of mucin genetics and evolution. In this chapter, we present practical protocols for identifying candidate mucin genes across species, annotating mucin exVNTRs from long-read genome assemblies, and genotyping exVNTR alleles in large short-read sequencing cohorts. We further outline analytical strategies for evaluating natural selection acting on mucin repeat domains. Together, these protocols enable systematic identification, structural resolution, and evolutionary analysis of mucin variation across species and human populations.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"732 ","pages":"569-588"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148382311","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
Characterization of O-glycoproteases for applications in mass spectrometry. o型糖蛋白酶在质谱分析中的应用
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-04-02 DOI: 10.1016/bs.mie.2026.03.004
Alexandra D Steigmeyer, Joann Chongsaritsinsuk, Stacy A Malaker
{"title":"Characterization of O-glycoproteases for applications in mass spectrometry.","authors":"Alexandra D Steigmeyer, Joann Chongsaritsinsuk, Stacy A Malaker","doi":"10.1016/bs.mie.2026.03.004","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.03.004","url":null,"abstract":"<p><p>This chapter describes practical workflows for the characterization and application of O-glycoproteases and mucinases in mass spectrometry-based analysis of mucin-domain glycoproteins. Dense O-glycosylation limits the effectiveness of conventional proteases, making specialized enzymes essential for generating informative peptides. We outline a stepwise strategy that begins with rapid gel-based assays to determine enzyme activity, glycan dependence, and sensitivity to sialylation. These qualitative experiments identify suitable substrates and guide optimization of enzyme to substrate ratios and digestion conditions. We then detail preparation of mucin samples for LC-MS/MS, including optional glycosidase treatment, controlled proteolysis, cleanup, and acquisition parameters that enable confident identification of glycopeptides. Particular emphasis is placed on interpreting fragmentation data and combining collisional and electron-based dissociation to determine peptide sequence and glycosite localization. Because automated searches remain error prone for O-glycopeptides, we provide guidelines for manual validation and for constructing cleavage motifs that incorporate both amino acid and glycan preferences. Together, these approaches establish a general framework for defining enzyme specificity and selecting appropriate tools for complex biological samples. Comprehensive characterization of mucin-degrading enzymes expands the analytical toolbox for studying mucin biology and improves the ability to map site-specific O-glycosylation with molecular precision.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"732 ","pages":"31-63"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148382378","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
Lipid imaging using bifunctional lipid probes. 脂质成像使用双功能脂质探针。
4区 生物学
Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-01-21 DOI: 10.1016/bs.mie.2026.01.019
Martin Buitrago-Arango, Chia-Yi Chou, André Nadler
{"title":"Lipid imaging using bifunctional lipid probes.","authors":"Martin Buitrago-Arango, Chia-Yi Chou, André Nadler","doi":"10.1016/bs.mie.2026.01.019","DOIUrl":"10.1016/bs.mie.2026.01.019","url":null,"abstract":"<p><p>Mechanistic lipid cell biology requires techniques to faithfully image cellular lipid localization and transport in a species-specific manner. This is only partially possible by employing fluorescent lipid analogues and lipid biosensors, creating a need for more precise techniques. Here we outline techniques for using bifunctional (clickable and crosslinkable) lipid probes to visualize individual lipid species in cells and quantify their inter-organelle dynamics. We provide a detailed overview with regard to the nature of the chemical reporter groups, their effects on biophysical lipid properties, provide experimental details for sample preparation, fluorescence imaging, image analysis strategies and data interpretation. We outline pitfalls, limitations and best practices for control experiments. The lipid imaging strategy is described here for immortalized cell lines, but can easily be adapted for other tissue culture systems.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"17-45"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326671","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
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