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Direct measurement of ATP13A2 polyamine-dependent ATPase activity following rapid purification of lysosomes. 快速纯化溶酶体后，直接测量ATP13A2多胺依赖性atp酶活性。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-17 DOI: 10.1016/bs.mie.2025.01.069
Christina Efthymiou, Sydney Drury, Kenneth Lee
{"title":"Direct measurement of ATP13A2 polyamine-dependent ATPase activity following rapid purification of lysosomes.","authors":"Christina Efthymiou, Sydney Drury, Kenneth Lee","doi":"10.1016/bs.mie.2025.01.069","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.069","url":null,"abstract":"<p><p>The P5B family P-type ATPase ATP13A2(PARK9) is a bona fide polyamine transporter resident in the endolysosomal compartment where it mediates the import of endocytosed polyamines from the lysosome lumen into the cytosol. Dysfunction of ATP13A2 can negatively impact cellular survival and genetic aberrations its coding gene are linked to a number of neurodegenerative disorders with devastating consequences. While there has been much progress in its structural characterization in vitro, our understanding of ATP13A2's mechanism of action and regulation in a native lysosomal setting remains incomplete. Here we describe our approach to measure the polyamine-dependent ATPase activity of lysosomal ATP13A2 following our newly developed method to rapidly capture and purify lysosomes from mammalian cells. This strategy enables the targeted functional interrogation of the lysosome-localized population of ATP13A2 specifically.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"201-210"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094393","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
Feruloyl-CoA synthetases and feruloyl-CoA hydratase/lyases: Expression, biochemical characterisation, and generation of vanillin from ferulic acid and lignocellulosic hydrolysates. 阿魏酸辅酶a合成酶和阿魏酸辅酶a水合酶/裂解酶：从阿魏酸和木质纤维素水解物中表达、生化特性和生成香兰素。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-16 DOI: 10.1016/bs.mie.2025.01.046
Victoria Sodré, Fabio Marcio Squina
{"title":"Feruloyl-CoA synthetases and feruloyl-CoA hydratase/lyases: Expression, biochemical characterisation, and generation of vanillin from ferulic acid and lignocellulosic hydrolysates.","authors":"Victoria Sodré, Fabio Marcio Squina","doi":"10.1016/bs.mie.2025.01.046","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.046","url":null,"abstract":"<p><p>Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a valuable aroma and flavour compound with diverse applications in the food, fragrance, and pharmaceutical industries. Currently, the majority of vanillin produced globally is chemically synthesised from fossil-derived resources, but biocatalytic production from plant biomass offers a sustainable alternative. Alkaline pretreatment of grass-derived biomass releases ferulic acid, which can be converted into vanillin through a two-step biotransformation catalysed by feruloyl-CoA synthetases (FCSs) and feruloyl-CoA hydratase/lyases (FCHLs). This article presents detailed methodologies for the expression, purification, and biochemical characterisation of FCSs and FCHLs sourced from a lignin-degrading microbial consortium. Additionally, it describes protocols for preparing alkaline pretreatment hydrolysates from sugarcane bagasse and implementing the coupled FCS/FCHL reaction for vanillin synthesis. Analytical techniques for monitoring substrates and products are also discussed. These methods aim to support researchers in advancing the biocatalytic production of vanillin from renewable plant biomass.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"716 ","pages":"341-359"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144294059","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
Heterologous expression, in vitro refolding and steady-state kinetics of fungal aryl-alcohol oxidase. 真菌芳基醇氧化酶的异源表达、体外重折叠及稳态动力学研究。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-11 DOI: 10.1016/bs.mie.2025.03.001
Ana Serrano, Juan Carro
{"title":"Heterologous expression, in vitro refolding and steady-state kinetics of fungal aryl-alcohol oxidase.","authors":"Ana Serrano, Juan Carro","doi":"10.1016/bs.mie.2025.03.001","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.03.001","url":null,"abstract":"<p><p>Aryl-alcohol oxidase (AAO) is an FAD-dependent enzyme belonging to the glucose-methanol-choline oxidoreductase superfamily. AAOs are secreted by fungi and play a fundamental role as auxiliary enzymes in the lignocellulolytic process. On the one hand, they produce H<sub>2</sub>O<sub>2</sub> that activates peroxidases, which directly oxidize lignin, and triggers Fenton reactions to produce reactive oxygen species that attack lignin and carbohydrates. On the other, it is now known that they can also produce hydroquinones that promote hydroxyl radical formation to foster lignin decay. Genomic studies have revealed the significance of this class of enzymes for ligninolytic fungi, being produced by both white and brown-rot species. In this chapter, we deal with the methodology for the expression of AAO in the heterologous host E. coli as inclusion bodies, its subsequent in vitro refolding to produce active and soluble enzyme, as well as the estimation of their bi-substrate steady-state kinetic parameters.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"716 ","pages":"381-402"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144294087","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
Merging RODEO with genomic enzymology workflows to guide RiPP discovery. 将RODEO与基因组酶学工作流程合并，以指导RiPP的发现。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-16 DOI: 10.1016/bs.mie.2025.01.057
Graham A Hudson, Sangeetha Ramesh
{"title":"Merging RODEO with genomic enzymology workflows to guide RiPP discovery.","authors":"Graham A Hudson, Sangeetha Ramesh","doi":"10.1016/bs.mie.2025.01.057","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.057","url":null,"abstract":"<p><p>Natural products (NPs) have played a pivotal role in medicine, providing treatments that have saved or enhanced an incalculable number of lives. Traditionally, NPs were discovered using microbial extracts in conjunction with the bioactivity/phenotype-driven \"grind and find\" methodology. While initially fruitful during the \"golden age\" of NP discovery, it was quickly realized this method is fraught with drawbacks; namely, rediscovery of known/widely distributed secondary metabolites. Bioinformatics-guided discovery of novel NPs is a promising approach to obviate these drawbacks. Ribosomally synthesized and post-translationally modified peptide (RiPP) NPs are uniquely suited for a bioinformatics-driven strategy since RiPPs are biosynthesized from a ribosomally-encoded precursor peptide that is collocated with tailoring enzymes in biosynthetic gene clusters. This chapter details how the bioinformatics tool Rapid ORF Description and Evaluation Online (RODEO) can be united with genomic enzymology workflows to rapidly expand knowledge of RiPP NPs and details several case studies of how RODEO was used to explore a known class of RiPP, identify the origins of a novel post-translational modification, and discover the founding members of a new class of RiPP. While RODEO and the methods detailed herein are focused on RiPP NPs, they are largely translatable to other NP classes, and we anticipate these methods can be broadly useful to researchers in NP discovery.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"717 ","pages":"29-65"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619075","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
Recombinant expression, purification, and characterization of human ATE1 arginyltransferase. 人ATE1精氨酸转移酶的重组表达、纯化和特性研究。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-05 DOI: 10.1016/bs.mie.2025.06.020
Thilini Abeywansha, Abigail Kim, Sahil Bhaskaran, Yi Zhang
{"title":"Recombinant expression, purification, and characterization of human ATE1 arginyltransferase.","authors":"Thilini Abeywansha, Abigail Kim, Sahil Bhaskaran, Yi Zhang","doi":"10.1016/bs.mie.2025.06.020","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.06.020","url":null,"abstract":"<p><p>This chapter presents a straightforward method for expressing and purifying recombinant human Arginyl-tRNA-protein transferase 1 (ATE1) from E. coli. ATE1 is an enzyme that catalyzes the transfer of arginine from arginyl-tRNA to the N-terminal or internal Asp or Glu residues of the substrate proteins, a process that regulates protein turnover or function. The approach enables the efficient purification of milligram-scale quantities of highly soluble, enzymatically active ATE1 with over 98 % purity. Additionally, we describe the procedures for validating human ATE1 activity through an arginylation assay followed by mass spectrometry. We also describe the quantification of ATE1-tRNA binding affinity using Electrophoretic Mobility Shift Assay (EMSA).</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"718 ","pages":"283-294"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961129","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
A general framework to analyze potential roles of tDRs in mammalian protein synthesis. 分析tDRs在哺乳动物蛋白质合成中的潜在作用的一般框架。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-05 DOI: 10.1016/bs.mie.2024.11.018
Nupur Bhatter, Pavel Ivanov
{"title":"A general framework to analyze potential roles of tDRs in mammalian protein synthesis.","authors":"Nupur Bhatter, Pavel Ivanov","doi":"10.1016/bs.mie.2024.11.018","DOIUrl":"10.1016/bs.mie.2024.11.018","url":null,"abstract":"<p><p>tRNA-derived RNAs (tDRs) are a heterogeneous class of small non-coding RNAs that have been implicated in numerous biological processes including the regulation of mRNA translation. A subclass of tDRs called tRNA-derived stress-induced RNAs (tiRNAs) have been shown to participate in translational control under stress where specific tiRNAs repress protein synthesis. Here, we use a prototypical tiRNA (5'-tiRNA<sup>Ala</sup>) that inhibits mRNA translation in vitro and in cells as a model to study potential roles of tDRs in translational control. Specifically, we propose to use commercially available and custom-made in vitro translation systems together with sensitive luciferase-based mRNA reporters as well as transfection studies to determine potential effects of a given tDR on various aspects of protein synthesis. We overview methods to probe the capacity of specific tDRs to target specific steps of mRNA translation initiation, the most regulated step in translational control. Using 5'-tiRNA<sup>Ala</sup> as an example, we analyze its effects on the integrity of the m<sup>7</sup>GTP (cap)-bound eIF4F complex and phosphorylation of eIF2α, the key regulatory molecule of the Integrated Stress Response. Using transfection studies, we also monitor whether tDRs can promote formation of stress granules (SGs), RNA granules are often formed in response to global translation repression in live cells. This simple workflow offers fast, scalable, and reliable analyses of a potential involvement of specific tDRs in the modulation of protein synthesis and provides initial hints on molecular mechanisms that underline such mRNA translation regulation.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"29-46"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425700","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
Methods for Cas13a expression and purification for use in CRISPR diagnostics. 用于CRISPR诊断的Cas13a表达和纯化方法。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-12 DOI: 10.1016/bs.mie.2025.01.030
Her Xiang Chai, Rebecca S Bamert, Gavin J Knott
{"title":"Methods for Cas13a expression and purification for use in CRISPR diagnostics.","authors":"Her Xiang Chai, Rebecca S Bamert, Gavin J Knott","doi":"10.1016/bs.mie.2025.01.030","DOIUrl":"10.1016/bs.mie.2025.01.030","url":null,"abstract":"<p><p>The threat of emerging infectious diseases (e.g., SARS-CoV-2 the RNA virus responsible for the COVID-19 pandemic) has highlighted the importance of accurate and rapid testing for screening, patient diagnosis, and effective treatment of infectious disease. Nucleic acid diagnostic tools such as qPCR are considered the gold standard, providing a sensitive, accurate, and robust method of detection. However, these conventional diagnostic platforms are resource intensive, limited in some applications, and are almost always confined to laboratory settings. With the increasing demand for low-cost, rapid, and accurate point-of-care diagnostics, CRISPR-based systems have emerged as powerful tools to augment detection capabilities. Of note is the potent RNA detection enzyme, Leptotrichia buccalis (Lbu) Cas13a, which is capable of rapid RNA detection in complex mixtures with or without pre-amplification. To support its wide-spread use, we describe a detailed method for the expression, purification, and validation of LbuCas13a for use in molecular diagnostics.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"225-244"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692822","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
Measuring double-strand break repair events in mammalian cells with multi-target CRISPR. 用多靶点CRISPR测量哺乳动物细胞中的双链断裂修复事件。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-07 DOI: 10.1016/bs.mie.2025.01.011
Alberto Marin-Gonzalez, Adam T Rybczynski, Roger S Zou, Taekjip Ha
{"title":"Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.","authors":"Alberto Marin-Gonzalez, Adam T Rybczynski, Roger S Zou, Taekjip Ha","doi":"10.1016/bs.mie.2025.01.011","DOIUrl":"10.1016/bs.mie.2025.01.011","url":null,"abstract":"<p><p>A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"1-22"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692807","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
Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies. 通过动力学结构研究可视化CRISPR-Cas9的构象景观。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.004
Grace N Hibshman, David W Taylor
{"title":"Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.","authors":"Grace N Hibshman, David W Taylor","doi":"10.1016/bs.mie.2025.01.004","DOIUrl":"10.1016/bs.mie.2025.01.004","url":null,"abstract":"<p><p>CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"41-53"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692837","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
Cloning and validating systems for high throughput molecular recording. 克隆和验证系统的高通量分子记录。
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-09 DOI: 10.1016/bs.mie.2025.01.015
Anqi Zhao, Michelle M Chan
{"title":"Cloning and validating systems for high throughput molecular recording.","authors":"Anqi Zhao, Michelle M Chan","doi":"10.1016/bs.mie.2025.01.015","DOIUrl":"10.1016/bs.mie.2025.01.015","url":null,"abstract":"<p><p>Molecular recording technologies record and store information about cellular history. Lineage tracing is one form of molecular recording and produces information describing cellular trajectories during mammalian development, differentiation and maintenance of adult stem cell niches, and tumor evolution. Our molecular recorder technology utilizes CRISPR-Cas9 barcode editing to generate mutations in genomically integrated, engineered DNA cassettes, which are read out by single-cell RNA sequencing and used to produce high-resolution lineage trees. Here, we describe optimized cloning and validation procedures to construct the molecular recorder lineage tracing system. We include information on considerations of technology design, cloning procedures, the generation of lineage tracing cell lines, and time course experiments to assess their performance.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"453-473"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692757","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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