Methods in enzymology最新文献_第8页

Yeast reconstituted translation assays for analysis of eIF5A function. 酵母重组翻译法分析eIF5A功能。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-22 DOI: 10.1016/bs.mie.2025.01.076

Byung-Sik Shin, Thomas E Dever

{"title":"Yeast reconstituted translation assays for analysis of eIF5A function.","authors":"Byung-Sik Shin, Thomas E Dever","doi":"10.1016/bs.mie.2025.01.076","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.076","url":null,"abstract":"Polyamines are critically important for protein synthesis. Through their positive ionic charge, polyamines readily bind to ribosomes, as well as to mRNAs and tRNAs. Moreover, the polyamine spermidine serves as a substrate for the synthesis of hypusine, an essential post-translational modification on the translation factor eIF5A. Though originally thought to function in translation initiation, eIF5A is now known to generally promote translation elongation and termination. Moreover, translation of certain motifs like polyproline show a greater dependency on eIF5A. In this chapter, we describe the biochemical assays we use to study eIF5A and its regulation. Owing to the complex nature of protein synthesis, these assays require the purification of over 10 translation factors plus ribosomes, tRNAs, and aminoacyl-tRNA synthetases. We describe the methods used to purify these components, to synthesize the mRNA templates for translation, and to resolve the translation products by electrophoretic thin-layer chromatography. With the recent identification of eIF5A as a key target for regulating the synthesis of polyamine synthesis and transport, and the recent identification of mutations in eIF5A causing a neurodevelopmental disorder, the assays described in this chapter will be useful in further elucidating the function and regulation of this enigmatic protein.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"155-182"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094376","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

Collection, preparation, and biobanking of clinical specimens for analysis in polyaminopathies. 多胺病临床标本的收集、制备和生物库分析。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-18 DOI: 10.1016/bs.mie.2025.01.073

Elizabeth A VanSickle, Chad R Schultz, André S Bachmann, Caleb P Bupp

{"title":"Collection, preparation, and biobanking of clinical specimens for analysis in polyaminopathies.","authors":"Elizabeth A VanSickle, Chad R Schultz, André S Bachmann, Caleb P Bupp","doi":"10.1016/bs.mie.2025.01.073","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.073","url":null,"abstract":"Polyaminopathies are a relatively new family of rare genetic syndromes recently described in the literature. These syndromes are involved in the biosynthesis of polyamines, which include putrescine, spermidine, and spermine. Polyamines are aliphatic molecular that are found in most life forms, including humans, and are essential for embryogenesis, organogenesis, and tumorigenesis. The five known polyaminopathies that have been described to date include Snyder-Robinson Syndrome (SRS), Bachmann-Bupp Syndrome (BABS), Faundes-Banka Syndrome (FABAS), as well as neurodevelopmental disorders associated with variants in DHPS and DOHH. These syndromes share many overlapping clinical phenotypes, including developmental delay, hypotonia, and intellectual disability. Here we describe details for identifying and obtaining high-quality biological samples from patients with polyaminopathies. This includes special considerations for the informed consent process and the collection and shipment of biological samples for patients with rare diseases, many of whom live in countries around the world. We also detail the technical protocols for the collection, processing, storage, and tracking of biological samples for downstream research analysis specific to research in polyaminopathies, as well as biobanking for future use.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"309-321"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094388","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

Development and characterization of a Drosophila model of Snyder-Robinson syndrome. Snyder-Robinson综合征的果蝇模型的发展和特征。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-27 DOI: 10.1016/bs.mie.2025.01.077

Xianzun Tao, R Grace Zhai

引用次数: 0

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

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":"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.","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

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":"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.","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

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":"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'-tiRNAAla) 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'-tiRNAAla as an example, we analyze its effects on the integrity of the m7GTP (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.","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

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

引用次数: 0