Methods in enzymology最新文献_第7页

Prime editing in bacteria with BacPE. 带BacPE的细菌启动编辑。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-20 DOI: 10.1016/bs.mie.2025.01.026

Hongyuan Zhang, Quanjiang Ji

引用次数: 0

In vitro functional analysis of plant tDRs. 植物tDRs的离体功能分析。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-03 DOI: 10.1016/bs.mie.2024.11.011

Christina Berrissou, Laurence Drouard

引用次数: 0

Genetic analyses of Myc and hypusine circuits in tumorigenesis. 肿瘤发生中Myc和hypusine回路的遗传分析。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-21 DOI: 10.1016/bs.mie.2025.02.005

Shima Nakanishi, John L Cleveland

{"title":"Genetic analyses of Myc and hypusine circuits in tumorigenesis.","authors":"Shima Nakanishi, John L Cleveland","doi":"10.1016/bs.mie.2025.02.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.02.005","url":null,"abstract":"A prominent metabolic pathway induced by MYC family oncoproteins in cancer is the polyamine-hypusine circuit, which post-translationally modifies a specific lysine residue of eukaryotic translation initiation factor 5 A (eIF5A) with a unique amino acid coined hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. This modification occurs in a two-step process, whereby the aminobutyl group of the polyamine spermidine is covalently linked to lysine-50 of eIF5A via deoxyhypusine synthase (DHPS) to form the intermediate deoxyhypusinated eIF5A, which is subsequently hydoxylated by deoxyhypusine hydroxylase (DOHH) to form the fully mature eIF5AHyp. As a result, eIF5AHyp is elevated in MYC-driven cancers. Recently it has become evident that eIF5AHyp (i) plays key roles in the development, progression and maintenance of tumors; and (ii) eIF5AHyp functions are often tissue/cell context-specific. Thus, it is important to mechanistically assess how eIF5AHyp affects normal cells and tumorigenesis using suitable in vivo and ex vivo models. In this chapter, we describe the methods used in our laboratory to assess the effects of MYC-polyamine-hypusine axis on the development and maintenance of MYC-driven B-cell lymphoma. The goals of this chapter are twofold. First, we discuss genetic and cell biological approaches that can be applied to assess roles of eIF5AHyp on lymphoma and normal B cell development. Second, we discuss methods that can be used to assess the roles of eIF5AHyp on the growth and maintenance of lymphoma. Collectively, these approaches provide a template that can be applied to evaluate roles of any putative regulator of the development and/or maintenance of lymphoma.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"1-17"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094217","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

Monitoring ODC activity and polyamines in Bachmann-Bupp syndrome patient biological samples. 监测巴赫曼-布普综合征患者生物样本中ODC活性和多胺。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-26 DOI: 10.1016/bs.mie.2025.01.071

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

{"title":"Monitoring ODC activity and polyamines in Bachmann-Bupp syndrome patient biological samples.","authors":"Chad R Schultz, Elizabeth A VanSickle, Caleb P Bupp, André S Bachmann","doi":"10.1016/bs.mie.2025.01.071","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.071","url":null,"abstract":"Polyamines are aliphatic molecules that include putrescine, spermidine, and spermine. Polyamines are present in most living organisms including humans. These positively charged molecules play important roles in cell physiology and pathology by contributing to embryonic cell development, regulation of cell division and, if overproduced, the stimulation of cancer cell proliferation and tumorigenesis. We recently discovered Bachmann-Bupp Syndrome (BABS); a rare neurodevelopmental disorder linked to de novo mutations in the ornithine decarboxylase 1 (ODC1) gene. ODC1 gene mutations that are linked to BABS always produce C-terminally truncated versions of the enzyme ornithine decarboxylase (ODC). These shortened ODC proteins remain enzymatically active and are not cleared by the proteasome, therefore leading to ODC protein accumulation in cells. ODC is a key enzyme of polyamine biosynthesis by converting ornithine to putrescine, and if accumulated, can lead to high putrescine levels in human cells including red blood cells (RBCs) and primary dermal fibroblasts. Here we describe how to quantitatively measure ODC enzymatic activity and the polyamines by a radiolabeled 14C-ornithine assay and by reverse phase (RP)-HPLC, respectively. While these methods have been developed decades ago, many publications provide incomplete protocols with omission of experimental details, which inadvertently can lead to mistakes, inconclusive results, and failed experiments. There is a growing number of laboratories that have become interested in exploring polyamines (in part due to metabolomics analyses in human health-related studies). The detailed protocols of this chapter provide step-by-step guidance detailing how to measure ODC activity and polyamines in human RBCs.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"257-270"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094311","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

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