Methods in enzymology最新文献_第2页

A fluorescence-based assay for measuring aminopropyltransferase activity. 一种基于荧光的测定氨基丙基转移酶活性的方法。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-11 DOI: 10.1016/bs.mie.2025.01.067

Pallavi Singh, Jae-Yeon Choi, Choukri Ben Mamoun

{"title":"A fluorescence-based assay for measuring aminopropyltransferase activity.","authors":"Pallavi Singh, Jae-Yeon Choi, Choukri Ben Mamoun","doi":"10.1016/bs.mie.2025.01.067","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.067","url":null,"abstract":"Polyamines (PAs) are small polycationic alkylamines that are essential for numerous cellular processes and found in all living cells. The three principal polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), have been shown to play crucial roles in cellular function and implicated in several diseases including infectious diseases, cancer and neurodegenerative disorders. As such, the enzymes involved in polyamine biosynthesis are promising targets for developing antimicrobial, antineoplastic and neuroprotective therapies. Aminopropyl transferases (APTs) are key enzymes in this pathway, catalyzing the formation of spermidine from putrescine and spermine from spermidine. While in most eukaryotes and prokaryotes, the spermidine synthase and spermine synthase activities are catalyzed by distinct enzymes, some organisms such as Plasmodium falciparum have a single enzyme, which catalyzes both reactions with varying efficiency. To date, efforts to inhibit APTs have focused primarily on substrate analogs, often with limited selectivity. A major challenge in discovering novel inhibitors has been the lack of an assay suitable for high-throughput chemical screening. We have recently developed DAB-APT, the first fluorescence-based assay for measuring APT activity, using 1,2-diacetyl benzene (DAB) which reacts with putrescine, spermidine, and spermine to form fluorescent conjugates, with fluorescence intensity correlating to carbon chain length. The DAB-APT assay has been validated using APT enzymes from Saccharomyces cerevisiae, and P. falciparum, and has been found to be suitable for high-throughput screening of large chemical libraries. This assay represents a significant advancement, offering a valuable tool for identifying potential inhibitors of APT enzymes and accelerating drug discovery efforts in cancer, neurobiology, and infectious diseases.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"363-388"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094329","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 bacterial transporters involved in the uptake of lignin-derived aromatic compounds. 参与木质素衍生芳香族化合物摄取的细菌转运体的表征。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-18 DOI: 10.1016/bs.mie.2025.01.053

Masaya Fujita, Naofumi Kamimura, Eiji Masai

引用次数: 0

Dye-decolorising peroxidase DyP1B from Pseudomonas fluorescens: Expression, reconstitution and reaction with polymeric lignin substrates. 荧光假单胞菌染料脱色过氧化物酶DyP1B：表达、重组和与聚合木质素底物的反应。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-09 DOI: 10.1016/bs.mie.2025.01.049

Rahman Rahmanpour, Timothy D H Bugg

引用次数: 0

Preface. 前言。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00184-3

引用次数: 0

Reductive amination: Methods for cell-free and whole-cell biocatalysis. 还原胺化：无细胞和全细胞生物催化的方法。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-09 DOI: 10.1016/bs.mie.2025.01.002

Vasilis Tseliou, Matteo Damian, Josemarco Mendoza-Avila, Marco Rabuffetti, Francesco G Mutti

引用次数: 0

How to engineer giant enzymes: A methodology for mutagenesis of polyketide synthases in native hosts. 如何设计巨型酶：原生宿主中聚酮合酶的诱变方法。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-07 DOI: 10.1016/bs.mie.2025.02.007

Susanna Kushnir, Uschi Hübner, Frank Schulz

引用次数: 0

Defining APOBEC-induced mutation signatures and modifying activities in yeast. 在酵母中定义apobecc诱导的突变特征和修饰活性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-02 DOI: 10.1016/bs.mie.2024.11.041

Tony M Mertz, Zachary W Kockler, Margo Coxon, Cameron Cordero, Atri K Raval, Alexander J Brown, Victoria Harcy, Dmitry A Gordenin, Steven A Roberts

{"title":"Defining APOBEC-induced mutation signatures and modifying activities in yeast.","authors":"Tony M Mertz, Zachary W Kockler, Margo Coxon, Cameron Cordero, Atri K Raval, Alexander J Brown, Victoria Harcy, Dmitry A Gordenin, Steven A Roberts","doi":"10.1016/bs.mie.2024.11.041","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.041","url":null,"abstract":"APOBEC cytidine deaminases guard cells in a variety of organisms from invading viruses and foreign nucleic acids. Recently, several human APOBECs have been implicated in mutating evolving cancer genomes. Expression of APOBEC3A and APOBEC3B in yeast allowed experimental derivation of the substitution patterns they cause in dividing cells, which provided critical links to these enzymes in the etiology of the COSMIC single base substitution (SBS) signatures 2 and 13 in human tumors. Additionally, the ability to scale yeast experiments to high-throughput screens allows use of this system to also investigate cellular pathways impacting the frequency of APOBEC-induced mutation. Here, we present validated methods utilizing yeast to determine APOBEC mutation signatures, genetic interactors, and chromosomal substrate preferences. These methods can be employed to assess the potential of other human APOBECs and APOBEC orthologs in different species to contribute to cancer genome evolution as well as define the pathways that protect the nuclear genome from inadvertent APOBEC activity during viral restriction.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"115-161"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144035720","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

cP-RNA-seq for tRNA half sequencing. cP-RNA-seq用于tRNA半测序。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.002

Megumi Shigematsu, Justin Gumas, Yohei Kirino

{"title":"cP-RNA-seq for tRNA half sequencing.","authors":"Megumi Shigematsu, Justin Gumas, Yohei Kirino","doi":"10.1016/bs.mie.2024.11.002","DOIUrl":"10.1016/bs.mie.2024.11.002","url":null,"abstract":"Although RNA-seq data are becoming more widely available for biomedical research, most datasets for short non-coding RNAs (sncRNAs) primarily focus on microRNA analysis using standard RNA-seq, which captures only sncRNAs with 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) ends. Standard RNA-seq fails to sequence sncRNAs with different terminal phosphate states, including tRNA halves, the most abundant class of tRNA-derived sncRNAs that play diverse roles in various biological processes. tRNA halves are produced through the endoribonucleolytic cleavage of mature tRNA anticodon loops. The responsible endoribonucleases, such as Angiogenin, commonly leave a 2',3'-cyclic phosphate (cP) at the 3'-end of 5'-tRNA halves and forms a 5'-OH end of 3'-tRNA halves, making them incompatible with standard RNA-seq. We developed a method named \"cP-RNA-seq\" that selectively amplifies and sequences tRNA halves and other cP-containing sncRNAs. Here we describe a detailed and recently updated cP-RNA-seq protocol. In this method, the 3'-end of all sncRNAs, except those containing a cP, are cleaved through periodate treatment after phosphatase treatment. Consequently, adaptor ligation and cDNA amplification steps are exclusively applied to cP-containing sncRNAs. Our cP-RNA-seq only requires commercially available reagents and is broadly applicable for the global identification of tRNA halves and other cP-containing sncRNA repertoires in various transcriptomes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"135-153"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11938272/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425655","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}

引用次数: 0

Assay for ribosome stimulation of angiogenin nuclease activity. 核糖体刺激血管生成素核酸酶活性的测定。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.007

Emily Sholi, Anna B Loveland, Andrei A Korostelev

{"title":"Assay for ribosome stimulation of angiogenin nuclease activity.","authors":"Emily Sholi, Anna B Loveland, Andrei A Korostelev","doi":"10.1016/bs.mie.2024.11.007","DOIUrl":"10.1016/bs.mie.2024.11.007","url":null,"abstract":"Angiogenin (RNase 5) is an unusual member of the RNase A family with very weak RNase activity and a preference for tRNA. The tRNAs cleaved by angiogenin are thought to have a variety of roles in cellular processes including translation reprogramming, apoptosis, angiogenesis, and neuroprotection. We recently demonstrated that angiogenin is potently activated by the cytoplasmic 80S ribosome. Angiogenin's binding to the ribosome rearranges the C-terminus of the protein, opening the active site for the cleavage of tRNA delivered to the ribosomal A site which angiogenin occupies. Here, we describe the biochemical procedure to test angiogenin's activation by the ribosome using the assay termed the Ribosome Stimulated Angiogenin Nuclease Assay (RiSANA). RiSANA can be used to test the activity of wild-type or mutant angiogenin, or other RNases, against different tRNAs and with different ribosome complexes. For example, given that angiogenin has been implicated in anti-microbial activity, we tested the ability of bacterial 70S ribosomes to stimulate angiogenin activity and found that the E. coli ribosome does not stimulate angiogenin. We also assayed whether angiogenin's closest homolog, RNase 4, could be stimulated by the ribosome, but unlike angiogenin this enzyme was not further activated by the ribosome. The RiSANA assay promises to reveal new aspects of angiogenin mechanism and may aid in the development of new diagnostic tools and therapeutics.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"381-404"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11839171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425704","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}

引用次数: 0

Analysis of metal-dependent DNA nicking activities by Cas endonucleases. 利用Cas内切酶分析金属依赖性DNA刻蚀活性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-07 DOI: 10.1016/bs.mie.2025.01.034

Giang T Nguyen, Akshara Raju, Dipali G Sashital

引用次数: 0