Methods in enzymology最新文献_第6页

Preface. 前言。

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Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00134-X

引用次数: 0

Purification of endogenous tDRs by hybridization-based pulldown. 基于杂交的下拉法纯化内源性tDRs。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.019

Yoshika Takenaka, Nana Kunii, Yasutoshi Akiyama

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Self-quenched tRNA reporters for imaging tRNA-derived RNA biogenesis. 用于成像tRNA衍生RNA生物发生的自猝灭tRNA报告器。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-03 DOI: 10.1016/bs.mie.2024.11.025

Guoping Li, Saumya Das

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Detection of mitochondrial tDRs in killifish embryos and other non-model organisms. 鳉鱼胚胎和其他非模式生物线粒体tDRs的检测。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-06 DOI: 10.1016/bs.mie.2024.11.012

Claire L Riggs, Gazal Kalyan, Amie Lt Romney, Jason E Podrabsky

{"title":"Detection of mitochondrial tDRs in killifish embryos and other non-model organisms.","authors":"Claire L Riggs, Gazal Kalyan, Amie Lt Romney, Jason E Podrabsky","doi":"10.1016/bs.mie.2024.11.012","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.012","url":null,"abstract":"In recent years a diversity of small noncoding RNAs have been identified that originate from the mitochondrial genome. These mitosRNAs are often dominated by tRNA-derived small RNAs (mito-tDRs). Differential expression of mito-tDRs is associated with responses to stress. They also appear to be expressed differentially during development and their expression may be enriched in stress-tolerant animals. Very little is currently known about roles or modes of action of these sequences, although they are implicated in a diversity of processes such as cell cycle regulation, mRNA stability, regulation of ROS production, and import of proteins into the mitochondrion. To better understand the various roles these sequences may play, it is critical that we understand their diversity, cellular location, and the context for their expression. This protocol outlines the methodologies used to detect mitosRNAs, including mito-tDRs, in embryos and cells of the annual killifish Austrofundulus limnaeus. We highlight critical steps in the isolation of RNA, creation of sequencing libraries, bioinformatics processing of sequence data, and methods for validation of expression that support a robust discovery pipeline for mitosRNAs even from species with incomplete reference genome sequences.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"283-311"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425659","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}

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Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs. 利用内源性修饰的trna衍生rna测定小rna相互作用的蛋白质组。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-03 DOI: 10.1016/bs.mie.2024.11.005

Vera Oberbauer, Aleksej Drino, Matthias R Schaefer

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Functional analysis of tRNA-derived small translational regulation. trna衍生小翻译调控的功能分析。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-03 DOI: 10.1016/bs.mie.2024.11.017

Dongjin Kim, Hak Kyun Kim, Mark A Kay

{"title":"Functional analysis of tRNA-derived small translational regulation.","authors":"Dongjin Kim, Hak Kyun Kim, Mark A Kay","doi":"10.1016/bs.mie.2024.11.017","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.017","url":null,"abstract":"Transfer RNAs (tRNAs) are short non-coding RNA molecules that play a crucial role in protein synthesis by carrying amino acids to ribosomes during translation. tRNAs are highly conserved and abundant across species, with each type categorized based on its anticodon sequence. Although traditionally viewed as essential for protein synthesis, tRNAs have been found to have additional roles in cell proliferation, tumor metastasis, and neuronal homeostasis. In addition, tRNAs are cleaved by ribonucleases to produce smaller fragments. These fragments have previously been referred to as tRNA fragments (tRF RNAs) or tRNA-derived small RNAs (tsRNAs). More recently a nomenclature has been but forward for all tRNA derived RNAs referred to as tDRs. We will use tsRNA and tDR interterchangeably. The tDRs are processed at specific sites in tRNAs and can be differentially expressed in various tissues and diseases, indicating their potential as unique non-coding RNAs with specific functions. In a previous study, we identified a 3'tDR, which can regulate the translation of a target mRNA by altering its secondary structure. This chapter provides a detailed protocol to analyze the tDR-mediated translational regulation based on several molecular methods.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"336-355"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425678","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

Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis. 直接、集合 FRET 方法监测人类 DNA 聚合酶 δ 全酶组装和 DNA 合成启动的瞬态动力学。

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Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-28 DOI: 10.1016/bs.mie.2024.08.002

Jessica L Norris, Mark Hedglin

{"title":"Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.","authors":"Jessica L Norris, Mark Hedglin","doi":"10.1016/bs.mie.2024.08.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.08.002","url":null,"abstract":"In humans, DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand replication, the initiation of leading strand DNA replication as well as most of the major DNA damage repair pathways. In each of these contexts, pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process that involves the PCNA clamp loader, replication factor C and, depending on the DNA synthesis pathway, the major single strand DNA-binding protein complex, replication protein A (RPA). In a recent report from our laboratory, we designed and utilized direct, ensemble Förster Resonance Energy Transfer approaches to monitor the transient state kinetics of pol δ holoenzyme assembly and initiation of DNA synthesis on P/T junctions engaged by RPA. In this chapter, we detail the original approaches and discuss adaptations that can be utilized to monitor fast kinetic reactions in the millisecond (ms) timescale. All approaches described in this chapter utilize a commercially-available fluorescence spectrophotometer, can be readily evolved for alternative DNA polymerases and P/T DNA substrates, and permit incorporation of protein posttranslational modifications, accessory factors, DNA covalent modifications, accessory factors, enzymes, etc. Hence, these approaches are widely accessible and broadly applicable for characterizing DNA polymerase holoenzyme assembly and initiation of DNA synthesis during any PCNA-dependent DNA synthesis pathway.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"271-309"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400652","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

In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01. 荧光假单胞菌 IP01 的三组分 Rieske 加氧酶--积烯二加氧酶的体外分析。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-12 DOI: 10.1016/bs.mie.2024.05.013

Niels A W de Kok, Hui Miao, Sandy Schmidt

{"title":"In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01.","authors":"Niels A W de Kok, Hui Miao, Sandy Schmidt","doi":"10.1016/bs.mie.2024.05.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.013","url":null,"abstract":"Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"167-192"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291291","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

Purification of functional recombinant human mitochondrial Hsp60. 纯化功能性重组人线粒体 Hsp60。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-31 DOI: 10.1016/bs.mie.2024.07.049

Celeste Weiss, Alberto G Berruezo, Shaikhah Seraidy, Avital Parnas, Igor Tascón, Iban Ubarretxena-Belandia, Abdussalam Azem

{"title":"Purification of functional recombinant human mitochondrial Hsp60.","authors":"Celeste Weiss, Alberto G Berruezo, Shaikhah Seraidy, Avital Parnas, Igor Tascón, Iban Ubarretxena-Belandia, Abdussalam Azem","doi":"10.1016/bs.mie.2024.07.049","DOIUrl":"10.1016/bs.mie.2024.07.049","url":null,"abstract":"The mitochondrial 60 kDa heat shock protein (mHsp60) is an oligomeric, barrel-like structure that mediates protein folding in cooperation with its cochaperonin Hsp10, in an ATP-dependent manner. In contrast to the extremely stable oligomeric structure of the bacterial chaperonin, GroEL, the human mHsp60 exists in equilibrium between single and double heptameric units, which dissociate easily to inactive monomers under laboratory conditions. Consequently, purification and manipulation of active mHsp60 oligomers is not straightforward. In this manuscript, we present an improved protocol for the purification of functional mHsp60, following its expression in bacteria. This method is based upon a previously published strategy that exploits the notorious instability of mHsp60 to purify the monomeric form, which is subsequently reconstituted to functional oligomers under controlled conditions. In our protocol, we use affinity chromatography on a Ni NTA-agarose resin as the initial step, facilitating purification of substantial amounts of highly pure active protein. The resulting Hsp60 is suitable for both functional and structural analyses, including crystallography and electron cryo-microscopy (cryo-EM) studies, to obtain high resolution structures of the mHsp60 oligomers alone and in various complexes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"423-440"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564646","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

Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach. 揭示半胱胺二氧化酶的机制：HPLC-MS测定与金属置换相结合的方法。

4区生物学

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-22 DOI: 10.1016/bs.mie.2024.05.018

Ran Duan, Jiasong Li, Aimin Liu

{"title":"Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.","authors":"Ran Duan, Jiasong Li, Aimin Liu","doi":"10.1016/bs.mie.2024.05.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.05.018","url":null,"abstract":"Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"703 ","pages":"147-166"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291211","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