Methods in enzymology最新文献_第8页

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

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":"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.","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

引用次数: 0

Design of fusion proteins for biocatalysis. 生物催化融合蛋白的设计。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-12 DOI: 10.1016/bs.mie.2025.01.013

Beyzanur Celebi, Janina Lawniczek, David Angelo V Guanzon, Anna Christina R Ngo

{"title":"Design of fusion proteins for biocatalysis.","authors":"Beyzanur Celebi, Janina Lawniczek, David Angelo V Guanzon, Anna Christina R Ngo","doi":"10.1016/bs.mie.2025.01.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.013","url":null,"abstract":"The use of enzymes to convert substrates into valuable products has been an integral part of biocatalysis. However, some reactions are energy-demanding that requires the use of NAD(P)H to proceed. This NAD(P)H can be costly impeding the progress of enzyme usage at a bigger scale. The rise of sophisticated cloning methods has allowed the possibility of constructing multi-enzyme complexes such as coupling NAD(P)H-requiring enzymes with NADH-regeneration systems such as formate dehydrogenases. This allows a more-efficient way to recycle co-factors or co-substrates with cheaper sacrificial substrate such as formate for formate dehydrogenases or glucose for glucose dehydrogenases. However, the design of fusion proteins requires careful attention especially on the peptide linker that will be used to connect two protein domains. The length and the property of the linker and even the orientation of the genes encoding for the proteins in the open reading frame can significantly affect the outcome of the fusion protein. In this chapter, we present a step-by-step procedure for the design of a fusion protein construct via Gibson assembly and how to design linker libraries from one construct using site-directed mutagenesis.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"393-406"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972168","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

Tungsten containing aldehyde oxidoreductase (AOR)-family enzymes; past, present and future production strategies. 含钨醛氧化还原酶家族酶；过去、现在和未来的生产策略。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2025.01.027

Deborah M Boes, Rob A Schmitz, Peter-Leon Hagedoorn

{"title":"Tungsten containing aldehyde oxidoreductase (AOR)-family enzymes; past, present and future production strategies.","authors":"Deborah M Boes, Rob A Schmitz, Peter-Leon Hagedoorn","doi":"10.1016/bs.mie.2025.01.027","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.027","url":null,"abstract":"The transition metals tungsten and molybdenum are the heaviest metals found in biological systems and are embedded in the cofactor of several metalloenzymes. As a result of their redox activity, they provide great catalytic power in these enzymes and facilitate chemical reactions that would not occur using only the functionalities of natural amino acids. For their functionality these enzymes depend on a metal cofactor, which consists of at least one metal binding pterin (MPT) and a tungsten or molybdenum ion, but the complete make-up of the cofactor differs per enzyme group. One of these enzyme groups comprises the AOR-family enzymes. These enzymes have the ability to oxidize a range of aldehyde substrates into their corresponding carboxylic acid products. Next to this, they are also the only known catalysts able to perform the thermodynamically challenging reduction reaction of carboxylic acids to aldehydes. These enzymes are currently obtained by purification from the hyperthermophilic archaeon Pyrococcus furiosus. This process, however, does not yield a large amount of enzyme, since it is naturally expressed at moderate levels. For that reason, other production methods need to be considered if the enzyme is to be used on a large scale. These alternatives include the use of a recombinant expression system. The recombinant expression of W-dependent enzymes in different host organisms, such as Escherichia coli, has already been attempted for different enzymes, but with varying success. This shows that more research on the production, and especially incorporation of the metal cofactor, is necessary to achieve a successful production and use of recombinant AOR-family enzymes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"313-336"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143972172","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

Programmable C-to-U editing to track endogenous proteins. 可编程C-to-U编辑跟踪内源性蛋白质。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-04-02 DOI: 10.1016/bs.mie.2024.11.039

Min Hao, Tao Liu

引用次数: 0

Engineering unspecific peroxygenases by structure-guided in vivo recombination of homologous protein blocks. 通过结构引导同源蛋白块的体内重组工程非特异性过氧酶。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-06 DOI: 10.1016/bs.mie.2025.01.008

Alejandro Beltran-Nogal, Ivan Mateljak, David Gonzalez-Perez, Miguel Alcalde

引用次数: 0

EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts. EndoVIA用于量化A-to-I编辑和绘制编辑转录本的亚细胞定位。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.029

Alexandria L Quillin, Benoît Arnould, Steve D Knutson, Tatiana F Flores, Jennifer M Heemstra

{"title":"EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts.","authors":"Alexandria L Quillin, Benoît Arnould, Steve D Knutson, Tatiana F Flores, Jennifer M Heemstra","doi":"10.1016/bs.mie.2024.11.029","DOIUrl":"10.1016/bs.mie.2024.11.029","url":null,"abstract":"Adenosine-to-inosine (A-to-I) editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a prevalent post-transcriptional modification that is vital for numerous biological functions. Given that this modification impacts global gene expression, RNA localization, and innate cellular immunity, dysregulation of A-to-I editing has unsurprisingly been linked to a variety of cancers and other diseases. However, our current understanding of the underpinning mechanisms that connect dysregulated A-to-I editing and disease processes remains limited. Widely used methods require RNA extraction and pooling that ultimately erases subcellular localization and cell-to-cell variation, which may be critical to understanding misregulation. To overcome these challenges, we recently developed Endonuclease V Immunostaining Assay (EndoVIA) to selectively detect and visualize A-to-I edited RNA in situ. In this chapter, we describe in detail how to prepare cell samples, stain A-to-I edited transcripts with EndoVIA, quantify global inosine abundance, and visualize the subcellular localization of inosine-containing RNAs at the single molecule level.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"99-130"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908505/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052987","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