Methods in enzymology最新文献_第10页

Purification of the lipid transfer protein tricalbin 3 in a native environment by using an amphiphilic copolymer. 用两亲性共聚物在天然环境中纯化脂质转移蛋白tricalbin 3。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-04 DOI: 10.1016/bs.mie.2025.11.020

Bingke Yang, Xin Bian

引用次数: 0

Synthesis and functional characterisation of small molecule activator of p300/CBP lysine acetyltransferase activity: Implications in spinal injury repair. p300/CBP赖氨酸乙酰转移酶活性小分子激活剂的合成和功能表征：在脊髓损伤修复中的意义。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-04 DOI: 10.1016/bs.mie.2026.05.027

Amrish Rai, Prajjval Mishra, Akash Kumar Singh, Priyanka Pandey, Tapas K Kundu

{"title":"Synthesis and functional characterisation of small molecule activator of p300/CBP lysine acetyltransferase activity: Implications in spinal injury repair.","authors":"Amrish Rai, Prajjval Mishra, Akash Kumar Singh, Priyanka Pandey, Tapas K Kundu","doi":"10.1016/bs.mie.2026.05.027","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.027","url":null,"abstract":"p300/CBP is a lysine acetyltransferase enzyme that modifies the lysine residue of histones by adding acetyl groups from acetyl-CoA. Dysregulation of p300/CBP KAT activity has been implicated in several neurological disorders. Pharmacological activation of p300/CBP via small-molecule provides a promising strategy to restore acetylation balance and promote neuronal repair. Our laboratory has reported that the p300/CBP-specific small molecule KAT activator TTK21 (N-[4-chloro-3-(trifluoromethyl)phenyl]-2-propoxy-benzamide), when conjugated to a glucose-derived carbon nanosphere (CSP), efficiently crossed the plasma membrane and induced acetylation. Surprisingly, its systemic administration in mice resulted in increased acetylation levels in the frontal cortex and hippocampus. Furthermore, it induced axon regeneration and improved functional recovery after spinal cord injury. Interestingly, oral administration of CSP-TTK21 also induced acetylation and promoted motor recovery in a rat model of spinal cord injury. In this chapter, we illustrate a method for the chemical synthesis of TTK21, glucose-derived carbon nanosphere (CSP), and their conjugate (CSP-TTK21). In addition, we systematically describe the methodologies for the chemical and biological characterization of the KAT activator, including the evaluation of histone acetylation in vitro and in vivo. Lastly, we present detailed experimental procedures to assess the therapeutic efficacy of CSP-TTK21 in models of spinal cord injury. Collectively, this chapter provides comprehensive protocols and practical guidance for the characterization and biological evaluation of p300/CBP activators for spinal cord injury repair.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"33-54"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795471","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

Analysis of copper toxicity by western blotting of secretory proteins translocated via the Sec61 channel. 通过Sec61通道转移的分泌蛋白的western印迹分析铜毒性。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-01 DOI: 10.1016/bs.mie.2026.05.028

Smriti Anand, Prateeksha Thakur, Vandana Anjana, Santoshi Acharjee, Rajshree Pal, Sugandh Sharma, Raghuvir Singh Tomar

{"title":"Analysis of copper toxicity by western blotting of secretory proteins translocated via the Sec61 channel.","authors":"Smriti Anand, Prateeksha Thakur, Vandana Anjana, Santoshi Acharjee, Rajshree Pal, Sugandh Sharma, Raghuvir Singh Tomar","doi":"10.1016/bs.mie.2026.05.028","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.028","url":null,"abstract":"As a trace element, copper plays a vital role in regulating cellular homeostasis by serving as a cofactor for various enzymes. Therefore, maintaining an adequate copper concentration inside the cell is crucial. Even moderate dysregulation of homeostasis can lead to cytotoxicity, protein aggregation, resulting in cell death. One of the significant cellular consequences in Saccharomyces cerevisiae is the inhibition of Sec61-mediated protein translocation in the secretory pathway, resulting in the accumulation of the cytosolic form of precursor secretory proteins, such as Gas1 and CPY, in the cytosol. The accumulation of unprocessed precursor proteins in the cytosol provides a method to read out translocon dysfunction. This chapter describes experimental approaches for analysing copper-induced translocation defects in yeast, with a primary focus on Western blot-based detection of precursor and mature secretory protein species. Given the high conservation of copper homeostasis and the secretory pathway across eukaryotes, these methods offer a robust framework for investigating the molecular mechanisms underlying copper toxicity and its impact on protein folding and maturation. This biochemical assay further enables the identification and characterization of chemical or genetic modulators that mitigate copper-induced translocation defects. Compounds that restore Sec61 function or improve ER targeting and maturation reduce the accumulation of precursor forms and promote the recovery of mature protein species. Thus, this approach provides a robust platform for assessing copper toxicity and for screening chemical agents that rescue ER translocation and protein maturation under metal stress conditions.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"253-278"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795481","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

Defining active and repressive chromatin states in neural crest cells using low-input CUT&RUN. 使用低输入CUT&RUN定义神经嵴细胞中活跃和抑制染色质状态。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-09 DOI: 10.1016/bs.mie.2026.05.048

Ayesha Nasreen, Sribas Chowdhury, Vivek T Natarajan

{"title":"Defining active and repressive chromatin states in neural crest cells using low-input CUT&RUN.","authors":"Ayesha Nasreen, Sribas Chowdhury, Vivek T Natarajan","doi":"10.1016/bs.mie.2026.05.048","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.048","url":null,"abstract":"The transition of neural crest cells (NCCs) from a multipotent state to lineage-restricted derivatives, including melanocytes, is governed by tightly regulated epigenetic mechanisms that orchestrate cell type specific gene expression programs. Histone post-translational modifications (PTMs), in particular, play an important role in modulating chromatin accessibility, enhancer activation, and transcription factor occupancy, thereby facilitating dynamic chromatin and transcriptional reprogramming required during development. However, profiling such chromatin states in rare and transient Neural Crest Cell (NCC) populations in vivo remains technically challenging. To address this, we present an optimized low-input Cleavage Under Targets and Release Using Nuclease (CUT&RUN) workflow tailored for fluorescence-activated cell sorting (FACS) isolated NCCs from zebrafish embryos. This approach enables high-resolution and low-background mapping of key histone modifications, including H3K27ac, H3K4me3, and H3K27me3, from limited cell numbers. Collectively, these methodologies provide a robust framework for dissecting chromatin state dynamics in developmental systems and can also offer insights into epigenetic dysregulation associated with disease.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"734 ","pages":"105-120"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148888051","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 short guide on in vivo transfection of flag-HDAC4 in dentate gyrus for study of adult neurogenesis in repetitive mild traumatic brain injury. 在齿状回体内转染flag-HDAC4用于重复性轻度创伤性脑损伤成人神经发生研究的简要指南。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-06 DOI: 10.1016/bs.mie.2026.05.058

Singh Ankit Satyaprakash, Nitesh Kumar Gupta, Humaira Farooq, Surya Pratap Singh, Rajaneesh Kumar Gupta

引用次数: 0

Bioorthogonal click-chemistry labeling and visualization of dual noncanonical amino acid-incorporated HIV-1 Env on intact virions. 完整病毒粒子上双非规范氨基酸结合HIV-1 Env的生物正交点击化学标记和可视化。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-27 DOI: 10.1016/bs.mie.2026.05.005

Narendra Kumar Gonepudi, Wang Xu, Harry Baffour Awuah, Maolin Lu

{"title":"Bioorthogonal click-chemistry labeling and visualization of dual noncanonical amino acid-incorporated HIV-1 Env on intact virions.","authors":"Narendra Kumar Gonepudi, Wang Xu, Harry Baffour Awuah, Maolin Lu","doi":"10.1016/bs.mie.2026.05.005","DOIUrl":"10.1016/bs.mie.2026.05.005","url":null,"abstract":"Site-specific fluorescent tagging enables visualization of protein conformational changes and molecular interactions on native biological assemblies. Using the HIV-1 envelope (Env) glycoprotein as an example, this protocol details a site-specific dual-fluorophore tagging strategy suitable for downstream single-molecule Förster resonance energy transfer (smFRET) studies. Genetic code expansion via amber (TAG stop codon) suppression, combined with click chemistry, offers a minimally invasive route to such labeling. However, the sequential incorporation of noncanonical amino acids (ncAAs) at two introduced amber codons often faces low efficiency and off-target readthrough at naturally occurring amber sites in the viral genome. To mitigate these technical constraints, the methods employ an engineered \"intact amber-free HIV-1 provirus\" in which endogenous TAG codons in essential viral genes are replaced with TAA. This design allows precise incorporation of ncAA trans-cyclooct-2-en-L-lysine (TCO*A) exclusively at two engineered amber sites within Env during virion production in HEK293T cells, followed by labeling via strain-promoted inverse electron-demand Diels-Alder cycloaddition with tetrazine-conjugated fluorophores. The method supports site-specific dual-click labeling for smFRET analysis and single-site labeling for live-cell imaging. The methods presented here provide stepwise procedures for click-chemistry labeling and visualization of Env on intact virions and can be easily adapted for single- or dual-site biorthogonal tagging of other viral and cellular proteins.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"167-191"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13305208/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148331081","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

Measurement of thiaminase I activity in biological samples: 4-nitrothiophenol (4-NTP) assays, optimization, and interpretation. 生物样品中硫胺酶I活性的测量：4-亚硝基苯酚（4-NTP）测定，优化和解释。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-07-24 DOI: 10.1016/bs.mie.2026.06.028

Katie A Edwards, Esther R Angert, Clifford E Kraft, Eileen A Randall, Patricia C Wolfe

{"title":"Measurement of thiaminase I activity in biological samples: 4-nitrothiophenol (4-NTP) assays, optimization, and interpretation.","authors":"Katie A Edwards, Esther R Angert, Clifford E Kraft, Eileen A Randall, Patricia C Wolfe","doi":"10.1016/bs.mie.2026.06.028","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.028","url":null,"abstract":"Thiaminase I enzymes catalyze the cleavage of thiamine (vitamin B1) and have been implicated in dietary and ecological thiamine deficiency syndromes affecting fish, wildlife, livestock, and other organisms. This chapter focuses on the colorimetric 4-nitrothiophenol (4-NTP) assay for thiaminase I activity and discusses alternative techniques. Protocols for sample collection, tissue homogenization, extraction, enzyme preservation, assay optimization, and data analysis are presented in a stepwise format. The application of the assay to bacterial culture supernatants, which offer valuable mechanistic insights at the microbial level, and to more complex tissue homogenates to better understand thiamine deficiency, is discussed. Factors influencing assay performance and interpretation, including endogenous thiamine carryover, endpoint versus kinetic measurements, and physiological relevance of measured in vitro activity, are considered. Emphasis is placed on data processing and normalization. Under the conditions employed, the assay has a limit of detection of 50 pmol 4-NTP degraded per minute, corresponding to 1 ng of thiaminase from Paenibacillus thiaminolyticus. The assay measures maximal catalytic capacity, though we recommend caution when translating thiaminase activities measured under substrate-saturating, elevated-temperature in vitro conditions using homogenized and de-compartmentalized samples to physiological thiamine degradation in vivo. In the latter, thiaminase-mediated thiamine degradation is constrained by substrate availability, an organism's body temperature, biochemical degradation processes, inhibitory factors, and physiological compartmentalization. Collectively, the approach and considerations presented here provide a framework for measuring and interpreting thiaminase I activity in complex biological matrices.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"257-299"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030760","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 structural dynamics of nascent bacterial riboswitches responding to TPP within transcriptional complexes. 在转录复合体中监测新生细菌核开关响应TPP的结构动力学。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-17 DOI: 10.1016/bs.mie.2026.05.043

Mirette Hashem, Patrick St-Pierre, Adrien Chauvier, J Carlos Penedo, Daniel A Lafontaine

{"title":"Monitoring structural dynamics of nascent bacterial riboswitches responding to TPP within transcriptional complexes.","authors":"Mirette Hashem, Patrick St-Pierre, Adrien Chauvier, J Carlos Penedo, Daniel A Lafontaine","doi":"10.1016/bs.mie.2026.05.043","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.043","url":null,"abstract":"This chapter describes a single-molecule fluorescence resonance energy transfer (smFRET) approach to monitor cotranscriptional RNA folding within native transcription complexes. The approach combines stepwise transcription by the Escherichia coli RNA polymerase (RNAP) with site-specific RNA labeling with a Cy3-Cy5 FRET pair via copper-free click chemistry. The chapter also details single molecule approaches to monitor the structural aspects of nascent transcripts and the analysis of observed RNA populations. Using the thiamin pyrophosphate (TPP)-sensing E. coli tbpA riboswitch as a model system, we detail procedures for DNA template preparation, RNA labeling and smFRET acquisition. Key advantages of the described smFRET approach include the use of native transcripts and the ability to monitor RNA structural changes within transcription complexes that are either stalled at naturally occurring pause sites or that are actively elongating along the DNA template. This method permits the study of cotranscriptional regulatory mechanisms taking place either in bacterial or eukaryotic elongation complexes and could be extrapolated to the use of any biochemical probes compatible with the click chemistry approach.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"465-491"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030768","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 metabolite-sensing riboswitches using in vivo reporter gene assays. 体内报告基因测定代谢物感应核蛋白开关的表征。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-09-01 DOI: 10.1016/bs.mie.2026.06.027

Anna Hübenthal, Matthias Mack

{"title":"Characterization of metabolite-sensing riboswitches using in vivo reporter gene assays.","authors":"Anna Hübenthal, Matthias Mack","doi":"10.1016/bs.mie.2026.06.027","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.06.027","url":null,"abstract":"Riboswitches are genetic regulatory elements based on RNA molecules that control the expression of genes. Most known metabolite-sensing riboswitches control expression of genes involved in anabolic reactions but a few also control catabolic reactions. In addition, riboswitches control the expression of genes encoding metabolite uptake systems. Binding of a specific small molecule ligand (<1500 Da) to the aptamer (\"binder\") part of the riboswitch typically results in repression of the downstream genes, in many cases by either transcriptional termination or inhibition of translation. The downstream expression control is supported by the so called \"expression platform\" which is part of the riboswitch. In this chapter the analysis of thiamine pyrophosphate (TPP)-responsive riboswitches using a specialized dual-luciferase reporter gene assay is exemplarily described and may serve as a blueprint to study other bacterial riboswitches. Also, the mode of action of the riboswitches can be determined by testing transcriptional as well as translational gene fusions using different test plasmids. The effect of candidate ligands which negatively interfere with riboswitch functions can be investigated using this assay, provided the chemicals are taken up by the test organism. The assay can be thus to identify and characterize novel compounds that have the capacity to act as antibiotics. As riboswitches constitute widespread genetic control elements, they represent novel targets for antimicrobial compounds.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"401-435"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030786","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

Mapping ligand-RNA interactions of thiamine analogues using transcriptome-wide sequencing. 利用转录组测序绘制硫胺素类似物的配体- rna相互作用。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-07-04 DOI: 10.1016/bs.mie.2026.06.022

Daphne A L van den Homberg, Stefan Crielaard, Willem A Velema

引用次数: 0