Methods in enzymology最新文献_第7页

Site-specific antibody labeling via endo-S2 mediated Fc glycan remodeling. 通过内切- s2介导的Fc聚糖重塑进行位点特异性抗体标记。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-25 DOI: 10.1016/bs.mie.2026.05.002

D Natasha Owitipana, Xianyang Wang, Xiao Zhang, Lai-Xi Wang

引用次数: 0

Generation and characterization of thiamin-biofortified plants. 生物强化硫胺素植物的产生与特性研究。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-06 DOI: 10.1016/bs.mie.2026.05.041

Aymeric Goyer, Keonny Cosek, David Handy

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Engineered nanopore biosensor for thiamine quantification: Data analysis. 工程纳米孔生物传感器硫胺素定量：数据分析。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-08-24 DOI: 10.1016/bs.mie.2026.07.020

Gerard Carrera I Cardona, Sofia B Relvas, Jan P Dobert, Carsten Wloka, Florian L R Lucas

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ThiN as a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. 作为古生菌硫胺素生物合成的转录抑制因子和催化酶的多功能结构域。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-18 DOI: 10.1016/bs.mie.2026.05.039

Nicholas Cox, Katherine R Weber, Julie A Maupin-Furlow

{"title":"ThiN as a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea.","authors":"Nicholas Cox, Katherine R Weber, Julie A Maupin-Furlow","doi":"10.1016/bs.mie.2026.05.039","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.039","url":null,"abstract":"Thiamine pyrophosphate (TPP), the active form of vitamin B1, is a cofactor required for catalyzing key reactions in carbohydrate and amino acid biosynthesis. TPP can be acquired through environmental salvage or de novo biosynthesis. Two separate pathways are used for the de novo biosynthesis of thiamine: one produces a sulfur-containing thiazole ring and the other a pyrimidine moiety, which are then joined and phosphorylated to form TPP. While the synthesis and regulation of thiamine are well characterized in certain bacteria, plants and fungi, these pathways are comparatively understudied in archaea. Recent work reveals archaeal thiamine metabolism to be chimeric, combining eukaryotic THI4-dependent thiazole formation with bacterial ThiC-mediated pyrimidine synthesis. Moreover, archaeal thiamine synthesis can be regulated by a ThiN-domain-dependent transcriptional repression mechanism distinct from riboswitch-based control. This chapter describes the experimental techniques that have been utilized for defining catalytic enzymes and transcriptional regulators of thiamine biosynthesis in archaea, using Haloferax volcanii as a model organism. Approaches include targeted gene deletion, complementation, site-directed mutagenesis (SDM), 3D-structural modeling, DNA regulatory motif identification, transcriptional reporter assays, and bioinformatic comparisons providing a versatile toolbox for functional and regulatory analysis of thiamine metabolism in extremophilic microorganisms.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"149-187"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030897","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

Protocol for the measurement of human brain transketolase activity. 人脑转酮醇酶活性测定规程。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-08-01 DOI: 10.1016/bs.mie.2026.07.012

Damon A Parkington, Albert Koulman, Sarah A Flowers, Gary E Gibson, Kerry S Jones

{"title":"Protocol for the measurement of human brain transketolase activity.","authors":"Damon A Parkington, Albert Koulman, Sarah A Flowers, Gary E Gibson, Kerry S Jones","doi":"10.1016/bs.mie.2026.07.012","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.07.012","url":null,"abstract":"Thiamine (vitamin B1) as thiamine diphosphate (or thiamine pyrophosphate) is a cofactor for enzymes involved in energy metabolism and is essential for brain function. Reliable biomarkers of thiamine status and function are necessary to understand thiamine's role in health and disease and provide mechanistic insights and to determine the efficacy of pharmacological interventions of thiamine analogues. The activity of the thiamine-dependent transketolase enzyme in erythrocytes is the established functional marker of whole-body thiamine status. In this chapter, we describe the analogous method optimized for the determination of transketolase activity in brain tissue and the calculation of the brain transketolase 'activity coefficient'. Provided are full method details for the preparation of brain material and instructions to perform the assay in 96-well plates using a microplate spectrophotometer. We provide details of suitable quality control materials and tips for troubleshooting. Instructions are also provided for the calculation of absolute basal transketolase activity. These protocols provide standardization for the determination of transketolase activity in human post mortem brain samples or from animal experiments and will be useful for researchers interested in the role of thiamine in brain-related conditions. Such protocols are essential to harmonize methods between laboratories for the benefit of comparisons between studies.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"735 ","pages":"217-238"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149030853","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

Metabolic labeling and targeting of dendritic cells. 树突状细胞的代谢标记和靶向。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-05-23 DOI: 10.1016/bs.mie.2026.05.016

Yusheng Liu, Hua Wang

{"title":"Metabolic labeling and targeting of dendritic cells.","authors":"Yusheng Liu, Hua Wang","doi":"10.1016/bs.mie.2026.05.016","DOIUrl":"10.1016/bs.mie.2026.05.016","url":null,"abstract":"Dendritic cells (DCs) play a central role in orchestrating immune responses, and methods that enable precise, modular, and non-invasive manipulation of DCs are essential for both mechanistic studies and translational applications. Here, we describe a protocol for the metabolic glycan labeling and bioorthogonal targeting of bone marrow-derived dendritic cells (BMDCs). This method leverages endogenous glycan biosynthetic pathways to incorporate azido-functionalized monosaccharides into DC surface glycans, followed by strain-promoted azide-alkyne cycloaddition (SPAAC) with diverse cargos conjugated with dibenzocyclooctyne (DBCO). The protocol details BMDC isolation and differentiation, optimization of azido-sugar labeling conditions, and subsequent in vitro and in vivo DBCO-cargo conjugation. Using flow cytometry and imaging-based validation, this approach enables efficient and reproducible surface functionalization of BMDCs while preserving cell viability and phenotype. The modular nature of the click chemistry step allows flexible attachment of fluorophores, proteins, or other functional molecules. Overall, this protocol provides a versatile platform for studying DC biology, enabling targeted modulation, imaging, and functional interrogation of DCs.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"731 ","pages":"285-300"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148331022","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

Synergetic inhibition of deubiquitinases and acetyltransferases to regulate tau acetylation and aggregation in SH-SY5Y cell line model of Alzheimer's disease. 去泛素酶和乙酰转移酶协同抑制阿尔茨海默病SH-SY5Y细胞系模型中tau乙酰化和聚集

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-06-06 DOI: 10.1016/bs.mie.2026.05.034

Basavaraju K C, Harshitha D K, Poornima Priyadarshini

{"title":"Synergetic inhibition of deubiquitinases and acetyltransferases to regulate tau acetylation and aggregation in SH-SY5Y cell line model of Alzheimer's disease.","authors":"Basavaraju K C, Harshitha D K, Poornima Priyadarshini","doi":"10.1016/bs.mie.2026.05.034","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.05.034","url":null,"abstract":"Tau protein homeostasis depends on a balance between ubiquitination and acetylation, two key post-translational modifications (PTMs) which simultaneously compete for lysine residues that govern tau stability. Importantly, ubiquitination signals of proteins for degradation through the 26S proteasome, whereas acetylation prevents ubiquitin attachment, leading to tau stabilization and an increased risk of aggregation. In Alzheimer's disease (AD), this balance is disrupted by upregulation of acetyltransferase activity, such as p300/CBP, and increased deubiquitinase (DUBs) activity, which together hinder proteasomal clearance and accumulation of pathological tau. Since these pathways are interconnected, targeting only one may be insufficient to address tau-mediated neurotoxicity. In this book chapter, we highlight the importance of a dual approach aimed at inhibiting both aberrant acetylation and deubiquitinases activity, thereby restoring ubiquitination and promoting tau degradation. In addition, the development of standardized cell culture models in the SH-SY5Y neuronal system will be crucial to evaluating the therapeutic potential of this synergetic inhibition strategy.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"733 ","pages":"297-319"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795486","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

Mechanistic insights on HDAC6 ZnF UBP-Tau interaction through biophysical and computational approaches. 通过生物物理和计算方法了解HDAC6 ZnF - UBP-Tau相互作用的机制。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2026-07-28 DOI: 10.1016/bs.mie.2026.07.009

Abhishek Ankur Balmik, Shweta Kishor Sonawane, Nalini Vijay Gorantla, Subashchandrabose Chinnathambi

{"title":"Mechanistic insights on HDAC6 ZnF UBP-Tau interaction through biophysical and computational approaches.","authors":"Abhishek Ankur Balmik, Shweta Kishor Sonawane, Nalini Vijay Gorantla, Subashchandrabose Chinnathambi","doi":"10.1016/bs.mie.2026.07.009","DOIUrl":"https://doi.org/10.1016/bs.mie.2026.07.009","url":null,"abstract":"Histone deacetylase 6 is a unique cytoplasmic deacetylase implicated in cellular functions such as microtubule dynamics, protein quality control, ubiquitin-mediated degradation and neurodegenerative disorders. The ZnF UBP (zinc finger ubiquitin binding protein) domain of HDAC6 is known to be directly modulate several cellular processes linked to neurodegeneration such as sequestering polyubiquitinated aggregates and regulating protein aggregate clearance mechanisms in neurons. Microtubule associated protein Tau (MAP Tau) undergoes aggregation in neurodegenerative conditions like Alzheimer's disease (AD) and several other tauopathies. Tau is a natively disordered protein which is functionally regulated by wide array of post-translational modifications (PTMs) as well as by interacting with several proteins. This methodological study aims to understand the molecular interaction between HDAC6 ZnF UBP domain and Tau protein in order to elucidate the role of HDAC6 ZnF UBP domain in Tau aggregation and stability. We employed an integrated biochemical, biophysical and computational workflow to characterize the interaction between HDAC6 ZnF UBP and Tau. NMR spectroscopy, isothermal titration calorimetry and pull-down assay with purified HDAC6 ZnF UBP and Tau proteins demonstrated direct interaction between the two, with interaction associated structural perturbations and favourable binding kinetics as observed in NMR and ITC respectively. Computational analyses further suggest the formation of Tau-HDAC6 ZnF UBP complex and provided underlying molecular interactions involved in the binding of these two proteins. The findings in this study helps to advance the current understanding of regulatory role of HDAC6 specifically in Tau biology and further provides a useful framework for investigating the modulation of aggregation prone proteins via protein-protein interaction in neurodegenerative diseases.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"734 ","pages":"79-103"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148888164","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

The ARTS toolset: Resistance-based genome mining for systematic prioritization of bioactive gene clusters. ARTS工具集：基于耐药性的基因组挖掘，对生物活性基因簇进行系统的优先排序。

4区生物学

Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-09-30 DOI: 10.1016/bs.mie.2025.08.023

Martina Adamek, Turgut Mesut Yılmaz, Semih Erdogmus, Sophie Moore, Nadine Ziemert

{"title":"The ARTS toolset: Resistance-based genome mining for systematic prioritization of bioactive gene clusters.","authors":"Martina Adamek, Turgut Mesut Yılmaz, Semih Erdogmus, Sophie Moore, Nadine Ziemert","doi":"10.1016/bs.mie.2025.08.023","DOIUrl":"10.1016/bs.mie.2025.08.023","url":null,"abstract":"Natural products, especially those produced by bacteria and fungi, have been a rich source of antibiotics and other medically important compounds. Advances in genome sequencing have revealed that many microorganisms harbor far more biosynthetic potential than previously known, but identifying which gene clusters are most likely to produce bioactive compounds remains a major challenge. One promising strategy is to look for genes that protect the producing organism from its own toxic products-so-called resistance genes-which often appear near the biosynthetic genes. In this chapter, we introduce the ARTS toolset, a collection of computational tools designed to identify such resistance-linked biosynthetic gene clusters in microbial genomes. ARTS 2.0 allows users to analyze bacterial genomes and metagenomes, ARTS-DB provides access to precomputed results from tens of thousands of genomes, and FunARTS adapts the approach for fungal genomes. We describe how each tool works and provide examples to guide their use, with additional online tutorial videos provided by the authors.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"730 ","pages":"35-60"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148005802","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

Use of small unilamellar vesicles to study the biochemical properties of the GRAM domain of GRAMD1. 利用小的单层囊泡研究了GRAMD1的GRAM结构域的生化特性。

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Methods in enzymology Pub Date : 2026-01-01 Epub Date: 2025-12-05 DOI: 10.1016/bs.mie.2025.11.014

Dylan Hong Zheng Koh, Yasunori Saheki

{"title":"Use of small unilamellar vesicles to study the biochemical properties of the GRAM domain of GRAMD1.","authors":"Dylan Hong Zheng Koh, Yasunori Saheki","doi":"10.1016/bs.mie.2025.11.014","DOIUrl":"10.1016/bs.mie.2025.11.014","url":null,"abstract":"Cellular membranes are composed of a diverse array of lipids, which are essential for maintaining membrane integrity and facilitating cellular signaling. While proteins often exhibit selective affinities for specific lipid species, the precise thresholds and determinants of these interactions need to be carefully studied to elucidate protein functions. Here, we employ an in vitro liposome-based assay to assess lipid-protein interactions using small unilamellar vesicles of defined lipid compositions. Specifically, we outline the methodology for purifying the GRAM domain of GRAMD1b/Aster-B protein, which has been previously characterized for its ability to detect accessible cholesterol and anionic lipids, including phosphatidylserine, at cellular membranes. We further detail the preparation of liposomes and demonstrate that the purified GRAM domain proteins bind preferentially to liposomes containing both cholesterol and anionic lipids, rather than to those containing either lipid alone, thereby confirming their role as co-incidence detectors. This finding highlights the utility of small unilamellar vesicles as a robust and versatile system to assess the lipid-binding preference of proteins, providing a complementary approach to cell-based assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"727 ","pages":"429-451"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326742","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