Methods in enzymology最新文献_第9页

Computational tools in rational metalloenzyme design. 合理金属酶设计中的计算工具。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-16 DOI: 10.1016/bs.mie.2025.06.036

Mohd Taher, Shyamalava Mazumdar

{"title":"Computational tools in rational metalloenzyme design.","authors":"Mohd Taher, Shyamalava Mazumdar","doi":"10.1016/bs.mie.2025.06.036","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.06.036","url":null,"abstract":"Biocatalytic processes are by far provide the best eco-friendly and sustainable ways to synthesize fine chemicals. Metalloenzymes are known to catalyse chemical reactions with exceptionally high regio- and enantioselectivity and with faster reaction kinetics. Metalloenzymes become more desirable than conventional chemical catalysts for the chemical transformation of substrates having multiple functional groups. Due to their high selectivity, metalloenzymes perform chemical modification at a particular site without hampering other more reactive functional groups on the substrates. One limitation of metalloenzymes is that they show a narrow substrate range and often do not catalyse the transformation of non-native substrates. Recent advancements in the field of protein engineering, such as 'directed evolution' and 'rational enzyme design', have substantially expanded the substrate horizon of metalloenzymes. This article describes computationally assisted enzyme design to expand the substrate scope of metalloenzymes. We have taken Cytochrome P450 as a model enzyme to explain the tools frequently used in rational design. We have discussed multiple sequence alignment, tunnels and channels analysis and molecular docking with detailed protocols. This article serves as a step-by-step guide for students learning these concepts.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"720 ","pages":"239-264"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145232843","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

Development of a flow cytometric method to evaluate the impact of N-terminal sequences on protein stability. 开发一种流式细胞术方法来评估n端序列对蛋白质稳定性的影响。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-16 DOI: 10.1016/bs.mie.2025.06.034

Sabyasachi Sen, Khai Khee Kho, Aditya M Kunjapur

引用次数: 0

DEGRONOPEDIA: A practical guide to identifying and targeting protein degrons. DEGRONOPEDIA：鉴定和靶向蛋白质degron的实用指南。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-05 DOI: 10.1016/bs.mie.2025.06.014

Natalia A Szulc, Wojciech Pokrzywa

引用次数: 0

Fluorescence polarization binding assays for the E3 ligase FEM1C. E3连接酶FEM1C的荧光极化结合试验。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-11 DOI: 10.1016/bs.mie.2025.06.002

Emma K Seipp, Rong Huang

引用次数: 0

Characterization of the E3 ligase KCMF1 as a ZZ/N-recognin of the autophagic Arg/N-degron pathway. 表征E3连接酶KCMF1作为自噬Arg/N-degron途径的ZZ/ n -识别。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-07-07 DOI: 10.1016/bs.mie.2025.06.010

Ji Su Lee, Min Ju Lee, Su Bin Kim, Yong Tae Kwon, Chang Hoon Ji

{"title":"Characterization of the E3 ligase KCMF1 as a ZZ/N-recognin of the autophagic Arg/N-degron pathway.","authors":"Ji Su Lee, Min Ju Lee, Su Bin Kim, Yong Tae Kwon, Chang Hoon Ji","doi":"10.1016/bs.mie.2025.06.010","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.06.010","url":null,"abstract":"In the Arg/N-degron pathway, Arg/N-degrons share the N-terminal (Nt) arginine (Nt-Arg) residue and hydrophobic amino acid residues that can be generated through Nt-arginylation by ATE1-encoded R-transferases (EC 2.3.2). In the ubiquitin-proteasome system (UPS), N-degrons are recognized by the UBR box of N-recognins that facilitate ubiquitination and proteasomal degradation. Arg/N-degrons also modulate the lysosomal degradation of proteins and other biomaterials via the autophagy-lysosome system (ALS). In this autophagic process, N-degrons function through their recognition by the ZZ-type zinc finger domain of the N-recognin p62/SQSTM1-1/Sequestosome-1. Recently, we identified the E3 ligase KCMF1 (potassium channel modulatory factor 1) as an autophagic N-recognin at the crossroads of the UPS and ALS. KCMF1 binds Nt-Arg and structurally related Nt-motifs through its ZZ domain, a structural equivalent to the ZZ domain of p62 as well as the UBR box of N-recognins. Under oxidative stress such as prolonged hypoxia where protein aggregates accumulate, the cysteine (Cys) residue at position 2 is Nt-exposed through the Nt-methionine (Nt-Met) excision and undergoes chemical oxidation into Cys sulfonic acid (CysO3) followed by Nt-arginylation. The resulting Arg-CysO3 N-degron binds KCMF1 to induce the assembly of lysine 63 (Lys63)-linked Ub chains, to which p62-type autophagic receptors bind via their Ub-associated (UBA) domain for autophagic degradation. Through this collaboration between the UPS and ALS, Arg-CysO3 N-degrons contribute the degradation of harmful protein species generated under cellular stresses. Here, we describe biochemical assays to characterize KCMF1 as an emerging N-recognin, including its interaction with synthetic N-degrons and its activity to undergo self-polymerization stimulated by N-degrons.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"719 ","pages":"211-235"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145138186","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

Genetic analyses of Myc and hypusine circuits in tumorigenesis. 肿瘤发生中Myc和hypusine回路的遗传分析。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-21 DOI: 10.1016/bs.mie.2025.02.005

Shima Nakanishi, John L Cleveland

{"title":"Genetic analyses of Myc and hypusine circuits in tumorigenesis.","authors":"Shima Nakanishi, John L Cleveland","doi":"10.1016/bs.mie.2025.02.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.02.005","url":null,"abstract":"A prominent metabolic pathway induced by MYC family oncoproteins in cancer is the polyamine-hypusine circuit, which post-translationally modifies a specific lysine residue of eukaryotic translation initiation factor 5 A (eIF5A) with a unique amino acid coined hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. This modification occurs in a two-step process, whereby the aminobutyl group of the polyamine spermidine is covalently linked to lysine-50 of eIF5A via deoxyhypusine synthase (DHPS) to form the intermediate deoxyhypusinated eIF5A, which is subsequently hydoxylated by deoxyhypusine hydroxylase (DOHH) to form the fully mature eIF5AHyp. As a result, eIF5AHyp is elevated in MYC-driven cancers. Recently it has become evident that eIF5AHyp (i) plays key roles in the development, progression and maintenance of tumors; and (ii) eIF5AHyp functions are often tissue/cell context-specific. Thus, it is important to mechanistically assess how eIF5AHyp affects normal cells and tumorigenesis using suitable in vivo and ex vivo models. In this chapter, we describe the methods used in our laboratory to assess the effects of MYC-polyamine-hypusine axis on the development and maintenance of MYC-driven B-cell lymphoma. The goals of this chapter are twofold. First, we discuss genetic and cell biological approaches that can be applied to assess roles of eIF5AHyp on lymphoma and normal B cell development. Second, we discuss methods that can be used to assess the roles of eIF5AHyp on the growth and maintenance of lymphoma. Collectively, these approaches provide a template that can be applied to evaluate roles of any putative regulator of the development and/or maintenance of lymphoma.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"1-17"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094217","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 ODC activity and polyamines in Bachmann-Bupp syndrome patient biological samples. 监测巴赫曼-布普综合征患者生物样本中ODC活性和多胺。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-26 DOI: 10.1016/bs.mie.2025.01.071

Chad R Schultz, Elizabeth A VanSickle, Caleb P Bupp, André S Bachmann

{"title":"Monitoring ODC activity and polyamines in Bachmann-Bupp syndrome patient biological samples.","authors":"Chad R Schultz, Elizabeth A VanSickle, Caleb P Bupp, André S Bachmann","doi":"10.1016/bs.mie.2025.01.071","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.071","url":null,"abstract":"Polyamines are aliphatic molecules that include putrescine, spermidine, and spermine. Polyamines are present in most living organisms including humans. These positively charged molecules play important roles in cell physiology and pathology by contributing to embryonic cell development, regulation of cell division and, if overproduced, the stimulation of cancer cell proliferation and tumorigenesis. We recently discovered Bachmann-Bupp Syndrome (BABS); a rare neurodevelopmental disorder linked to de novo mutations in the ornithine decarboxylase 1 (ODC1) gene. ODC1 gene mutations that are linked to BABS always produce C-terminally truncated versions of the enzyme ornithine decarboxylase (ODC). These shortened ODC proteins remain enzymatically active and are not cleared by the proteasome, therefore leading to ODC protein accumulation in cells. ODC is a key enzyme of polyamine biosynthesis by converting ornithine to putrescine, and if accumulated, can lead to high putrescine levels in human cells including red blood cells (RBCs) and primary dermal fibroblasts. Here we describe how to quantitatively measure ODC enzymatic activity and the polyamines by a radiolabeled 14C-ornithine assay and by reverse phase (RP)-HPLC, respectively. While these methods have been developed decades ago, many publications provide incomplete protocols with omission of experimental details, which inadvertently can lead to mistakes, inconclusive results, and failed experiments. There is a growing number of laboratories that have become interested in exploring polyamines (in part due to metabolomics analyses in human health-related studies). The detailed protocols of this chapter provide step-by-step guidance detailing how to measure ODC activity and polyamines in human RBCs.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"257-270"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094311","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

Yeast reconstituted translation assays for analysis of eIF5A function. 酵母重组翻译法分析eIF5A功能。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-22 DOI: 10.1016/bs.mie.2025.01.076

Byung-Sik Shin, Thomas E Dever

{"title":"Yeast reconstituted translation assays for analysis of eIF5A function.","authors":"Byung-Sik Shin, Thomas E Dever","doi":"10.1016/bs.mie.2025.01.076","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.076","url":null,"abstract":"Polyamines are critically important for protein synthesis. Through their positive ionic charge, polyamines readily bind to ribosomes, as well as to mRNAs and tRNAs. Moreover, the polyamine spermidine serves as a substrate for the synthesis of hypusine, an essential post-translational modification on the translation factor eIF5A. Though originally thought to function in translation initiation, eIF5A is now known to generally promote translation elongation and termination. Moreover, translation of certain motifs like polyproline show a greater dependency on eIF5A. In this chapter, we describe the biochemical assays we use to study eIF5A and its regulation. Owing to the complex nature of protein synthesis, these assays require the purification of over 10 translation factors plus ribosomes, tRNAs, and aminoacyl-tRNA synthetases. We describe the methods used to purify these components, to synthesize the mRNA templates for translation, and to resolve the translation products by electrophoretic thin-layer chromatography. With the recent identification of eIF5A as a key target for regulating the synthesis of polyamine synthesis and transport, and the recent identification of mutations in eIF5A causing a neurodevelopmental disorder, the assays described in this chapter will be useful in further elucidating the function and regulation of this enigmatic protein.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"155-182"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094376","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

Collection, preparation, and biobanking of clinical specimens for analysis in polyaminopathies. 多胺病临床标本的收集、制备和生物库分析。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-18 DOI: 10.1016/bs.mie.2025.01.073

Elizabeth A VanSickle, Chad R Schultz, André S Bachmann, Caleb P Bupp

{"title":"Collection, preparation, and biobanking of clinical specimens for analysis in polyaminopathies.","authors":"Elizabeth A VanSickle, Chad R Schultz, André S Bachmann, Caleb P Bupp","doi":"10.1016/bs.mie.2025.01.073","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.073","url":null,"abstract":"Polyaminopathies are a relatively new family of rare genetic syndromes recently described in the literature. These syndromes are involved in the biosynthesis of polyamines, which include putrescine, spermidine, and spermine. Polyamines are aliphatic molecular that are found in most life forms, including humans, and are essential for embryogenesis, organogenesis, and tumorigenesis. The five known polyaminopathies that have been described to date include Snyder-Robinson Syndrome (SRS), Bachmann-Bupp Syndrome (BABS), Faundes-Banka Syndrome (FABAS), as well as neurodevelopmental disorders associated with variants in DHPS and DOHH. These syndromes share many overlapping clinical phenotypes, including developmental delay, hypotonia, and intellectual disability. Here we describe details for identifying and obtaining high-quality biological samples from patients with polyaminopathies. This includes special considerations for the informed consent process and the collection and shipment of biological samples for patients with rare diseases, many of whom live in countries around the world. We also detail the technical protocols for the collection, processing, storage, and tracking of biological samples for downstream research analysis specific to research in polyaminopathies, as well as biobanking for future use.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"309-321"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094388","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

Development and characterization of a Drosophila model of Snyder-Robinson syndrome. Snyder-Robinson综合征的果蝇模型的发展和特征。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-27 DOI: 10.1016/bs.mie.2025.01.077

Xianzun Tao, R Grace Zhai

引用次数: 0