Alexandria N. Van Scoyk , Orlando Antelope , Donald E. Ayer , Randall T. Peterson , Anthony D. Pomicter , Shawn C. Owen , Michael W. Deininger
{"title":"赖氨酸脱乙酰酶 sirtuin 活性的生物发光测定","authors":"Alexandria N. Van Scoyk , Orlando Antelope , Donald E. Ayer , Randall T. Peterson , Anthony D. Pomicter , Shawn C. Owen , Michael W. Deininger","doi":"10.1016/j.chembiol.2024.10.006","DOIUrl":null,"url":null,"abstract":"<div><div>Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine toward maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple conditions, including cancer. Measuring sirtuins’ activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed “SIRT<em>ify</em>”, a high-sensitivity assay for measuring sirtuin activity <em>in vitro</em> and <em>in vivo</em>. SIRT<em>ify</em> is based on a split-version of the NanoLuc luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine-acylations and confirm the effects of sirtuin modulators. SIRT<em>ify</em> quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.</div></div>","PeriodicalId":265,"journal":{"name":"Cell Chemical Biology","volume":"31 11","pages":"Pages 2002-2014.e4"},"PeriodicalIF":6.6000,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Bioluminescence assay of lysine deacylase sirtuin activity\",\"authors\":\"Alexandria N. Van Scoyk , Orlando Antelope , Donald E. Ayer , Randall T. Peterson , Anthony D. Pomicter , Shawn C. Owen , Michael W. Deininger\",\"doi\":\"10.1016/j.chembiol.2024.10.006\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine toward maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple conditions, including cancer. Measuring sirtuins’ activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed “SIRT<em>ify</em>”, a high-sensitivity assay for measuring sirtuin activity <em>in vitro</em> and <em>in vivo</em>. SIRT<em>ify</em> is based on a split-version of the NanoLuc luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine-acylations and confirm the effects of sirtuin modulators. SIRT<em>ify</em> quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.</div></div>\",\"PeriodicalId\":265,\"journal\":{\"name\":\"Cell Chemical Biology\",\"volume\":\"31 11\",\"pages\":\"Pages 2002-2014.e4\"},\"PeriodicalIF\":6.6000,\"publicationDate\":\"2024-11-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cell Chemical Biology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2451945624004410\",\"RegionNum\":1,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell Chemical Biology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2451945624004410","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Bioluminescence assay of lysine deacylase sirtuin activity
Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine toward maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple conditions, including cancer. Measuring sirtuins’ activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed “SIRTify”, a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine-acylations and confirm the effects of sirtuin modulators. SIRTify quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.
Cell Chemical BiologyBiochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
14.70
自引率
2.30%
发文量
143
期刊介绍:
Cell Chemical Biology, a Cell Press journal established in 1994 as Chemistry & Biology, focuses on publishing crucial advances in chemical biology research with broad appeal to our diverse community, spanning basic scientists to clinicians. Pioneering investigations at the chemistry-biology interface, the journal fosters collaboration between these disciplines. We encourage submissions providing significant conceptual advancements of broad interest across chemical, biological, clinical, and related fields. Particularly sought are articles utilizing chemical tools to perturb, visualize, and measure biological systems, offering unique insights into molecular mechanisms, disease biology, and therapeutics.