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.

中文翻译：

---

赖氨酸脱酰基酶 sirtuin 活性的生物发光测定


赖氨酸酰化可以指导蛋白质功能、定位和相互作用。Sirtuins 脱酰嗣赖氨酸以维持细胞稳态，它们的异常表达有助于多种疾病的发病机制，包括癌症。测量 sirtuins 的活性对于探索其作为治疗靶点的潜力至关重要，但准确定量具有挑战性。我们开发了“SIRTify”，这是一种用于测量体外和 体内 sirtuin 活性 的高灵敏度检测方法。SIRTify 基于 NanoLuc 荧光素酶的分裂版本，该酶由一个截短的、催化失活的 N 端部分 （LgBiT） 组成，该部分与高亲和力 C 端肽 （p86） 互补以形成活性荧光素酶。p86 内两个赖氨酸的酰化破坏了与 LgBiT 的结合并减轻了发光。去乙酰化酶的脱酰基可重建 p86 并恢复结合，产生与 sirtuin 活性成比例的发光信号。测量结果准确反映了报道的 sirtuin 对赖氨酸酰化的特异性，并证实了 sirtuin 调节剂的作用。SIRTify 可量化赖氨酸脱酰化动力学，并可能适用于监测其他翻译后修饰。