A novel fluorescent assay for ubiquitin isopeptide bond cleavage

描述(由申请人提供)：随着泛素和类泛素途径的重要性增加，对检测这些途径中涉及的酶的活性的需求越来越大。目前，唯一高通量测量泛素和泛素样异肽酶活性的方法依赖于非生理性泛素偶联物。使用最广泛的是Ub-AMC。在本实验中，泛素的C末端与一个小的荧光团融合。当被泛素异肽酶切割时，荧光增强。这种分析形式不代表生理事件，这可以解释为什么许多异肽酶不能裂解这种结合。虽然通过蛋白质印迹法可以用商业泛素链等生理底物来测量异肽酶的活性，但只有在测试少量样本的情况下，这才是一个可行的选择。例如，为了筛选小分子或天然产物中的异肽酶抑制剂，SDS-PAGE和Western blotting是不可接受的方法。出于这些原因，我们建议开发一种新的方法来测量具有生理底物的泛素异肽酶活性。该检测方法适用于高通量筛选，不会受到目前泛素异肽酶检测方法的限制。简而言之，野生型泛素的C末端与两种不同的泛素连接，这两种泛素只含有赖氨酸48或赖氨酸63，可用于异肽键的形成和荧光标记。在体外，野生型泛素通过异肽键与另一种泛素结合，得到双泛素，它将包含优化的内部猝灭荧光对。我们将通过展示赖氨酸48或63异肽键选择性脱泛素酶的裂解来确定新型双泛素底物的生理作用。利用生理底物高通量定量泛素异肽酶活性的方法的建立代表了这些关键细胞酶研究的重大进展。 与公共健康相关：泛素修饰蛋白质在许多细胞过程中起着重要作用。在过去的十年里，泛素研究领域取得了爆炸性的增长。从靶蛋白中去除泛素的酶是非常重要的药物靶点。需要更好的分析方法来测量这些酶的活性，这些酶具有高度的特异性和生理相关性。使用生理底物的分析方法的发展代表了对这一重要的细胞酶的研究的重大进展，也是这一提议的主题。

DESCRIPTION (provided by applicant): As the prominence of the ubiquitin and ubiquitin-like pathways increases the need for assays to measure the activity of the enzymes involved in these pathways grows. Currently the only high throughput methods for measuring ubiquitin and ubiquitin-like isopeptidase activity rely on non-physiological ubiquitin conjugates. The most widely used is Ub-AMC. In this assay the C- terminus of ubiquitin is fused to a small fluorophore. Upon cleavage by an ubiquitin isopeptidase there is an increase in fluorescence. This assay format does not represent a physiological event which may explain why many isopeptidases are unable to cleave this conjugate. Although it is possible to measure isopeptidase activity with physiological substrates such as commercially available ubiquitin chains by western blotting this is only a viable option if a small number of samples are being tested. For instance in order to screen small molecules or natural products for inhibitors of isopeptidases SDS-PAGE and western blotting are unacceptable methods. For these reasons we propose to develop a novel assay for measuring ubiquitin isopeptidase activity with physiological substrates. This assay will be amenable to high throughput screening and will not suffer from the limitations shared by current ubiquitin isopeptidase assays. Briefly, C-terminus of wild type ubiquitin is conjugated to two different ubiquitins, which contain only lysine 48 or lysine 63, available for isopeptide bond formation and fluorescent labeling. In vitro conjugation wild type ubiquitin, via an isopeptide bond to another ubiquitin results in di-ubiquitin that will contain optimized internally quenched fluorescent pairs. We will establish physiological role of the novel di-ubiquitin substrates by demonstrating cleavage with lysine 48 or 63 isopeptide bond selective de-ubiquitylases. The generation of high throughput assays for quantifying ubiquitin isopeptidase activity using physiological substrates represents a major advancement in the study of these crucial cellular enzymes. PUBLIC HEALTH RELEVANCE: Modification of proteins by ubiquitin plays important roles in many cellular processes. In the last decade there has been an explosive growth in the field of ubiquitin research. The enzymes that remove ubiquitin from target proteins are very important drug targets. There is a need for better assays to measure the activity of the enzymes, which are highly specific and physiologically relevant. The development of assays using physiological substrates represents a major advancement in the study of this important group of cellular enzymes and the topic of this proposal.