A novel fluorescent assay for ubiquitin isopeptide bond cleavage

描述（由申请人提供）：随着泛素和泛素样途径的重要性增加，对检测这些途径中涉及的酶活性的需求也在增加。目前，测量泛素和泛素样异肽酶活性的唯一高通量方法依赖于非生生性泛素偶联物。使用最广泛的是Ub-AMC。在这个实验中，泛素的C端融合成一个小的荧光团。在被泛素异肽酶切割后，荧光增强。这种分析格式不能代表生理事件，这可以解释为什么许多异肽酶不能切割这种缀合物。虽然有可能用生理底物（如市售的泛素链）通过western blotting来测量异肽酶活性，但这只是在测试少量样品时可行的选择。例如，为了筛选异肽酶抑制剂的小分子或天然产物，SDS-PAGE和western blotting是不可接受的方法。基于这些原因，我们建议开发一种新的测定泛素异肽酶活性的生理底物的方法。该检测将适用于高通量筛选，并且不会受到当前泛素异肽酶检测所共有的限制。简而言之，野生型泛素的c端与两种不同的泛素结合，这两种泛素仅含有赖氨酸48或赖氨酸63，可用于异肽键形成和荧光标记。在体外偶联野生型泛素，通过异肽键到另一个泛素产生二泛素，将包含优化的内部淬灭荧光对。我们将通过展示与赖氨酸48或63异肽键选择性去泛素化酶的裂解来确定新的二泛素底物的生理作用。利用生理底物定量测定泛素异肽酶活性的高通量测定方法的产生代表了这些关键细胞酶研究的重大进展。

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.