A novel fluorescent assay for ubiquitin isopeptide bond cleavage

泛素异肽键裂解的新型荧光测定

基本信息

批准号：
8200131
负责人：
James Strickler
金额：
$ 47.27万
依托单位：
LIFESENSORS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-05 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8200131
关键词：
Accounting Alzheimer&apos s Disease Amides Amidohydrolases Applications Grants Autoimmune Diseases Autophagocytosis Biological Assay Biological Factors Biology Blast Cell C-terminal Cell physiology Chemicals Cleaved cell Communicable Diseases Development Disease Distal Drug Delivery Systems Dyes Enzymes Event Excision Exhibits Fluorescence Fluorescence Resonance Energy Transfer Fluorescent Dyes Genes Glycine Goals Grant Growth Half-Life Human Human Genome Hydrolase Hydrolysis In Vitro Individual Label Lead Link Lysine Mails Malignant Neoplasms Measures Methods Nature Pathway interactions Peptide Hydrolases Phase Phosphorylation Physiological Play Polyubiquitin Positioning Attribute Post-Translational Protein Processing Process Production Proteins Regulation Research Role Sales Sampling Screening procedure Series Signal Transduction Specificity Substrate Specificity System Technology Testing UBD protein Ubiquitin Ubiquitin C Ubiquitination Variant Western Blotting Work amino group assay development carboxylate cell growth regulation commercialization drug discovery enzyme activity fluorophore high throughput screening inhibitor/antagonist isopeptidase liquid chromatography mass spectrometry meetings multicatalytic endopeptidase complex novel protein degradation receptor recycling small molecule success ubiquitin-protein ligase web site

项目摘要

DESCRIPTION (provided by applicant): In the last decade there has been an explosive growth in the field of ubiquitin research, with approximately 530 human genes predicted to encode enzymes involved in the conjugation and deconjugation of ubiquitin. Of these 95 encode for deubiquitylases (DUBs). In order understand the biology of these enzymes better, there is a need for better assays to measure the most physiologically relevant activity of the enzymes. All the currently available high throughput methods for measuring deubiquitylase activity rely on C-terminal amidohydrolase activity (involved in processing the precursors of ubiquitin) rather than the isopeptidase activity involved in ubiquitin deconjugation (important in regulating various cellular processes) of DUBs. The most widely used substrate, Ub-AMC has a small fluorophore attached to the C-terminus of ubiquitin via an amide bond. Hydrolysis of this C-terminal amide bond by a deubiquitylase leads to an increase in fluorescence. This assay format does not adequately mimic the more important physiological event - deconjugation of ubiquitin via isopeptidase activity. Furthermore, many DUBs do not possess C-terminal amidohydrolytic activity and hence are unable to cleave conjugates like Ub-AMC. Although it is possible to measure isopeptidase activity with physiological substrates such as commercially available ubiquitin chains by SDS-PAGE, western blotting or LC/MS, such options are viable only if a small number of samples are being tested. For screening small molecules or natural products for inhibitors of isopeptidases these methods are unacceptable. In Phase I we developed a novel fluorescent assay for measuring the actual isopeptidase activity of the DUBs with substrates that are more relevant to physiological conditions. This assay is amenable to high throughput screening and does not suffer from the limitations shared by current DUB assays. Briefly, we have created a series of diUb molecules in which one Ub chain is derivatized with a fluorescence quenching dye and the second Ub moiety carrys a fluorophore. The two Ubs are joined by an isopeptide bond linking the C-terminus of one Ub to either Lys48 (K48) or Lys63 (K63) of the second Ub. Following hydrolysis of this isopeptide bond by a DUB, FRET-quenching is released and the resulting increase in fluorescence is directly proportional to DUB activity. We have validated these substrates using DUBs which only cleave the K48- or K63-linkage. In the current grant application, we propose to extend this technology to the remaining 5 Lys's in ubiquitin and create a panel of substrates encompassing all of ubiquitin isopeptide linkages. These substrates will greatly enhance our understanding of DUB activity and selectivity and enable high throughput screening campaigns using physiologically relevant substrates. 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 substrates that are physiologically relevant, the topic of this proposal, represents a major advancement in the study of this important group of cellular enzymes.

描述（由申请人提供）：在过去的十年中，泛素研究领域爆炸性增长，预计大约530种人体基因编码涉及泛素结合和解偶的酶。在这95个用于去渗酶（DUB）的编码中。为了更好地了解这些酶的生物学，需要更好的测定方法来测量酶最相关的活性。所有目前可用的高吞吐量方法用于测量去泛素地酶活性的所有依赖于C末端氨基氢化酶活性（涉及加工泛素的前体），而不是参与泛素解偶联偶联（在调节各种细胞过程中很重要）的异肽酶活性。 UB-AMC使用最广泛的底物，其荧光团通过酰胺键附着在泛素的C末端。通过去泛素地酶对这种C末端酰胺键的水解导致荧光增加。这种测定格式不能充分模仿更重要的生理事件 - 通过异肽酶活性对泛素的脱糖化。此外，许多配音都不具有C末端酰胺水解活性，因此无法像UB-AMC那样裂解结合物。尽管可以通过SDS-PAGE，Western blotting或LC/MS来测量生理底物（例如商业上泛素链）的生理底物，但仅当测试少量样品时才可行。为了筛选小分子或天然产物以用于异肽酶抑制剂，这些方法是不可接受的。在第一阶段，我们开发了一种新型的荧光测定，用于测量与生理条件更相关的底物的配音的实际异肽酶活性。该测定法适合高吞吐量筛选，并且不会遭受当前配音分析所共有的局限性。简而言之，我们创建了一系列DIUB分子，其中一个UB链被荧光淬灭染料衍生而来，第二个UB部分携带荧光团。这两个UB与第二个UB的Lys48（K48）或Lys63（K63）的IB的C末端连接在一起。通过DUB水解这种异肽键后，释放了鞭打液化，荧光的增加与DUB活性成正比。我们已经使用仅裂解K48或K63链接的DUB验证了这些底物。在当前的赠款应用程序中，我们建议将此技术扩展到泛素中的其余5个LYS，并创建一个包含所有泛素无菌链接的基板面板。这些底物将大大增强我们对DUB活动和选择性的理解，并使用与生理相关的底物实现高吞吐量筛查运动。公共卫生相关性：泛素修饰蛋白质在许多细胞过程中都起着重要作用。在过去的十年中，泛素研究领域爆炸性增长。从靶蛋白中去除泛素的酶是非常重要的药物靶标。需要更好的测定方法来测量酶的活性，这些酶高度具体且在生理上相关。使用生理上相关的底物开发测定，该提案的主题代表了对这一重要的细胞酶研究的主要进步。