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Development of proteasome adaptors to catalytically deplete specific proteins from cells

开发蛋白酶体接头以催化消耗细胞中的特定蛋白质

基本信息

批准号：
9253359
负责人：
ANDREAS MATOUSCHEK
金额：
$ 18.63万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-04 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9253359
关键词：
Acceleration Adaptor Signaling Protein Adverse effects Affect Affinity Animal Model Antibodies Bcr-Abl tyrosine kinase Behavior Binding Biochemical Biochemistry Biological Assay Cancer cell line Carcinogens Cell Culture Techniques Cell Nucleus Cell Survival Cell model Cells Cellular Stress Characteristics Charge Chimeric Proteins Cytosol Development Drug Receptors Effectiveness Elements Engineering Eukaryotic Cell Fab Immunoglobulins Feeds Future Goals HIV Head Human Human Papillomavirus In Vitro Link Mediating Messenger RNA Methodology Methods Oncogenic Oncoproteins PSMD10 gene Pharmaceutical Preparations Population Positioning Attribute Post-Translational Protein Processing Primary Cell Cultures Process Proteasome Binding Protein Biosynthesis Proteins Proteolysis Proto-Oncogene Proteins c-myc RNA Interference RNA Processing Reaction Time Reagent Recombinant Antibody Research Research Personnel Resistance Signal Pathway Small RNA Specificity Substrate Domain System TP53 gene Tail Technology Testing Therapeutic Tumor Suppressor Proteins Ubiquitin Validation Viral Fusion Proteins Viral Oncogene Viral Proteins Work arm base design drug development experimental study feeding flexibility genetic regulatory protein molecular targeted therapies multicatalytic endopeptidase complex protein degradation public health relevance purge receptor response small hairpin RNA small molecule inhibitor therapeutic development therapeutic target tool ubiquitin ligase uptake

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a versatile tool to deplete specific proteins from the cytosol and nucleus of eukaryotic cells rapidl and effectively. The project will test the feasibility of a strategy based on proteasome adaptors, which we call degradons, built on a recombinant antibody platform. The proteasome adaptors or degradons will target proteins for destruction by the cellular Ubiquitin-Proteasome-System (UPS). Degradons should be useful as a research tool by allowing the researcher to remove specific proteins from cells to determine their function. They could also serve as a therapeutic approach to deplete toxic proteins such as oncogene products from cells. Degradons represents a new strategy to control cellular protein concentrations that is complementary to RNAi technology and could be used independently or in combination with RNAi approaches. Where RNAi interferes with protein synthesis, degradons induce protein destruction. Thus, degradons are not limited by natural protein turnover rates and can be specific for post-translationally modified forms of proteins. Degradon design builds on the detailed mechanistic understanding of proteasome biochemistry that has been developed over the past ten years and the maturation of experimental strategies to develop high affinity and specificity interaction agents, most importantly antibody technology. The degradon strategy is inspired by natural components of the UPS, the UbL-UBA proteins that serve as substrate adaptors, as well as viral proteins and oncogenes that act by subverting the UPS to purge tumor suppressor proteins from cells. Degradons will consist of two interaction domains, a proteasome binding domain derived from natural substrate receptors, viral proteins or oncogenes, and a target recognition domain, mostly derived from a recombinant antibody reagent. The two domains will be linked by a flexible, degradation-resistant arm. Degradons will position the bound target optimally for destruction but themselves escapes proteolysis to act catalytically. Degradons will be delivered to cells using methods developed for antibody-based drugs. We will test degradon design on three target proteins representing major classes of regulatory proteins and important oncogens. They are the kinase Bcr-Abl, the transcription factor Myc, and the ubiquitin ligase Mdm2, which controls p53 concentrations. Sets of systematically engineered adaptors will first be characterized biochemically in an in vitro degradation system and the best designs will then be optimized in mammalian culture cells and cancer cell lines. Degradons will be tested by themselves in comparison to RNAi and combination of RNAi and degradons will explore synergistic effects between both methods of protein depletion. Finally, delivery of degradons as a protein drug by receptor-mediated uptake will be tested. If degradons show promise as a protein depletion agent, future experiments will test their effectiveness in primary cell cultures and animal models.

描述（由申请人提供）：本项目的长期目标是开发一种多功能工具，以快速有效地从真核细胞的胞质溶胶和细胞核中去除特定蛋白质。该项目将测试基于蛋白酶体衔接子（我们称之为降解子）的策略的可行性，该策略建立在重组抗体平台上。蛋白酶体衔接子或降解子将靶向蛋白质以被细胞泛素-蛋白酶体系统（UPS）破坏。降解子应该是一种有用的研究工具，允许研究人员从细胞中去除特定的蛋白质以确定它们的功能。它们也可以作为一种治疗方法，从细胞中消耗有毒蛋白质，如致癌基因产物。降解子代表了一种控制细胞蛋白质浓度的新策略，它与RNAi技术互补，可以单独使用或与RNAi方法结合使用。当RNAi干扰蛋白质合成时，降解子诱导蛋白质破坏。因此，降解子不受天然蛋白质周转率的限制，并且可以对蛋白质的后修饰形式具有特异性。降解设计建立在对蛋白酶体生物化学的详细机制理解的基础上，该机制在过去十年中得到了发展，并且实验策略成熟，以开发高亲和力和特异性相互作用剂，最重要的是抗体技术。降解子策略的灵感来自UPS的天然组分，作为底物衔接子的UbL-UBA蛋白，以及通过破坏UPS来清除细胞中的肿瘤抑制蛋白的病毒蛋白和癌基因。降解子将由两个相互作用结构域组成，一个是源自天然底物受体、病毒蛋白或致癌基因的蛋白酶体结合结构域，另一个是主要源自重组抗体试剂的靶标识别结构域。两个结构域通过一个灵活的、抗降解的臂连接起来，降解子将结合的靶分子定位在最佳的位置，以便于破坏，但它们本身逃避蛋白水解而起催化作用。降解子将使用为基于抗体的药物开发的方法递送到细胞。我们将在代表主要调节蛋白和重要致癌基因的三种靶蛋白上测试降解子设计。它们是激酶Bcr-Abl、转录因子Myc和控制p53浓度的泛素连接酶Mdm 2。首先将在体外降解系统中对系统工程改造的衔接子进行生物化学表征，然后在哺乳动物培养细胞和癌细胞系中优化最佳设计。将单独测试降解子与RNAi的比较，RNAi和降解子的组合将探索两种蛋白质消耗方法之间的协同效应。最后，将测试通过受体介导的摄取作为蛋白质药物的降解物的递送。如果降解剂显示出作为蛋白质消耗剂的前景，未来的实验将在原代细胞培养和动物模型中测试其有效性。