Identification and characterization of linkage-specific ubiquitin binding element

连接特异性泛素结合元件的鉴定和表征

基本信息

批准号：
8394560
负责人：
James Strickler
金额：
$ 19.95万
依托单位：
LIFESENSORS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-21 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8394560
关键词：
Affect Affinity Alzheimer&apos s Disease Autoimmune Process Autophagocytosis Avidity Binding C-terminal Cells Chemicals Collaborations Communicable Diseases Complement Complex Mixtures Complication Consensus Coupled DNA Repair Detection Diagnostic Discrimination Disease Distal Elements Endocytosis Enzymes Event Excision Exhibits Fishes Generations Glycine Goals Grant Half-Life Human Human Genome Image Immune response Inflammation Knowledge Link Lysine Malignant Neoplasms Measures Mediating Modification Nature Nerve Degeneration Neurologic Oligonucleotides Pathway interactions Pharmaceutical Preparations Phase Phosphorylation Physiological Play Polyubiquitin Population Post-Translational Protein Processing Process Property Protein Binding Domain Protein Microchips Proteins Proteomics Reagent Recycling Regulation Relative (related person)Research Support Role Series Signal Transduction Specificity Staining method Stains System Time Ubiquitin Ubiquitin-Conjugating Enzymes Ubiquitination amino group carboxylate cell growth regulation drug discovery glycosylation isopeptidase meetings multicatalytic endopeptidase complex nervous system disorder novel protein complex protein degradation protein function protein transport receptor receptor recycling success tool ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Identification, quantification, and isolation of low abundance proteins from complex mixtures is, at best, a difficult task. This is especially the case for proteins carrying a post-translational modification (PTM) that affects their half-life or regulatory properties. Examples of such PTMs include phosphorylation, glycosylation (especially O-GlcNAcylation), and ubiquitinylation. In many instances, one PTM can abrogate or enhance other PTMs on the same protein providing exquisite mechanisms for control of the protein's activity. "Fishing" a protein with one particular PTM out of the pool of possible modifie proteins becomes nearly impossible without selective tools. A further complication in the case of ubiquitinylation is the presence of multiple types of Ub-Ub linkages in polyubiquitin chains. Ubiquitin (Ub) is attached, via isopeptide bonds, to lysine residues in the target protein. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs, again through the formation of isopeptide bonds between the C- terminus of one Ub and any of seven (7) lysines in the target Ub. The general consensus in the field is that chains with different linkages convey different meanings to the cell and hence, determine the ultimate fate of the protein, be it degradation, translocation, phosphorylation, etc. The precise information encoded in different chain linkages is largely unknown due to the lack of specific reagents that recognize different linkages. The goal of this proposal is to develop tools that allow the selective identification, quantification, and isolation of proteins modified by polyubiquitin chains containing different linkages. This will be accomplished using information encoded in the human genome that allows the cell to discriminate between different linkages, i.e. Ub-binding domains (UbDs). In Phase I, we will identify and characterize novel UbDs exhibiting, at least, partial selectivity. In Phase II we will use these UbDs to construct higher avidity reagents capable of linkage-specific discrimination. Both ubiquitinylation and de- ubiquitinylation have been linked to cancer, inflammation and neurological diseases; hence, the tools developed in this grant will have a major impact on our ability to dissect these disease processes. PUBLIC HEALTH RELEVANCE: Ubiquitinylation and de-ubiquitinylation are post-translational modifications that affect the function of many proteins and whose dysregulation has been implicated in many disease processes from cancer to inflammation to neurological degeneration. Study of these modifications is difficult due to their low abundance and the complexity of polyubiquitin chains. This grant proposes to generate specific reagents that will distinguish between different forms of polyubiquitin, enabling their detection and isolation from complex mixtures. Use of these tools will dramatically enhance our understanding of many diseases.

描述（由申请人提供）：从复杂混合物中鉴定、定量和分离低丰度蛋白质充其量是一项艰巨的任务。尤其是这种情况用于携带影响其半衰期或调节特性的翻译后修饰 (PTM) 的蛋白质。此类 PTM 的实例包括磷酸化、糖基化（尤其是 O-GlcNA 酰化）和泛素化。在许多情况下，一种 PTM 可以消除或增强同一蛋白质上的其他 PTM，从而提供控制蛋白质活性的精细机制。如果没有选择性工具，从可能的修饰蛋白质库中“钓取”具有特定 PTM 的蛋白质几乎是不可能的。泛素化的另一个复杂之处是多泛素链中存在多种类型的 Ub-Ub 连接。泛素 (Ub) 通过异肽键连接至靶蛋白中的赖氨酸残基。然后，这些 Ub 部分可以再次通过在一个 Ub 的 C 末端与目标 Ub 中的七 (7) 个赖氨酸中的任何一个之间形成异肽键，作为额外 Ub 缀合的底物。该领域的普遍共识是，具有不同链接的链条向细胞传达不同的含义，从而决定蛋白质的最终命运，无论是降解、易位、磷酸化等。由于缺乏识别不同连接的特定试剂，不同链连接中编码的精确信息在很大程度上是未知的。该提案的目标是开发能够选择性鉴定、定量和分离由含有不同连接的多聚泛素链修饰的蛋白质的工具。这将利用人类基因组中编码的信息来实现，该信息允许细胞区分不同的连接，即 Ub 结合域 (UbD)。在第一阶段，我们将识别和表征至少表现出部分选择性的新型 UbD。在第二阶段，我们将使用这些 UbD 构建能够进行连锁特异性区分的更高亲合力试剂。泛素化和去泛素化都与癌症、炎症和神经系统疾病有关；因此，这笔赠款中开发的工具将对我们剖析这些疾病过程的能力产生重大影响。公共健康相关性：泛素化和去泛素化是影响许多蛋白质功能的翻译后修饰，其失调与从癌症到炎症再到神经退行性疾病的许多疾病过程有关。由于多泛素链的丰度低且复杂，对这些修饰的研究很困难。该资助计划生产能够区分不同形式的多聚泛素的特定试剂，从而能够从复杂的混合物中检测和分离它们。使用这些工具将极大地增强我们对许多疾病的了解。