Recognition of non-ubiquitin signals at the proteasome

蛋白酶体上非泛素信号的识别

• 批准号: 8187399
• 负责人: DAVID FUSHMAN
• 金额: $ 35.81万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8187399
• 关键词: ATP-Dependent Proteases; Address; Affinity; Architecture; Attitude; Binding; Binding Sites; Biochemical; Biological; Cell Survival; Cell physiology; Cells; Complement; Complex; Coupling; Defect; Degradation Pathway; Disease; Dose; Drug Design; Employee Strikes; Eukaryotic Cell; Event; Goals; Immune; In Vitro; Inflammatory; Knowledge; Ligands; Malignant Neoplasms; Maps; Mediating; Molecular; Mono-S; Mutation; Myopathy; Nature; Neurodegenerative Disorders; Outcome; Pathway interactions; Pharmaceutical Preparations; Polyubiquitin; Process; Property; Proteasome Binding; Proteasome Inhibition; Proteins; Proteome; Proteomics; Regulation; Relative (related person); Research; Research Proposals; Resolution; Role; Signal Pathway; Signal Transduction; Site; Structure; Structure-Activity Relationship; Surface; Testing; Ubiquitin; Ubiquitin Like Proteins; base; cancer cell; design; developmental disease; multicatalytic endopeptidase complex; mutant; nanomachine; protein B; protein complex; receptor; scaffold; stoichiometry; three dimensional structure; wasting

DESCRIPTION (provided by applicant): The proteasome is one of the largest protein complexes to be found in cells. This multi-subunit ATP-dependent protease controls numerous cellular processes by degrading carefully selected targets. Proteasome function is essential for all eukaryotic cells. Defects in the proteasomal degradation pathway are associated with various diseases, including cancers, neurodegenerative disorders, developmental disorders, immune and inflammatory disorders, and muscle wasting. How proteins destined for degradation are recognized by the proteasome and then degraded is only partially understood. Precise knowledge of proteasome subunit architecture, their interactions with each other, and associations with transient auxiliary factors is critical if we are to understand its mode of action. Such information would also serve as the basis for ongoing drug design efforts. Targeting of polyubiquitinated substrates to the proteasome is mediated by several shuttle proteins (e.g., Dsk2, Rad23, Ddi1), all of which contain a ubiquitin-like (UBL) domain that interacts directly with several proteasomal subunits. Of these subunits, the primary UBL-receptor is apparently Rpn1. The goal of the proposed research is to understand how the proteasome differentiates between ubiquitin and ubiquitin-like signals. We will do so by characterizing, at the biochemical and structural levels, the nature and the strength of interactions involved in recognition of the UBL signals by the proteasome. We will map UBL-binding regions on Rpn1, determine the three-dimensional structure of specialized UBL-receptor regions at an atomic level resolution and characterize the nature and the strength of these interactions both structurally and biochemically. We will compare recognition of UBLs to understand how they differ among themselves, and how as a group they pose a distinct signal from ubiquitin which binds to designated receptors. We will address the ability of the proteasome to differentiate between ubiquitin and ubiquitin-like proteins, and the apparent redundancy in signaling by polyubiquitin and UBLs. All structural predictions and pairs-wise interactions will be verified through biological manipulations and proteomic screens. We will complement the in vitro characterization with verification of interactions in cells, test the outcome of mutants designed to abolish or strengthen pair-wise interactions that we identify, alter hierarchy of proteasome targeting, and look at the broad proteomic role of each shuttle, UBL signal, or receptor individually. This project relies on a variety of experimental approaches (biochemical, biophysical, structural (NMR), and proteomic) to reveal the structure-function relationship of the proteasome with its substrates, targets, and signals. These studies will provide a detailed picture of molecular events involved in the recognition of the UBL signals by the proteasome, triggering subsequent events at the proteasome leading to substrate degradation. PUBLIC HEALTH RELEVANCE: The ubiquitin-proteasome proteolytic pathway is the principal regulatory mechanism that influences a variety of vital cellular events by degrading carefully selected targets. Defects in proteasomal degradation are associated with a variety of diseases, including cancers, neurodegenerative disorders, developmental disorders, immune and inflammatory disorders, and muscle wasting. This research will extend our understanding of the molecular mechanisms of recognition and regulation in this pathway which is the target for extensive drug design efforts.

描述（由申请人提供）：蛋白酶体是细胞中最大的蛋白质复合物之一。这种多亚基ATP依赖性蛋白酶通过降低精心选择的目标来控制许多细胞过程。蛋白酶体功能对于所有真核细胞都是必不可少的。蛋白酶体降解途径的缺陷与各种疾病有关，包括癌症，神经退行性疾病，发育障碍，免疫和炎症性疾病以及肌肉浪费。只有部分理解蛋白酶体并降解的蛋白质如何被蛋白酶体识别。如果我们要了解其作用方式，那么对蛋白酶体亚基结构，它们相互相互作用以及与瞬态辅助因素的相互作用以及与瞬态辅助因素的关联至关重要。这些信息还将作为正在进行的药物设计工作的基础。靶向多泛素化的底物对蛋白酶体的靶向是由几种穿梭蛋白（例如DSK2，RAD23，DDI1）介导的，所有蛋白质都包含一个直接与多个蛋白酶体亚基相互作用的泛素样（UBL）结构域。在这些亚基中，主要的UBL受体显然是RPN1。拟议的研究的目的是了解蛋白酶体如何区分泛素和泛素样信号。我们将通过在生化和结构水平上表征蛋白酶体识别UBL信号所涉及的相互作用的性质和强度来做到这一点。我们将在RPN1上绘制UBL结合区域，确定原子水平分辨率的专用UBL受体区域的三维结构，并在结构和生化上表征这些相互作用的性质和强度。我们将比较对UBL的识别，以了解它们之间的不同之处，以及作为一组群体如何与泛素产生与指定受体结合的明显信号。我们将解决蛋白酶体分化泛素和泛素样蛋白的能力，以及通过多泛素和UBL的信号传导的明显冗余。所有结构预测和对的相互作用将通过生物操纵和蛋白质组学筛选来验证。我们将通过对细胞中相互作用的验证进行补充体外表征，测试旨在废除或增强我们识别的成对相互作用的突变体的结果，这些突变体我们识别，改变蛋白酶体靶向的层次结构，并查看每种途径，UBL信号或单独受体的宽阔蛋白质组织作用。该项目依靠多种实验方法（生化，生物物理，结构（NMR）和蛋白质组学）来揭示蛋白酶体与其底物，靶标和信号的结构功能关系。这些研究将为蛋白酶体识别UBL信号所涉及的分子事件提供详细的图片，从而触发蛋白酶体的随后事件，从而导致底物降解。 公共卫生相关性：泛素 - 蛋白酶体蛋白水解途径是主要的调节机制，它通过降低精心选择的靶标而影响各种重要的细胞事件。蛋白酶体降解的缺陷与多种疾病有关，包括癌症，神经退行性疾病，发育障碍，免疫和炎症性疾病以及肌肉浪费。这项研究将扩展我们对这一途径中识别和调节的分子机制的理解，这是广泛的药物设计工作的目标。