Structural and biochemical studies of protein PARylation and PARylation-dependent

蛋白质 PARylation 和 PARylation-dependent 的结构和生化研究

• 批准号: 8448609
• 负责人: Wenqing Xu
• 金额: $ 27.43万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448609
• 关键词: Adenosine Diphosphate Ribose; Apoptosis; Binding; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Catalysis; Cell Survival; Cells; Complex; Coupled; Crystallography; Development; Disease; Drug Targeting; Enzymes; Family; Goals; Homologous Gene; Hydrolysis; In Vitro; Ischemic Brain Injury; Malignant Neoplasms; Molecular; Mono-S; Play; Poly Adenosine Diphosphate Ribose; Polymers; Post-Translational Protein Processing; Property; Protein Analysis; Proteins; Protocols documentation; Recombinants; Regulation; Research; Resolution; Role; Signal Transduction; Structure; System; Testing; Therapeutic; Ubiquitin-Conjugating Enzymes; Ubiquitination; Work; base; human JTB protein; inhibitor/antagonist; mutant; novel; poly ADP-ribose glycohydrolase; poly ADPR glycohydrolase inhib; poly(ADP-ribose) protein complex; prevent; protein degradation; reconstitution; research study; three dimensional structure; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Protein poly (ADP-ribosyl) ation (PARylation) is an important posttranslational modification that regulates cell-survival and cell-death programs, and is being recognized as playing a role in an increasing number of other biological functions. Recent observations suggest that, in some cases, PARylation of a protein serves as a signal for its ubiquitination and degradation. Axin is the first example in which the PARylation-dependent ubiquitination and degradation has been clearly demonstrated. Our collaborators have recently identified RNF146 as a key protein for ubiquitinating PARylated Axin. RNF146 contains only two conserved domains, a RING domain that is likely an E3 ubiquitin ligase, and a WWE domain with unknown function. Now our biochemical analyses demonstrate that the RNF146 WWE domain is a specific poly (ADP- ribose) (PAR) binding domain. Strikingly, most WWE-domain containing proteins also contain an E3 ubiquitin ligase domain (either a RING or a HECT domain). This suggests that there may be a family of E3 ubiquitin ligases that use their WWE domains to specifically recognize PARylated proteins as their ubiquitination substrates. In this proposal, we have two major goals. The first is to further characterize the specific RNF146 WWE-PAR interaction, to reveal the structural basis of this recognition through structural determination, and to analyze the molecular mechanism underlying PARylation-dependent ubiquitination by RNF146. These studies will provide a paradigm for understanding protein PARylation and PARylation-dependent ubiquitination. The second goal is to determine crystal structures of poly (ADP-ribose) glycohydrolase (PARG), the only known enzyme responsible for PAR degradation in the cell and a drug target for cancer and other diseases, and its complexes with the PARG substrate and inhibitors. This line of research will be critical not only for understanding the molecular mechanism of PARG catalysis, regulation, but also for developing better PARG inhibitors. The availability of specific PARG inhibitors will have a significant impact on efforts to explore the biological role of protein PARylation in addition to their potential therapeutic value.

描述（由申请人提供）：蛋白质聚（adp -核糖基）化（PARylation）是一种重要的翻译后修饰，可调节细胞生存和细胞死亡程序，并被认为在越来越多的其他生物学功能中发挥作用。最近的观察表明，在某些情况下，蛋白质的PARylation作为其泛素化和降解的信号。轴蛋白是第一个例子，其中paryl依赖性泛素化和降解已被清楚地证明。我们的合作者最近发现RNF146是泛素化PARylated Axin的关键蛋白。RNF146仅包含两个保守结构域，一个可能是E3泛素连接酶的RING结构域和一个功能未知的WWE结构域。现在我们的生化分析表明，RNF146 WWE结构域是一个特异性的聚（ADP-核糖）（PAR）结合结构域。引人注目的是，大多数含有wwe结构域的蛋白质也含有E3泛素连接酶结构域（RING或HECT结构域）。这表明可能存在一个E3泛素连接酶家族，该家族使用其WWE结构域特异性识别PARylated蛋白作为泛素化底物。在这项提议中，我们有两个主要目标。一是进一步表征RNF146 WWE-PAR的特异性相互作用，通过结构测定揭示这种识别的结构基础，分析RNF146 paryl依赖性泛素化的分子机制。这些研究将为理解蛋白质PARylation和PARylation依赖性泛素化提供一个范例。第二个目标是确定聚（adp -核糖）糖水解酶（PARG）的晶体结构，这是唯一已知的在细胞中负责PAR降解的酶，也是癌症和其他疾病的药物靶点，以及它与PARG底物和抑制剂的复合物。这项研究不仅对了解PARG催化、调控的分子机制至关重要，而且对开发更好的PARG抑制剂也至关重要。除了潜在的治疗价值外，特异性PARG抑制剂的可用性将对探索蛋白质PARylation的生物学作用产生重大影响。