Structural and biochemical studies of protein PARylation and PARylation-dependent

描述(申请人提供)：蛋白质多聚(ADP-核糖基化)(PARylation)是一种重要的翻译后修饰，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功能中发挥作用。最近的观察表明，在某些情况下，蛋白质的PAR化是其泛素化和降解的信号。Axin是第一个清楚地证明依赖于PAR化的泛素化和降解的例子。我们的合作者最近发现RNF146是泛素化PARylated Axin的关键蛋白质。RNF146只包含两个保守结构域，一个可能是E3泛素连接酶的环状结构域，以及一个功能未知的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)结合域。值得注意的是，大多数含有WWE结构域的蛋白质还含有E3泛素连接酶结构域(环或Hect结构域)。这表明可能存在一个E3泛素连接酶家族，它们利用其WWE结构域来特异性地识别PARylated蛋白作为其泛素化底物。在这项建议中，我们有两个主要目标。首先，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，并分析RNF146依赖PAR化的泛素化的分子机制。这些研究将为理解蛋白质的PAR化和依赖于PAR化的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，PARG是已知的唯一负责细胞内PAR降解的酶，也是癌症和其他疾病的药物靶点，及其与PARG底物和抑制剂的复合体。这一系列的研究不仅对于理解PARG催化、调控的分子机制，而且对于开发更好的PARG抑制剂都是至关重要的。特异性PARG抑制剂的可获得性除了具有潜在的治疗价值外，还将对探索蛋白质PAR化的生物学作用产生重大影响。

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.