Structural and biochemical studies of protein PARylation and PARylation-dependent

描述（由申请人提供）：蛋白质聚（ADP-核糖基）化（PAR化）是一种重要的翻译后修饰，可调节细胞存活和细胞死亡程序，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PAR化作为其泛素化和降解的信号。Axin是第一个明确证明PAR化依赖的泛素化和降解的例子。我们的合作者最近发现RNF 146是泛素化PAR化Axin的关键蛋白。RNF 146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，以及一个功能未知的WWE结构域。目前我们的生化分析表明RNF 146 WWE结构域是一个特异性的聚腺苷二磷酸核糖（PAR）结合结构域。引人注目的是，大多数含有WWE结构域的蛋白质还含有E3泛素连接酶结构域（RING或HECT结构域）。这表明可能存在E3泛素连接酶家族，其使用其WWE结构域特异性识别PAR化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。首先是进一步表征RNF 146 WWE-PAR相互作用的特异性，通过结构测定揭示这种识别的结构基础，并分析RNF 146依赖PAR化的泛素化的分子机制。这些研究将为理解蛋白质PAR化和PAR化依赖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是唯一已知的负责细胞中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。这一系列的研究不仅对于理解PARG催化、调节的分子机制，而且对于开发更好的PARG抑制剂都是至关重要的。特异性PARG抑制剂的可用性将对探索蛋白质PAR化的生物学作用以及其潜在治疗价值的努力产生重大影响。 公共卫生相关性：我们将集中在蛋白质PAR化和PAR化依赖的泛素化的结构和生化分析，这是许多生物过程的关键。我们还将研究PARG的结构和调节，PARG是调节蛋白质PARylation的关键酶。PARG抑制剂可用于预防缺血性脑损伤和其他治疗目的。

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. PUBLIC HEALTH RELEVANCE: We will focus on the structural and biochemical analysis of protein PARylation and PARylation- dependent ubiquitination, which is crucial for many biological processes. We will also work on the structure and regulation of PARG, a key enzyme regulating protein PARylation. PARG inhibitors may be useful for preventing ischemic brain injury and other therapeutic purposes.