Function & Regulation of two Novel PARPs, tankyrase 1&2

描述(申请人提供)：本研究的目的是了解两种新的聚(ADP-核糖)聚合酶Tankyrase 1和2的功能。Tankyrase含有一个催化PARP结构域和多个蛋白质相互作用结构域。与传统的PAP不同，它们不参与DNA修复。它们参与端粒凝聚和端粒长度调节，但也存在于高尔基体和有丝分裂纺锤体极。因此，Tankyrase参与了有可能进行治疗干预的过程，例如葡萄糖运输、染色体分离和端粒维持。我们的目标是了解tankyrase如何在如此多样化的生物过程中发挥作用。以前的研究表明，tankyrase可以促进非常大的蛋白质复合体的形成和解离。因此，它们可能是一种新型的支架蛋白，它使用聚(ADP-核糖)来调节蛋白质复合体在不同细胞位置的组装/拆卸。我们将结合生物化学和遗传学研究来探索这一假说，以检验每一种Tankyrase的作用、作用机制和调控。体内的工作将在哺乳动物细胞和遗传上易受影响的鸡DT40细胞中进行。目的1研究Tankyrase多聚(ADP-核糖基)和寡聚的作用机制。体外研究将考察SAM和ANK结构域在催化和聚合中的作用。目标2将确定tankyrase结合如何影响其相互作用伙伴IRAP和NUMA的活性。我们将检测体外多聚(ADP-核糖基)的效果和体内阻止tankyrase结合的效果。AIM 3将通过基因破坏和/或替换来描述tankyrase 1和2的功能。我们将使用基因破坏来确定tankyrase 1和2是否具有单独的功能，并通过突变来确定单个结构域对整体功能的贡献。目的4研究Tankyrase PARP活性的体内调节。我们将检查体内Tankyrase聚(ADP-核糖基)的水平，以及这是否受磷酸化的调节。

DESCRIPTION (provided by applicant): The goal of this research is to understand the function of tankyrase 1 and 2, two novel poly(ADP-ribose) polymerases. Tankyrases contain a catalytic PARP domain and multiple protein-interaction domains. Unlike conventional PARPs, they are not involved in DNA repair. They participate in telomere cohesion and telomere length regulation, but they are also found at Golgi and mitotic spindle poles. Thus, tankyrases are involved in processes that have the potential for therapeutic intervention; e.g. glucose transport, chromosome segregation and telomere maintenance. Our goal is to understand how tankyrases function in such a diverse array of biological processes. Previous studies indicate that tankyrases can promote the formation and dissociation of very large protein complexes. Thus, they may be a novel type of scaffolding protein that uses poly(ADP-ribosyl)ation to regulate the assembly/disassembly of protein complexes at various cellular locations. We will explore this hypothesis using a combination of biochemical and genetic studies to examine the role of each tankyrase, their mechanism of action, and regulation. The in vivo work will be performed with both mammalian cells and genetically tractable chicken DT40 cells. Aim 1 will characterize the mechanism of tankyrase poly(ADP-ribosyl)ation and oligomerization. In vitro studies will examine the role of the SAM and ANK domains in catalysis and polymerization. Aim 2 will determine how tankyrase binding affects the activities of its interaction partners IRAP and NuMA. We will examine the effect of in vitro poly(ADP-ribosyl) ation and the in vivo effect of preventing tankyrase binding. Aim 3 will delineate tankyrase 1 and 2 function via gene disruption and/or replacement. We will use gene disruption to determine whether tankyrase 1 and 2 have separate functions and mutants to define the contribution of individual domains to overall function. Aim 4 will investigate the in vivo regulation of tankyrase PARP activity. We will examine the in vivo levels of tankyrase poly(ADP-ribosyl)ation and whether this is regulated by phosphorylation.