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

描述（由申请人提供）：本研究的目的是了解端锚聚合酶1和2（两种新型聚（ADP-核糖）聚合酶）的功能。端锚聚合酶含有催化PARP结构域和多个蛋白质相互作用结构域。与传统的PARP不同，它们不参与DNA修复。它们参与端粒的凝聚和端粒长度的调节，但它们也被发现在高尔基体和有丝分裂纺锤体极。因此，端锚聚合酶参与具有治疗干预潜力的过程;例如葡萄糖转运、染色体分离和端粒维持。我们的目标是了解端锚聚合酶如何在如此多样化的生物过程中发挥作用。先前的研究表明端锚聚合酶可以促进非常大的蛋白质复合物的形成和解离。因此，它们可能是一种新型的支架蛋白，其使用聚（ADP-核糖基）化来调节蛋白质复合物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讨这一假设，以研究每种端锚聚合酶的作用，其作用机制和调控。将使用哺乳动物细胞和遗传上易处理的鸡DT 40细胞进行体内工作。目的1研究端锚聚合酶聚腺苷二磷酸核糖基化和寡聚化的机理。体外研究将检查SAM和ANK结构域在催化和聚合中的作用。目的2将确定端锚聚合酶结合如何影响其相互作用伴侣IRAP和NuMA的活性。我们将检查体外聚（ADP-核糖基）化的效果和防止端锚聚合酶结合的体内效果。目的3将描述端锚聚合酶1和2通过基因破坏和/或替换的功能。我们将使用基因破坏来确定端锚聚合酶1和2是否具有单独的功能和突变体来定义单个结构域对整体功能的贡献。目的4研究端锚聚合酶PARP活性的体内调控。我们将研究端锚聚合酶聚（ADP-核糖基）化的体内水平，以及这是否受磷酸化调节。

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.