Role of ZIC and GLI Protein-protein Interactions in Human Brain Disorders

描述（由申请人提供）：小脑锌指（ZIC）基因突变和胶质瘤相关癌基因同源物（GLI）与人类多种衰弱性先天性脑部疾病有关。特别是，这些蛋白的异常在诸如Dandy-Walker畸形、前脑畸形和Greig头多并指综合征等模式障碍中起着关键作用，对智力和预期寿命都有影响。这些基因在疾病状态中的含义已在文献中以多种方式得到证实。例如，Dandy-Walker的可行小鼠模型是通过融合zic1和ZIC4的突变创建的，删除了每个zic1和ZIC4的锌指结构域。然而，在人类中导致这些表型的精确突变仍然难以捉摸。对Dandy-Walker和其他患者进行检查的尝试未能发现ZIC和GLI蛋白的新生突变与疾病状态之间存在简单的因果关系。在其他情况下，即使存在导致特定疾病的突变，观察到的表型之间也存在实质性的异质性。我们从理论上认为，这两个问题都是由于对ZIC功能和交互模式的不完全理解而产生的。直到最近，锌指蛋白（如ZIC和GLI）的典型模型是蛋白质中的单个手指彼此之间不相互作用，而只与DNA相互作用。然而，最近的研究已经确定，ZIC和GLI蛋白可能不按照规范模型起作用。相反，它们是新发现的一类Cys2His2蛋白的成员，称为两指或相互作用手指蛋白，这是一个以手指之间的相互作用为特征的蛋白质子集。此外，最近已经证明，ZIC和GLI不仅在手指与手指之间相互作用，而且在蛋白质-蛋白质相互作用中相互作用，这些相互作用仍有待研究。这些新的观察结果，综合起来，使我们假设突变破坏了ZIC-ZIC和ZIC-GLI的相互作用，而不是单独的单个或配对突变，是与这些基因相关的模式障碍的原因，特别是在Dandy-Walker综合征中。因此，我们提出了一项为期两年的研究，我们将通过酵母双杂交筛选测试ZIC蛋白之间是否存在相互作用，通过等温滴定量热法（ITC）确认ZIC-ZIC和ZIC-GLI之间的相互作用，并确定与疾病状态相关的突变是否会破坏ZIC-ZIC和ZIC-GLI之间的相互作用。我们的希望是，这项工作不仅将导致对与ZIC和GLI基因相关的疾病的分子机制的理解，而且还将导致更好的预后和诊断临床测试，这将为早期干预创造机会，并为患有这些疾病的儿童带来更健康和更长的生命。公共卫生相关性：该项目将确定蛋白质-蛋白质相互作用在人类发育过程中表现出的一系列破坏性先天性脑疾病中所起的作用。根据我们最近对一类具有独特相互作用结构域的锌指蛋白的发现，我们将研究这些蛋白的突变如何导致或促成疾病状态，为未来的预后和诊断工具奠定基础，这些工具将允许早期干预发育模式障碍，如Dandy-Walker畸形、全前额畸形、Grieg头多指综合征等。

DESCRIPTION (provided by applicant): Mutations in Zinc Finger in the Cerebellum (ZIC) genes and the Glioma-associated Oncogene Homolog (GLI) have been linked to multiple debilitating, congenital brain disorders in humans. In particular, abnormalities in these proteins play a critical role in patterning disorders such as the Dandy-Walker Malformation, holoprosencephaly, and Greig Cephalopolysyndactyly Syndrome, with ramifications both for intellectual capacity and life expectancy. The implication of these genes in disease states has been proven in a variety of ways in the literature. For example, viable mouse models for Dandy-Walker are created through a mutation that fuses ZIC1and ZIC4, deleting zinc-finger domains from each. The precise mutations that cause these phenotypes in humans, however, have remained elusive. Attempts to examine Dandy-Walker and other patients have failed to uncover de novo mutations in ZIC and GLI proteins in a simple, causal relationship to the disease state. In other cases, substantial heterogenity exists between observed phenotypes even when mutations that cause a given disorder are present. We theorize that both of these issues arise from an incomplete understanding of ZIC functionality and interaction patterns. Until recently, the canonical model of zinc-finger proteins such as ZIC and GLI was that individual fingers in the protein never interacted with one another, but only with DNA. Recent research, however, has established that ZIC and GLI proteins likely do not function according to canonical models. Instead, they are members of a newly identified class of Cys2His2 proteins called two-finger or interacting-finger proteins, a subset characterized by interactions between fingers in a single protein. Furthermore, it has recently been proven that ZIC and GLI interact not only internally, from finger to finger, but with each other, in protein- protein interactions that remain understudied. These new observations, taken collectively, lead us to hypothesize that the disruption of ZIC-ZIC and ZIC-GLI interactions by mutations, and not single or paired mutations alone, are causal in the patterning disorders associated with these genes, particularly in Dandy-Walker Syndrome. We therefore propose a two-year study in which we will test whether interactions exist between ZIC proteins via yeast two-hybrid screens, confirm ZIC-ZIC and ZIC-GLI interactions via Isothermal Titration Calorimetry (ITC), and determine if mutations associated with disease states disrupt interactions between ZIC-ZIC and ZIC- GLI. Our hope is that the work will lead not only to an understanding of the molecular mechanisms underlying disorders associated with ZIC and GLI genes, but also to better prognostic and diagnostic clinical tests that will create opportunities for early intervention and lead to healthier and longer lives for children with some of these disorders. PUBLIC HEALTH RELEVANCE: This project will determine the role protein-protein interactions play in a set of devastating congenital brain disorders that manifest during human development. Guided by our recent findings on a class of zinc-finger proteins with a unique interaction domain, we will examine how mutation in these proteins causes or contributes to disease states, laying the foundation for future prognostic and diagnostic tools that will permit early intervention in developmental patterning disorders such as the Dandy-Walker Malformation, holoprosenchephaly, Grieg Cephalopolysyndactyly Syndrome, and others.