Molecular and Biochemical Basis of SMAD4 Mutation in Myhre Syndrome

项目摘要： Myhre综合征是一种越来越多的诊断罕见的疾病，是由杂合子获得性功能 SMAD 4中Ile 500密码子处的致病性变体。作为一种结缔组织疾病， Myhre综合征是一种危及生命的多器官进行性纤维化。SMAD 4基因编码一个关键的 TGF/BMP/SMAD信号通路的组成部分，参与了许多细胞过程，在两个 成熟的有机体和发育中的胚胎。SMAD 4基因的突变或缺失已被证明是 导致各种疾病，包括遗传性疾病和癌症。有趣的是，Myhre综合征只是 与SMAD 4中Ile 500的错义突变相关。的不变位置和限制谱 Myhre综合征致病突变支持Ile 500的改变具有独特和特异性的假设。 SMAD 4功能的影响，导致Myhre综合征。 像许多其他罕见疾病一样，Myhre综合征仍然严重缺乏研究。特别是，没有 由于对这种疾病的机制缺乏深入的了解， SMAD 4突变起作用。在这项提案中，我们将利用我们最近开发的Myhre综合征小鼠 模型来检验Ile 500处的SMAD 4突变改变其结构和功能完整性的假设， 有助于增加SMAD 4蛋白水平、增强SMAD 4转录活性并重新连接 分子间相互作用在SA 1中，我们将检查SMAD 4-Ile 500突变是否对其基因表达具有内在影响。 蛋白质稳定性为此，我们将定量SMAD 4、调节性SMAD和它们的相对蛋白水平。 在疾病表现之前，新生小鼠的磷酸化状态。多个器官 将检查来自3个胚层的发育衍生物。在SA 2中，我们将询问SMAD 4-Ile 500是否 突变增强SMAD 4转录活性并重新连接其分子间相互作用。根据 SMAD 4、Ile 500的晶体结构接近蛋白质表面。该区域的溶剂暴露位置 并且其高电荷特性使其成为蛋白质-蛋白质相互作用的可能候选物。此外，Ile 与SMAD 4激活结构域相邻。我们将使用皮肤成纤维细胞作为功能验证的细胞系统， 测量SMAD信号传导转录活性（荧光素酶测定，转录组学分析）并定位SMAD 4 通过亲和纯化-质谱法（AP-MS）测定相互作用体。 这项工作将提供初步的实验数据，以发展一个合理的机制，因果关系的SMAD 4 Myhre综合征疾病中的突变，加速开发新型治疗干预措施的关键 for disease疾病progression进展.

Project Summary: Myhre syndrome is an increasingly diagnosed rare disease that is caused by a heterozygous gain-of-function pathogenic variant in SMAD4 at the codon for Ile500. As a connective tissue disorder, the core symptom of Myhre syndrome is life-threatening progressive fibrosis in multiple organs. SMAD4 gene encodes a crucial component of the TGF/BMP/SMAD signaling pathway that is involved in many cellular processes in both the adult organism and the developing embryo. Mutations or deletions in the SMAD4 gene have been shown to result in various disorders including hereditary diseases and cancers. Intriguingly, Myhre syndrome is only associated with the missense mutations at Ile500 in SMAD4. The invariant location and restricted spectrum of Myhre syndrome-causative mutations support the hypothesis that alteration in Ile500 has distinct and specific consequences on SMAD4 function, resulting in Myhre syndrome. Like many other rare diseases, Myhre syndrome remains significantly understudied. Particularly, there is no available treatment for this disease due to a poor in-depth understanding of the mechanism by which the SMAD4 mutation acts. In this proposal, we will capitalize on our recently developed Myhre syndrome mouse model to test a hypothesis that SMAD4 mutation at Ile500 alters its structural and functional integrity, contributing to the increased SMAD4 protein level, enhanced SMAD4 transcriptional activity, and rewired intermolecular interaction. In SA1, we will examine if SMAD4-Ile500 mutation exhibits an intrinsic effect on its protein stability. To this end, we will quantify the relative protein level of SMAD4, regulatory SMADs, and their phosphorylated status in newborn mice before the disease manifestation. Multiple organs that are developmentally derived from 3 germ layers will be examined. In SA2, we will interrogate if SMAD4-Ile500 mutation enhances SMAD4 transcriptional activity and rewires its intermolecular interaction. According to the crystal structure of SMAD4, Ile500 is close to the protein surface. The solvent-exposed location of this region and its highly charged character make it a likely candidate for protein-protein interactions. In addition, Ile is adjacent to the SMAD4 activation domain. We will use skin fibroblasts as a proof-of-function cellular system to measure SMAD signaling transcriptional activity (luciferase assay, transcriptomic profiling) and to map SMAD4 interactome by Affinity Purification-Mass Spectrometry (AP-MS). This work will provide initial experimental data to develop a plausible mechanism of the causal role of SMAD4 mutation in Myhre syndrome disease, a key for expediting the development of novel therapeutic interventions for disease progression.