Molecular and Biochemical Basis of SMAD4 Mutation in Myhre Syndrome

项目总结： Myhre综合征是一种越来越被诊断为罕见的疾病，它是由杂合子功能获得引起的。 Ile500密码子Smad4致病变异。作为一种结缔组织疾病，其核心症状 迈尔综合征是一种危及生命的多器官进行性纤维化。Smad4基因编码一种至关重要的 转化生长因子/骨形态发生蛋白/SMAD信号通路的组成部分，参与许多细胞过程 成体和发育中的胚胎。Smad4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，迈尔综合征只是 与Smad4基因Ile500位错义突变相关。的不变位置和限制谱 Myhre综合征致病突变支持Ile500基因改变具有明显和特异性的假说 对Smad4功能的影响，导致Myhre综合征。 像许多其他罕见疾病一样，迈尔综合征的研究仍然很少。尤其是，没有 由于对这种疾病的机制缺乏深入的了解，这种疾病的现有治疗方法 Smad4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 模型来检验一种假设，即Ile500上的Smad4突变会改变其结构和功能的完整性， 有助于提高Smad4蛋白水平，增强Smad4转录活性，并重新连接 分子间相互作用。在SA1中，我们将检查Smad4-Ile500突变是否在其 蛋白质稳定性。为此，我们将量化Smad4的相对蛋白质水平、调节的Smad及其 新生小鼠在疾病表现前的磷酸化状态。多个器官是 从3个胚层发育而来的将被检查。在SA2中，我们将询问Smad4-Ile500 突变增强了Smad4的转录活性，并重新连接了它的分子间相互作用。根据 Smad4、Ile500的晶体结构靠近蛋白质表面。该区域暴露在溶剂中的位置 其高度带电的特性使其很可能成为蛋白质相互作用的候选者。此外，Ile是 与Smad4激活域相邻。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信号转录活性(荧光素酶测定、转录图谱)并定位Smad4 亲和纯化-质谱法(AP-MS)分析相互作用组。 这项工作将提供初步的实验数据，以开发Smad4的因果作用的可信机制 Myhre综合征的突变是加速开发新的治疗干预措施的关键 对于疾病的发展。

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.