Molecular and Biochemical Basis of SMAD4 Mutation in Myhre Syndrome

Myhre 综合征 SMAD4 突变的分子和生化基础

基本信息

批准号：
10723414
负责人：
Hongmei Mou
金额：
$ 16.49万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10723414
关键词：
Adult Affinity Chromatography Amino Acids Aortic coarctation Apoptosis Biochemical Biological Assay Biopsy Blood Brain Cell physiology Characteristics Charge Clinical Codon Nucleotides Connective Tissue Diseases Data Development Diagnosis Disease Disease Progression Embryo Embryonic Development Exhibits Eye Fibroblasts Fibrosis Genes Genetic Diseases Genetic Transcription Germ Layers Half-Life Heart Hereditary Disease Hereditary Malignant Neoplasm Heterozygote Human Iatrogenesis Individual Intratracheal Intubation Isoleucine Knock-in Mouse Knowledge Life Ligands Location Luciferases Lung MADH4 gene Maps Mass Spectrum Analysis Measures Membrane Proteins Missense Mutation Modeling Molecular Mus Mutation Myhre syndrome Newborn Infant Operative Surgical Procedures Organ Organism Pathogenesis Pathogenicity Pathologic Pathway interactions Patients Phenotype Phosphorylation Physiological Physiological Processes Post-Translational Protein Processing Proliferating Proteins Quality of life Rare Diseases Reporting Role Sampling Signal Pathway Signal Transduction Site Skin Solvents Stenosis Structure Symptoms System Testing Time Tissues Transforming Growth Factor beta Ubiquitination Variant Work body system causal variant cell type gain of function gain of function mutation intermolecular interaction medical examination migration mouse model novel therapeutic intervention organ growth prevent protein protein interaction response transcriptomic profiling

项目摘要

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.

项目摘要： MYHRE综合征是越来越多的罕见疾病，是由杂合功能获得的 ILE500的密码子的SMAD4中的致病变体。作为结缔组织障碍，是 MYHRE综合征是威胁生命的多个器官进行性纤维化。 Smad4基因编码至关重要 TGF/BMP/SMAD信号通路的组成部分，该途径涉及这两个细胞过程成人有机体和发育中的胚胎。 SMAD4基因中的突变或缺失已显示为导致各种疾病，包括遗传性疾病和癌症。有趣的是，Myhre综合征只是与Smad4中ILE500的错义突变有关。不变的位置和受限频谱 MYHRE综合征疾病突变支持以下假设：ILE500的改变具有独特的特异性 SMAD4功能的后果，导致MYHRE综合征。像许多其他罕见疾病一样，MYHRE综合征仍然深入研究。特别是，没有由于对这种疾病的可用治疗，由于对这种机制的深入了解不足 SMAD4突变作用。在此提案中，我们将利用我们最近开发的MYHRE综合征鼠标测试一个假设的模型，即ILE500的SMAD4突变改变了其结构和功能完整性，有助于提高的SMAD4蛋白水平，增强的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突变是否对其固有影响蛋白质稳定性。为此，我们将量化SMAD4，调节性SMAD及其的相对蛋白质水平疾病表现之前，新生小鼠的磷酸化状态。多个器官将检查源自3种细菌层的发展。在SA2中，我们将询问SMAD4-EIL500是否突变增强了SMAD4的转录活性，并将其重新分子间相互作用重新布线。根据 Smad4的晶体结构，Ile500靠近蛋白质表面。该区域的溶剂暴露位置它的高电荷特征使其成为蛋白质 - 蛋白质相互作用的候选者。此外，我是毗邻SMAD4激活域。我们将使用皮肤成纤维细胞作为功能证明的蜂窝系统测量SMAD信号转录活性（荧光素酶测定，转录组分析）和映射SMAD4 通过亲和力纯化 - 质量光谱法（AP-MS）进行互动组。这项工作将提供最初的实验数据，以开发出Smad4因果作用的合理机制 MYHRE综合症疾病突变，这是加快新型治疗干预措施发展的关键用于疾病进展。