Molecular and Biochemical Basis of SMAD4 Mutation in Myhre Syndrome

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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.