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Biochemical and cellular mechanisms linking actin mutations to visceral myopathy

将肌动蛋白突变与内脏肌病联系起来的生化和细胞机制

基本信息

批准号：
10491143
负责人：
ROBERT O HEUCKEROTH
金额：
$ 70.21万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-28 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10491143
关键词：
3-Dimensional Actin-Binding Protein Actins Acute Adult Affect Awareness Biochemical Biochemistry Birth Bladder Cardiomyopathies Catheterization Cell Line Cell Nucleus Cell Shape Cells Cellular biology Child Childhood Chimeric Proteins Chronic Congenital Abnormality Contractile Proteins Cytoskeleton Data Data Set Disease Drug Screening Engineering Functional disorder Gastroenterologist Gastrostomy Gene Expression Generations Genes Goals Human Ileostomy Image Analysis Impairment Individual Induced Mutation Intestinal Pseudo-Obstruction Intestines Intravenous Lead Life Link Lovastatin Methods Microfilaments Missense Mutation Molecular Muscle Weakness Muscle function Mutation Myopathy Patients Persons Physiological Point Mutation Post-Translational Protein Processing Property Protein Isoforms Proteins Recombinants Reporting Role Sensorineural Hearing Loss Sirolimus Smooth Muscle Smooth Muscle Actin Staining Method Smooth Muscle Myocytes Structure Symptoms Syndrome System Testing Uterus Variant Vascular Diseases Visceral Visceral Myopathies analysis pipeline base cell motility cell type colon growth design disease-causing mutation human pluripotent stem cell improved in utero innovation motility disorder mutant novel therapeutics nutrition open innovation polymerization prevent protein folding quantitative imaging reduce symptoms scaffold skeletal stem cell differentiation stem cells success transcription factor transplantation medicine

项目摘要

Project Summary: Our ultimate goal is to find new ways to improve smooth muscle function in people with visceral myopathy, a disease defined by profound bowel, bladder and uterine smooth muscle dysfunction. Bowel dysfunction, called myopathic Chronic Intestinal Pseudo-Obstruction (CIPO), is often treated by intravenous nutrition. Bladder weakness often requires catheterization. When symptoms start in utero, colon growth is minimal, causing Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS). Only ~20% of people with MMIHS survive to adulthood. Current treatments may reduce symptoms but are not based on disease mechanisms. Recent data show that 44% of people with MMIHS/CIPO have heterozygous point mutations in gamma smooth muscle actin (ACTG2), one of 6 actin isoforms. Actin isoforms have distinct roles in cells, and while actin is well studied, ACTG2 is barely studied. Myopathy-causing ACTG2 mutations are spread throughout the actin structure. This suggests variant-specific disease mechanisms that could benefit from variant-specific therapies. To design such therapies, we need a deep understanding of how individual variants cause disease. We therefore pursue an integrated strategy, combining biochemical, structural, cellular and stem cell approaches to determine how ACTG2 mutations cause visceral myopathy. Technical breakthroughs and extensive preliminary data set the groundwork for success. In Aim 1, we develop new ways to express recombinant human actin in human cells, without tags and featuring natural post-translational modifications. This major innovation opens the way to biochemical studies of ACTG2, and should also facilitate studies of variants of other actin isoforms causing skeletal myopathy, cardiomyopathy, vascular disease, sensorineural hearing loss, and congenital malformations. Using recombinant ACTG2, we will study the biochemical-structural properties of disease-causing ACTG2 variants, and their interactions with key Actin- Binding Proteins (ABPs) that regulate actin assembly. To determine how mutations affect cell biology (Aim 2), we express wild-type or mutant ACTG2 in human Intestinal Smooth Muscle Cells (hISMC). We selected hISMC because disease-causing ACTG2 variants might alter interactions with ABPs or depend on cell-type specific post-translational modifications. Our innovative quantitative image analysis pipeline already revealed how the most common ACTG2 mutation (R257C) affects the actin cytoskeleton and cell biology. We will now use this strategy to study other ACTG2 mutations. Some mutations might also cause myopathy by preventing the MRTF-A transcription factor from entering the nucleus to induce contractile gene expression and smooth muscle differentiation. To test this hypothesis, we invented a new way to convert human Pluripotent Stem Cells (hPSCs) to visceral smooth muscle-like cells (Aim 3) and made cell lines expressing disease-causing ACTG2 variants. Our cross-disciplinary, integrated strategy should clarify mechanisms of ACTG2 mutation-induced visceral myopathy, leading to mutation-specific drug screening strategies and new therapies.

项目总结：我们的最终目标是找到新的方法来改善患者的平滑肌功能