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：
10363282
负责人：
ROBERT O HEUCKEROTH
金额：
$ 70.68万
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/10363282
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 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.

项目摘要：我们的最终目标是找到改善患有以下疾病的人的平滑肌功能的新方法内脏肌病，一种由严重的肠、膀胱和子宫平滑肌功能障碍定义的疾病。肠功能障碍，称为肌病性慢性假性肠梗阻 (CIPO)，通常通过以下方法治疗静脉营养。膀胱无力通常需要导尿。当症状在子宫内出现时，结肠生长极小，导致巨囊菌小结肠肠蠕动功能减退综合征 (MMIHS)。仅~20% 的 MMIHS 患者存活至成年。目前的治疗可能会减轻症状，但并非基于疾病机制。最近的数据显示，44% 的 MMIHS/CIPO 患者有杂合点 γ 平滑肌肌动蛋白 (ACTG2) 发生突变，ACTG2 是 6 种肌动蛋白亚型之一。肌动蛋白亚型具有不同的作用在细胞中，虽然肌动蛋白已得到充分研究，但 ACTG2 却很少被研究。引起肌病的 ACTG2 突变是分布在整个肌动蛋白结构中。这表明变异特异性疾病机制可能会受益来自变体特异性疗法。为了设计这样的疗法，我们需要深入了解个体如何变异会导致疾病。因此，我们追求综合战略，将生物化学、结构、细胞以及干细胞方法来确定 ACTG2 突变如何导致内脏肌病。技术的突破和广泛的初步数据为成功奠定了基础。在目标 1 中，我们开发新方法在人体细胞中表达重组人肌动蛋白，无标签且具有天然翻译后特征修改。这一重大创新为 ACTG2 的生化研究开辟了道路，也应该促进研究导致骨骼肌病、心肌病、血管疾病的其他肌动蛋白亚型变体，感音神经性听力损失和先天性畸形。使用重组 ACTG2，我们将研究致病 ACTG2 变体的生化结构特性及其与关键肌动蛋白的相互作用调节肌动蛋白组装的结合蛋白 (ABP)。为了确定突变如何影响细胞生物学（目标 2），我们在人肠平滑肌细胞 (hISMC) 中表达野生型或突变型 ACTG2。我们选择了 hISMC，因为引起疾病的 ACTG2 变异可能会改变与 ABP 的相互作用或取决于细胞类型特定的翻译后修饰。我们创新的定量图像分析流程已经揭晓最常见的 ACTG2 突变 (R257C) 如何影响肌动蛋白细胞骨架和细胞生物学。我们现在将使用此策略来研究其他 ACTG2 突变。一些突变也可能通过预防而导致肌病 MRTF-A转录因子进入细胞核诱导收缩基因表达并平滑肌细胞肌肉分化。为了验证这一假设，我们发明了一种转化人类多能干细胞的新方法 (hPSC) 转化为内脏平滑肌样细胞（目标 3），并制作表达致病 ACTG2 的细胞系变体。我们的跨学科、综合策略应阐明 ACTG2 突变诱导的机制内脏肌病，导致突变特异性药物筛选策略和新疗法。