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

• 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
• 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

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：我们的最终目标是寻找新的方法来改善患有患者的平滑肌功能 内脏肌病是一种由肠，膀胱和子宫平滑肌功能障碍定义的疾病。 肠功能障碍，称为肌病性慢性肠道伪obstruction（CIPO），经常被治疗 静脉营养。膀胱无力通常需要导管插入术。症状从子宫开始时，结肠 生长很小，导致megacystis microcolon肠道综合症综合征（MMIHS）。只有〜20％ 有MMIH的人幸存到成年。当前治疗可能会减轻症状，但不是基于 疾病机制。最近的数据表明，有44％的MMIHS/CIPO患者具有杂合点 γ平滑肌肌动蛋白（ACTG2）的突变，这是6种肌动蛋白同工型之一。肌动蛋白同工型具有不同的作用 在细胞中，尽管对肌动蛋白进行了充分的研究，但几乎没有研究ACTG2。引起肌病的ACTG2突变是 遍及整个肌动蛋白结构。这表明可能受益的变异特异性疾病机制 来自特异性疗法。为了设计这种疗法，我们需要深入了解个人 变体引起疾病。因此，我们采用了综合策略，结合了生化，结构，细胞 和干细胞方法以确定ACTG2突变如何引起内脏肌病。技术的 突破性和广泛的初步数据为成功奠定了基础。在AIM 1中，我们开发了新的方式 在人类细胞中表达重组人肌动蛋白，没有标签并具有自然翻译后 修改。这项重大创新为ACTG2的生化研究开辟了道路，也应促进 研究其他肌动蛋白同工型的变体，导致骨骼肌病，心肌病，血管疾病，血管疾病， 感官听力损失和先天性畸形。使用重组ACTG2，我们将研究 引起疾病的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）。确定突变如何影响细胞生物学（AIM 2）， 我们在人肠平滑肌细胞（HISMC）中表达野生型或突变体ACTG2。我们选择了 HISMC，因为引起疾病的ACTG2变体可能会改变与ABP的相互作用或依赖细胞类型 特定的翻译后修改。我们的创新定量图像分析管道已经显示 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。我们现在会 使用此策略研究其他ACTG2突变。有些突变也可能通过防止 MRTF-A转录因子从进入细胞核以诱导收缩基因表达和光滑 肌肉分化。为了检验这一假设，我们发明了一种转换人类多能干细胞的新方法 （HPSC）到内脏平滑肌样细胞（AIM 3），并使表达引起疾病ACTG2的细胞系 变体。我们的跨学科，综合策略应阐明ACTG2突变引起的机制 内脏肌病，导致特异性药物筛查策略和新疗法。