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.

项目概述：我们的最终目标是找到新的方法来改善人的平滑肌功能， 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，称为肌病性慢性肠假性梗阻（锡波），通常通过以下方法治疗： 静脉营养膀胱无力通常需要导尿。当症状开始在子宫，结肠 生长是最小的，引起巨囊肿小结肠肠发育不全综合征（MMIHS）。只有~20% 的MMIHS患者能活到成年。目前的治疗方法可能会减轻症状，但不是基于 疾病机制。最近的数据显示，44%的MMIHS/锡波患者具有杂合子点， γ平滑肌肌动蛋白（ACTG 2）突变，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，虽然肌动蛋白得到了很好的研究，但ACTG 2几乎没有研究。引起肌病的ACTG 2突变是 遍布肌动蛋白结构。这表明变异特异性疾病机制， 变异特异性疗法的好处为了设计这样的疗法，我们需要深入了解个人如何 变异导致疾病。因此，我们采取综合战略，将生物化学、结构、细胞 和干细胞方法来确定ACTG 2突变如何导致内脏肌病。技术 突破性进展和广泛的初步数据为成功奠定了基础。在目标1中，我们开发新的方法， 在人细胞中表达重组人肌动蛋白，没有标签，具有天然的翻译后 修改.这一重大创新为ACTG 2的生物化学研究开辟了道路， 对引起骨骼肌病、心肌病、血管疾病 感音神经性听力损失和先天性畸形。使用重组ACTG 2，我们将研究 致病ACTG 2变体的生物化学结构特性，以及它们与关键肌动蛋白的相互作用， 调节肌动蛋白装配的结合蛋白质类。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ACTG 2。我们选择 hISMC，因为致病ACTG 2变体可能改变与ABP的相互作用或依赖于细胞类型 特定的翻译后修饰。我们创新的定量图像分析管道已经揭示了 最常见的ACTG 2突变（R257 C）如何影响肌动蛋白细胞骨架和细胞生物学。我们现在将 使用这种策略来研究其他ACTG 2突变。一些突变也可能通过阻止 MRTF-A转录因子进入细胞核诱导收缩基因表达和平滑 肌肉分化为了验证这一假设，我们发明了一种新的方法来转换人类多能干细胞， 将hPSC（hPSC）转化为内脏平滑肌样细胞（Aim 3），并制备表达致病ACTG 2的细胞系 变体。我们的跨学科综合策略应该阐明ACTG 2突变诱导的细胞凋亡的机制。 内脏肌病，导致突变特异性药物筛选策略和新疗法。