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: 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
• 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

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有糖尿病的人的平滑肌肉功能 内脏肌病，一种以严重的肠道、膀胱和子宫平滑肌功能障碍为特征的疾病。 肠功能障碍，称为肌源性慢性假性肠梗阻(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术。当症状开始于子宫、结肠 生长很小，导致巨结肠肠道动力减退综合症(MMIHS)。只有~20% 患有MMIHS的人可以活到成年。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/CIPO患者存在杂合点 6种肌动蛋白亚型中的一种--γ-平滑肌肌动蛋白(ACTG2)的突变。肌动蛋白亚型有不同的作用 在细胞中，虽然肌动蛋白研究得很好，但对ACTG2的研究很少。导致肌病的ACTG2突变是 遍布肌动蛋白结构。这表明变种特有的疾病机制可能会受益 从变种特有的疗法中。要设计这样的疗法，我们需要深刻理解个体如何 变异会导致疾病。因此，我们追求一种综合战略，将生化、结构、细胞 以及干细胞方法来确定ACTG2突变是如何导致内脏肌病的。技术 突破和广泛的初步数据为成功奠定了基础。在目标1中，我们开发了新的方法 在人细胞中表达重组人肌动蛋白，无标签，具有自然翻译后功能 修改。这一重大创新为ACTG2的生化研究开辟了道路，也应该有助于 其他肌动蛋白亚型变异导致骨骼肌病、心肌病、血管疾病的研究， 感音神经性耳聋和先天畸形。利用重组ACTG2，我们将研究 致病ACTG2变异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(ABPs)。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，因为致病的ACTG2变异体可能改变与ABPs的相互作用或取决于细胞类型 具体的翻译后修改。我们创新的定量图像分析流水线已经揭示 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。我们现在就会 使用这个策略来研究其他ACTG2突变。一些突变还可能通过阻止 MRTF-A转录因子从进入细胞核诱导收缩基因表达并平滑 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化人类多能干细胞 (HPSCs)至内脏平滑肌样细胞(AIM 3)，并使表达致病ACTG2的细胞系 变种。我们的跨学科、集成策略应该阐明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的治疗方法。