Use of novel methods to study the biochemical mechanisms of ACTG2 mutations in visceral myopathy

使用新方法研究内脏肌病中ACTG2突变的生化机制

• 批准号: 10533420
• 负责人: Rachel Ceron
• 金额: $ 0.25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10533420
• 关键词: Abdominal Pain; Actin-Binding Protein; Actins; Actomyosin; Adopted; Affect; Affinity; Air; Amino Acids; Binding; Biochemical; Biochemistry; Biological Assay; Birth; Bladder; Bladder Dysfunction; Cells; Child Care; Childhood; Chronic; Collaborations; Complex; Constipation; Contracts; Disease; Dominant-Negative Mutation; Eating; Excision; F-Actin; Filament; Fluorescence; G Actin; Generations; Genes; Goals; Growth; Human; Impairment; In Vitro; Individual; Institutes; Intestinal Pseudo-Obstruction; Intestines; Intravenous; Label; Laboratories; Lasers; Life; Life Expectancy; Malnutrition; Measures; Medicine; Methods; Missense Mutation; Muscle; Muscle Contraction; Muscle Weakness; Muscle function; Mutation; Myosin ATPase; Operative Surgical Procedures; Organ Transplantation; Pain; Pathogenicity; Patients; Pennsylvania; Persons; Physicians; Polymers; Post-Translational Protein Processing; Process; Protein Isoforms; Proteins; Publishing; Pyrenes; Quality of life; Recombinants; Research Personnel; Scientist; Smooth Muscle; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; Smooth Muscle Myosins; Structure; Symptoms; Syndrome; Testing; Training; Urination; Uterus; Variant; Visceral; Visceral Myopathies; Vomiting; Work; abdominal distension; base; disease-causing mutation; experimental study; feeding; globular protein; high throughput screening; high-throughput drug screening; improved; in vitro Assay; in vivo; insight; monomer; motility disorder; mutant; mutant mouse model; novel; novel strategies; nutrition; polymerization; rare condition; stem cells; treatment strategy

Project Summary The goal of this proposal is to study the mechanisms by which mutations in gamma smooth muscle actin (ACTG2) cause visceral myopathy by building upon newly developed methods to purify wildtype and mutant ACTG2 proteins. Visceral myopathies are life-threatening diseases characterized by weakness of smooth muscle in the bowel, bladder and uterus. Visceral smooth muscle weakness causes abdominal distension, vomiting, urination difficulties, abdominal pain and malnutrition. Current treatments of visceral myopathy, including surgical bowel resection and intravenous nutrition, are directed at symptoms and do not target underling disease mechanisms. Despite current treatments, people suffering from visceral myopathy are hospitalized for a significant portion of their life, undergo multiple invasive surgeries and often die in childhood. Approximately half of patients with visceral myopathy have heterozygous mutations in ACTG2. Actin is a highly conserved protein that forms contractile filaments with myosin in muscle tissue. The mechanisms by which ACTG2 mutations cause visceral myopathy are currently unknown. One major obstacle to studying ACTG2 mutations is the lack of methods for producing recombinant actin that is pure and has proper post-translational modifications. This work builds on recent unpublished advances to purify recombinant wildtype and mutant ACTG2 proteins for in vitro biochemical characterization and involves optimization of novel purification methods to study a representative set of uncharacterized ACTG2 variants associated with visceral myopathy. Studies will use purified wildtype and mutant ACTG2 proteins in multiple in vitro assays to characterize the effects of ACTG2 mutations on 1) actin polymerization and filament stability, 2) interaction with relevant actin-binding proteins that regulate ACTG2 dynamics in vivo, and 3) the ability of smooth muscle myosin to generate force on ACTG2 filaments. In addition, this work will determine if heterozygous ACTG2 mutations exert dominant negative effects on wildtype ACTG2 via assays with mixtures of wildtype and mutant proteins. The ultimate goal of this work is to determine the biochemical mechanisms through which ACTG2 mutations cause disease. This work is needed to develop mechanism-based treatments to improve the life expectancy and quality of life for individuals suffering from visceral myopathy. Studies are performed in the laboratories of Dr. Robert Heuckeroth, Dr. Roberto Dominguez and Dr. Michael Ostap who have established a close collaboration to study ACTG2. Dr. Dominguez is an actin structure and biochemistry expert. Dr. Ostap is an expert on myosin interactions with actin. Dr. Heuckeroth is an expert on bowel motility disorders including visceral myopathy and cares for children with ACTG2 mutations. This work is enhanced by parallel studies of these same ACTG2 mutations in cultured smooth muscle cells, stem cells converted to smooth muscle, and (soon) an ACTG2 mutant mouse model. Many Pennsylvania Muscle Institute collaborators also facilitate broad-based, mechanistic training that is ideal for a young physician-scientist planning to become an independent investigator.

项目摘要 这项提议的目的是研究γ平滑肌肌动蛋白突变的机制， （ACTG 2）通过建立新开发的纯化野生型和突变型的方法而引起内脏肌病 ACTG2蛋白。内脏肌病是一种危及生命的疾病， 肠道膀胱和子宫的肌肉内脏平滑肌无力导致腹胀， 呕吐、排尿困难、腹痛和营养不良。内脏肌病的当前治疗， 包括外科肠切除术和静脉营养，针对症状， 潜在的疾病机制。尽管目前的治疗，患有内脏肌病的人， 他们生命中很大一部分时间都在住院，经历了多次侵入性手术，并经常在儿童时期死亡。 大约一半的内脏肌病患者在ACTG2中存在杂合突变。肌动蛋白是一种高度 在肌肉组织中与肌球蛋白形成收缩丝的保守蛋白质。的机制 ACTG2突变导致内脏肌病目前尚不清楚。研究ACTG2的一个主要障碍 突变的主要原因是缺乏生产纯的重组肌动蛋白的方法， 修改.这项工作建立在最近未发表的进展，纯化重组野生型和突变体 用于体外生物化学表征的ACTG 2蛋白，并涉及新型纯化方法的优化 研究一组代表性的与内脏肌病相关的未表征的ACTG2变体。研究将 在多个体外试验中使用纯化的野生型和突变型ACTG2蛋白来表征ACTG2的作用 1）肌动蛋白聚合和细丝稳定性，2）与相关肌动蛋白结合蛋白的相互作用， 调节体内ACTG 2动力学，和3）平滑肌肌球蛋白对ACTG 2产生力的能力 细丝。此外，这项工作将确定杂合ACTG2突变是否会产生显性负效应， 通过使用野生型和突变体蛋白质的混合物的测定对野生型ACTG2的影响。这项工作的最终目标是 确定ACTG2突变导致疾病的生化机制。这项工作是必要的 开发以机制为基础的治疗方法，以提高患者的预期寿命和生活质量， 内脏肌病研究在Robert Heuckeroth博士和Roberto博士的实验室进行 Dominguez和Michael Ostap博士建立了密切的合作来研究ACTG2。多明格斯医生 是肌动蛋白结构和生物化学专家。奥斯塔普博士是肌球蛋白与肌动蛋白相互作用的专家。博士 Heuckeroth是肠道动力障碍（包括内脏肌病）方面的专家， ACTG2突变。这项工作通过在培养的光滑细胞中对这些相同的ACTG 2突变进行平行研究而得到加强 肌肉细胞，干细胞转化为平滑肌，以及（很快）ACTG2突变小鼠模型。许多 宾夕法尼亚肌肉研究所的合作者还促进了基础广泛的机械训练， 一位年轻的科学家兼医生，计划成为一名独立调查员。