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

基本信息

批准号：
10337038
负责人：
Rachel Ceron
金额：
$ 4.68万
依托单位：
UNIVERSITY OF PENNSYLVANIA
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：
2021-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10337038
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

项目摘要

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.

项目摘要该提案的目的是研究γ平滑肌肌动蛋白突变的机制（ACTG2）通过建立新开发的方法来净化野生型和突变体来引起内脏肌病 ACTG2蛋白。内脏肌病是威胁生命的疾病，其特征是光滑的弱点肠子，膀胱和子宫中的肌肉。内脏平滑肌无力导致腹部伸展，呕吐，排尿困难，腹痛和营养不良。当前的内脏肌病治疗，包括手术肠切除和静脉营养，针对症状，不针对基础疾病机制。尽管目前治疗，患有内脏肌病的人是住院的一部分生活，接受多次侵入性手术，并且经常在童年时代死亡。大约一半的内脏肌病患者在ACTG2中有杂合突变。肌动蛋白是一个高度保守的蛋白质在肌肉组织中与肌球蛋白形成收缩丝。所在的机制 ACTG2突变导致内脏肌病目前未知。研究ACTG2的一个主要障碍突变是缺乏产生纯净且具有适当翻译后重组肌动蛋白的方法修改。这项工作是基于净化重组野生型和突变体的最近未发表的进步 ACTG2蛋白用于体外生化表征，并涉及优化新型纯化方法研究与内脏肌病相关的一组代表性的未表征的ACTG2变体。研究将在多种体外测定中使用纯化的野生型和突变体ACTG2蛋白来表征ACTG2的影响 1）肌动蛋白聚合和丝稳定性的突变，2）与相关肌动蛋白结合蛋白的相互作用调节体内ACTG2动力学，3）平滑肌肌球蛋白对ACTG2产生力的能力细丝。此外，这项工作将确定杂合ACTG2突变是否产生显着的负面影响在WildType ActG2上通过野生型和突变蛋白的混合物测定。这项工作的最终目标是确定ACTG2突变引起疾病的生化机制。需要这项工作开发基于机制的治疗以改善苦难的个人的预期寿命和生活质量来自内脏肌病。研究是在罗伯特·海克罗斯（Robert Heuckeroth）博士罗伯托（Roberto）博士的实验室进行的 Dominguez和Michael Ostap博士建立了与ACTG2研究的密切合作。 Dominguez博士是肌动蛋白结构和生物化学专家。 Ostap博士是与肌动蛋白肌球蛋白相互作用的专家。博士 Heuckeroth是肠运动障碍的专家，包括内脏肌病和照顾儿童 ACTG2突变。通过对培养的平滑中这些相同ACTG2突变的平行研究，可以增强这项工作肌肉细胞，干细胞转化为平滑肌，（很快）ACTG2突变小鼠模型。许多宾夕法尼亚肌肉研究所合作者还促进了基于广泛的机械培训，非常适合年轻的医师科学家计划成为一名独立研究者。