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：
10521300
负责人：
Rachel Ceron
金额：
$ 5.02万
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/10521300
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 Heterozygote Hospitalization Human Impairment In Vitro Individual 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 Myocytes Smooth Muscle Myosins Structure Symptoms Syndrome Testing Training Urination Uterus Variant Visceral Visceral Myopathies Vomiting Work abdominal distension 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 profilin 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.

项目概要该提案的目的是研究伽玛平滑肌肌动蛋白突变的机制 (ACTG2) 通过利用新开发的方法纯化野生型和突变体引起内脏肌病 ACTG2 蛋白。内脏肌病是危及生命的疾病，其特征是平滑肌无力肠、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。目前内脏肌病的治疗方法，包括手术肠切除和静脉营养，都是针对症状而不是针对潜在的疾病机制。尽管目前有治疗方法，但患有内脏肌病的人仍他们一生的大部分时间都在医院度过，接受多次侵入性手术，并且常常在童年时期死亡。大约一半的内脏肌病患者存在 ACTG2 杂合突变。肌动蛋白是一种高度与肌肉组织中的肌球蛋白形成收缩丝的保守蛋白。其机制 ACTG2突变导致内脏肌病目前尚不清楚。研究ACTG2的一大障碍突变是缺乏生产纯净且具有适当翻译后功能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纯化重组野生型和突变体的进展之上 ACTG2 蛋白用于体外生化表征并涉及新型纯化方法的优化研究一组与内脏肌病相关的代表性未表征的 ACTG2 变异。研究将在多种体外测定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，以及 3) 平滑肌肌球蛋白对 ACTG2 产生作用力的能力细丝。此外，这项工作将确定杂合的 ACTG2 突变是否会产生显性负面影响通过使用野生型和突变蛋白的混合物进行测定来对野生型 ACTG2 进行检测。这项工作的最终目标是确定 ACTG2 突变导致疾病的生化机制。这项工作是需要的开发基于机制的治疗方法，以改善患者的预期寿命和生活质量来自内脏肌病。研究在 Robert Heuckeroth 博士、Roberto 博士的实验室进行 Dominguez 和 Michael Ostap 博士建立了密切的合作关系来研究 ACTG2。多明格斯博士是肌动蛋白结构和生物化学专家。 Ostap 博士是肌球蛋白与肌动蛋白相互作用方面的专家。博士。 Heuckeroth 是肠道动力障碍（包括内脏肌病）方面的专家，并关心患有以下疾病的儿童 ACTG2 突变。通过对培养的光滑细胞中这些相同的 ACTG2 突变进行平行研究，这项工作得到了加强。肌肉细胞、转化为平滑肌的干细胞以及（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基础广泛的机械训练，这对于年轻的医师科学家计划成为一名独立研究者。