Cofilin-2: Molecular Function and it's Role in Myopathies

Cofilin-2：分子功能及其在肌病中的作用

• 批准号: 8101006
• 负责人: PANKAJ B AGRAWAL
• 金额: $ 12.65万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8101006
• 关键词: ACTA1 gene; Accounting; Actins; Age; Alleles; Biochemical; Biopsy; Cell Line; Cell physiology; Cells; Code; Cytokinesis; Endocytosis; Environment; Exhibits; Exons; Family; Family member; Fostering; Gene Expression; Gene Mutation; Gene Silencing; Genes; Goals; Hereditary Disease; Human; Insecta; Knock-out; Life; Locomotion; Messenger RNA; Microarray Analysis; Missense Mutation; Molecular; Muscle; Muscle Development; Muscle Weakness; Muscle hypotonia; Mutate; Mutation; Myopathy; Nemaline Myopathies; Neonatology; Neuromuscular Diseases; Organism; Paper; Parents; Patients; Pattern; Phenotype; Physiological; Process; Proteins; Proteomics; Role; Siblings; Skeletal Muscle; Techniques; Thin Filament; Tropomyosin; Troponin T; Work; clinical Diagnosis; cofilin; cofilin 2; depolymerization; human subject; knock-down; mouse model; mutant; nebulin; novel; polymerization; protein function; research study; skeletal; small hairpin RNA

DESCRIPTION (provided by applicant): The long-term goals of this project are to understand the role of Cofilin-2 (CFL2) in skeletal muscle and its mutation in congenital myopathies (CMs). CMs are characterized by relatively non-progressive skeletal muscle weakness, hypotonia presenting in early life. Nemaline Myopathy (NM), the most common type, is a genetic disorder with nemaline bodies present in the muscle and mutations identified in five different genes that constitute the thin filament. In a recent breakthrough, I have identified a sixth gene, CFL2, mutated in NM. Two siblings with clinical diagnosis of typical NM and non-specific congenital myopathy born to consanguineous parents carry a homozygous missense mutation c.103G>A (A35T), and exhibit drastically reduced levels of cofilin-2 protein in their muscles. In contrast, the amount of CFL2 mRNA is increased about 8 fold compared to normal controls. Cofilin-2 belongs to AC group of proteins that include cofilin-1, cofilin-2 and actin depolymerization factor. These proteins function by causing actin severance and depolymerization, and are essential in a wide range of cellular functions including locomotion, cytokinesis, endocytosis, and muscle development. This is the first identified case of a human mutation in the AC group of genes. The molecular mechanism of NM/congenital myopathy caused by this mutation needs to be understood. In addition, cofilin-2 function in skeletal muscle needs to be elucidated. In Aim 1, we will screen all NM/other CM cases for CFL2 mutations and characterize the transcriptional profile associated with these mutations to understand downstream effects. In Aim 2, we will evaluate the biochemical and functional consequences of the identified mutation. In Aim 3, CFL2 expression in C2C12 cells will be knocked down using shRNA techniques and functional consequences will be evaluated. In Aim 4, conventional Cfl2 knockout and c.103G>A mutation knockin mouse models will be generated. This project will introduce me to a variety of techniques including microarray analysis, gene-silencing and creating knockout/knockin mouse models. It willl give me the opportunity to work in an intellectually stimulating environment, where I will approach scientific questions in novel ways fostering my transition to independence. My sponsor Dr Beggs, collaborators/advisors Dr Darras, Dr Dormitzer, Dr Kohane, Dr Kunkel and Dr Maciver, and my Neonatology Division Chief Dr Kourembanas will help me in this process.

描述（由申请人提供）：该项目的长期目标是了解 Cofilin-2 (CFL2) 在骨骼肌中的作用及其在先天性肌病 (CM) 中的突变。 CM 的特征是生命早期出现的相对非进行性骨骼肌无力、肌张力减退。线状肌病 (NM) 是最常见的类型，是一种遗传性疾病，肌肉中存在线状体，并且在构成细丝的五个不同基因中发现了突变。在最近的一项突破中，我发现了第六个基因，CFL2，在 NM 中发生突变。近亲父母所生的两个临床诊断为典型 NM 和非特异性先天性肌病的兄弟姐妹携带纯合错义突变 c.103G>A (A35T)，并且肌肉中的 cofilin-2 蛋白水平显着降低。相反，与正常对照相比，CFL2 mRNA 的量增加了约 8 倍。 Cofilin-2属于AC类蛋白质，包括cofilin-1、cofilin-2和肌动蛋白解聚因子。这些蛋白质通过引起肌动蛋白断裂和解聚来发挥作用，并且对于多种细胞功能（包括运动、胞质分裂、内吞作用和肌肉发育）至关重要。这是首个发现的人类 AC 组基因突变的病例。需要了解这种突变引起的 NM/先天性肌病的分子机制。此外，需要阐明 cofilin-2 在骨骼肌中的功能。在目标 1 中，我们将筛查所有 NM/其他 CM 病例的 CFL2 突变，并表征与这些突变相关的转录谱，以 了解下游影响。在目标 2 中，我们将评估已识别突变的生化和功能后果。在目标 3 中，将使用 shRNA 技术敲低 C2C12 细胞中的 CFL2 表达，并评估功能后果。在目标 4 中，将生成常规 Cfl2 敲除和 c.103G>A 突变敲入小鼠模型。这个项目将向我介绍各种技术，包括微阵列分析、基因沉默和创建基因敲除/敲入小鼠模型。它将让我有机会在一个激发智力的环境中工作，在那里我将以新颖的方式处理科学问题，从而促进我向独立的过渡。我的资助者 Beggs 博士、合作者/顾问 Darras 博士、Dormitzer 博士、Kohane 博士、Kunkel 博士和 Maciver 博士，以及我的新生儿科主任 Kourembanas 博士将在此过程中为我提供帮助。