ANO5 Functions and Mechanisms of LGMD2L

ANO5 LGMD2L 的功能和机制

• 批准号: 9907852
• 负责人: Steven Foltz
• 金额: $ 6.45万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907852
• 关键词: Address; Adult; Affect; Athletic; Behavior; Binding; Biochemical; Biological; Biology; Cell fusion; Cell membrane; Cell surface; Cells; Cellular biology; Clinical; Complement; Data; Defect; Disease; Enzymes; Event; Family member; Genes; Genetic; Genetic Diseases; Goals; Hip region structure; Impairment; Individual; Injury; Life; Lifting; Light; Limb structure; Limb-Girdle Muscular Dystrophies; Link; Lipid Bilayers; Lipids; Locomotion; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Monitor; Movement; Mus; Muscle; Muscle Fibers; Muscle function; Muscular Atrophy; Muscular Dystrophies; Mutation; Myalgia; Myoblasts; Myopathy; Natural regeneration; Pathogenesis; Pathologic; Pathology; Patients; Phenotype; Phosphatidylserines; Phospholipids; Physiological; Process; Proteins; Proteomics; Public Health; Quality of life; Respiration; Role; Shoulder; Signaling Molecule; Site; Skeletal Muscle; Technology; Testing; Time; Tissues; Translating; Uncertainty; Variant; Work; anoctamin 5; base; effective therapy; experimental study; in vivo; insight; knock-down; lens; muscle regeneration; muscle strength; new therapeutic target; novel; novel strategies; overexpression; phospholipid scramblase; protein expression; regenerative; repaired; therapeutic target; therapy development; treatment strategy; virtual

PROJECT SUMMARY Limb-girdle muscular dystrophies (LGMDs) are a debilitating group of diseases characterized by progressive weakness of the proximal limb muscles (typically the hips and shoulders). LGMD2L, caused by recessive mutations in Anoctamin-5 (ANO5), is a common but poorly understood subtype of LGMD. The clinical course of LGMD2L often advances from difficulties in performing stressful muscle activities (e.g. heavy lifting, athletics) and generalized myalgia to loss of ambulation. The disease represents a clear and significant burden to patient quality of life. Currently, no effective treatments exist for LGMD2L; furthermore, very little is known about ANO5 function (and consequently the pathogenesis of LGMD2L), making the identification of therapeutic targets virtually impossible. Recent work has uncovered an unexpected role for other Anoctamin family members as Ca2+-activated phospholipid scramblases (Ca2+-PLSases), enzymes that facilitate movement of lipids from one leaflet of the plasma membrane bilayer to the other. This process is important for externalizing phosphatidylserine (PtdSer), which serves as a signaling molecule in a variety of physiological contexts including fusion of myoblasts to form multi-nucleated myotubes. Fusion is a central step in muscle differentiation but is impaired ANO5-/- myoblasts, which may be a key aspect of LGMD2L. While ANO5 has not been definitively characterized as a PLSase, it is highly homologous to other ANO PLSases and, as with fusion, Ca2+-dependent phospholipid scrambling (Ca2+-PLS) is reduced in cells lacking ANO5. ANO5 is thus circumstantially linked to PLS, but some evidence suggests that it may not be a resident plasma membrane protein, which would be hard to reconcile with a role as a PLSase. Furthermore, other PLSases are expressed in muscle and could be responsible for pro-fusogenic PtdSer exposure during muscle differentiation; it is likewise possible that a specific PLS-independent mechanism is used by muscle to regulate PtdSer on the outer leaflet. In order to investigate the link between ANO5, PLS, and fusion, muscle-expressed PLSases will be knocked down in a certain type of muscle precursor cell (myoblasts), which will then be induced to differentiate. Additionally, the ability of individual PLSases to overcome the fusion-deficit in ANO5-/- cells will be examined in overexpression studies (Aim 1). A proteomics-based approach will be employed to clarify role(s) of ANO5 in muscle (Aim 2). This will complement Aim 1 by elucidating ANO5 binding partners that help to mediate its relationship to PLS; more importantly, it will provide an unbiased lens through which to view ANO5 biology and LGMD2L pathology. The long-term goal of this work is to identify potential therapeutic targets for ANO5-MD by determining disease mechanisms.

项目概要 肢带型肌营养不良症 (LGMD) 是一组使人衰弱的疾病，其特征是进行性进展 近端肢体肌肉无力（通常是臀部和肩膀）。 LGMD2L，由隐性遗传引起 Anoctamin-5 (ANO5) 突变是一种常见但人们知之甚少的 LGMD 亚型。临床病程 LGMD2L 通常因执行有压力的肌肉活动（例如举重、运动）时遇到困难而进展 以及全身肌痛导致无法行走。该疾病给患者带来了明显而重大的负担 生活质量。目前，LGMD2L 尚无有效治疗方法；此外，人们对 ANO5 知之甚少 功能（以及 LGMD2L 的发病机制），从而确定治疗靶点 几乎不可能。最近的研究揭示了 Anoctamin 家族其他成员的意想不到的作用： Ca2+ 激活的磷脂扰乱酶 (Ca2+-PLSases)，一种促进脂质从磷脂中移动的酶 质膜双层的小叶到另一个。这个过程对于外部化很重要 磷脂酰丝氨酸 (PtdSer)，在多种生理环境中充当信号分子，包括 成肌细胞融合形成多核肌管。融合是肌肉分化的核心步骤，但 ANO5-/- 成肌细胞受损，这可能是 LGMD2L 的一个关键方面。虽然 ANO5 尚未最终确定 作为 PLSase 的特征，它与其他 ANO PLSase 高度同源，并且与融合一样，Ca2+依赖性 缺乏 ANO5 的细胞中磷脂扰乱 (Ca2+-PLS) 减少。因此，ANO5 与 PLS，但一些证据表明它可能不是常驻质膜蛋白，这很难 与 PLSase 的角色相协调。此外，其他 PLSase 在肌肉中表达，可能是 负责肌肉分化过程中促融合的 PtdSer 暴露；同样有可能的是，一个特定的 肌肉使用 PLS 独立机制来调节外叶上的 PtdSer。为了调查 ANO5、PLS 和融合、肌肉表达的 PLSase 之间的联系将在某种类型的 肌肉前体细胞（成肌细胞），然后被诱导分化。另外，个人能力 将在过表达研究中检查克服 ANO5-/- 细胞融合缺陷的 PLSase（目标 1）。一个 将采用基于蛋白质组学的方法来阐明 ANO5 在肌肉中的作用（目标 2）。这将补充 目标 1：阐明有助于调节 ANO5 与 PLS 关系的 ANO5 结合伙伴；更重要的是，它将 提供一个公正的视角来观察 ANO5 生物学和 LGMD2L 病理学。长期目标是 这项工作是通过确定疾病机制来确定 ANO5-MD 的潜在治疗靶点。