ANO5 Functions and Mechanisms of LGMD2L

ANO5 LGMD2L 的功能和机制

• 批准号: 9761057
• 负责人: Steven Foltz
• 金额: $ 6.37万
• 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761057
• 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.

项目摘要 肢带型肌营养不良症（LGMD）是一组以进行性肌营养不良为特征的使人衰弱的疾病。 近端肢体肌肉（通常是臀部和肩部）的无力。LGMD2L，由隐性遗传引起 Anoctamin-5（ANO5）突变是LGMD的常见但知之甚少的亚型。的临床过程 LGMD2L通常在进行压力性肌肉活动（例如举重，田径）时遇到困难 以及全身性肌痛到截肢这种疾病对患者来说是一个明显而重大的负担。 生活质量目前，对LGMD2L没有有效的治疗方法;此外，对ANO5知之甚少 功能（因此LGMD2L的发病机制），使治疗靶点的鉴定 几乎不可能最近的研究发现了Anoctamin家族其他成员的一个意想不到的作用， Ca2 +-激活的磷脂乱序酶（Ca2 +-PLSases），促进脂质从一个细胞中移动的酶， 细胞膜双层的小叶到另一个。这个过程对于外化 磷脂酰丝氨酸（PtdSer），其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， 受损的ANO5-/-成肌细胞，这可能是LGMD2L的一个关键方面。虽然ANO5还没有被确定 作为一种PLS酶，它与其他ANO PLS酶高度同源，并且与融合一样，是Ca2+依赖性的。 磷脂紊乱（Ca2 +-PLS）在缺乏ANO5的细胞中减少。因此，ANO5与以下因素有间接联系： PLS，但一些证据表明，它可能不是一个居民质膜蛋白，这将是困难的 来协调作为一个PLSase的角色此外，其他PLS酶在肌肉中表达，并且可以在肌肉中表达。 负责促融合PtdSer暴露在肌肉分化过程中;同样可能的是， PLS非依赖性机制由肌肉用于调节外叶上的PtdSer。为了研究 ANO5、PLS和融合之间的联系，肌肉表达的PLS酶将在某种类型的 肌肉前体细胞（成肌细胞），然后诱导分化。此外，个人能力 将在过表达研究中检查克服ANO5-/-细胞中融合缺陷的PLS酶（目的1）。一 将采用基于蛋白质组学的方法来阐明ANO5在肌肉中的作用（目的2）。这将补充 目的1通过阐明有助于介导其与PLS关系的ANO5结合伴侣;更重要的是，它将 提供了一个无偏见的透镜，通过它来观察ANO5生物学和LGMD2L病理学。的长期目标 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。