The Role of SNAP29, a Novel Desmoplakin Interactor, in Cardiac Pathophysiology

SNAP29（一种新型桥粒斑蛋白相互作用物）在心脏病理生理学中的作用

• 批准号: 8597618
• 负责人: JASON R PELLMAN
• 金额: $ 3.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8597618
• 关键词: Address; Adenoviruses; Adult; Amino Acid Sequence; Animals; Arrhythmia; Binding; Biological Models; Body Weight; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Clinical Data; Congenital Heart Defects; Data; Defect; Desmosomes; Development; Disease; Distal; Echocardiography; Electrocardiogram; Electron Microscopy; Employee Strikes; Exhibits; Functional disorder; Gene Targeting; Genetic; Goals; Heart; Heart Diseases; Histological Techniques; Human; Hybrids; Intercalated disc; Knockout Mice; Lead; Link; Magnetic Resonance Imaging; Mechanics; Mediating; Membrane Fusion; Molecular; Morphology; Mus; Mutation; Myocardium; Neurologic Manifestations; Pathogenesis; Pathway interactions; Patients; Peptide Sequence Determination; Phenotype; Process; Proteins; Role; Skin; Staining method; Stains; Structure; Surface; Syndrome; Testing; Transmission Electron Microscopy; Ventricular Arrhythmia; Ventricular Dysfunction; Vesicle; Weaning; Weight; Western Blotting; Yeasts; Zebrafish; base; cDNA Library; cardiogenesis; cell type; desmocollin; desmoglein; desmoplakin; heart function; improved; in vitro Model; knock-down; mouse model; mutant; novel; overexpression; plakoglobin; plakophilins; protein transport; public health relevance; research study; sudden cardiac death; therapeutic target; trafficking

DESCRIPTION (provided by applicant): Arrhythmogenic cardiomyopathy (AC) is a genetic based heart disease characterized by ventricular dysfunction, fibrofatty replacement of the myocardium and ventricular arrhythmias leading to sudden cardiac death. Recent human clinical data has revealed that only 40% of AC patients harbor mutations in components of the desmosome (i.e. desmoplakin (DSP), desmoglein, desmocollin, plakoglobin, plakophilin), an intercellular structure that establishes a mechanical link between adjacent cells. The remaining causative mutations are largely unknown, suggesting that identification and characterization of novel desmosomal interacting proteins is of utmost importance. To address this challenge, we set out to identify novel desmosome associated proteins by performing a yeast-2-hybrid screen with DSP as bait in an adult human heart cDNA library. In this screen, we identified synaptosomal associated protein 29 (SNAP29), a vesicular trafficking protein with roles in mediating membrane fusion, though its function in the heart has not been established. In humans, SNAP29 mutations are associated with the desmosomal disease, CEDNIK syndrome, which includes neurological manifestations as well as skin defects associated with desmosomal instability. Our preliminary data show that SNAP29 colocalizes with DSP at the distal ends of cells where desmosomes are located in adult mouse and human hearts, suggesting that this interaction occurs in the intact heart. In hearts of DSP cardiac specific knockout (DSP-cKO) mice which exhibit features of AC, SNAP29 is reduced in the junctional fraction indicating a functional interaction between SNAP29 and DSP in the heart. Conversely, using hearts from our recently generated SNAP29 global knockout (SNAP29-gKO) mice which exhibit preweaning lethality, increased heart weight to body weight ratios, and skin defects, there is a reduction in DSP in the junctional fraction providing striking evidence for a functional relationship between SNAP29, DSP, and the desmosome. The importance of SNAP29 in heart function is further supported by our experiments knocking down SNAP29 in zebrafish which result in cardiac defects and arrhythmias. We hypothesize that SNAP29 is a novel component of the cardiac desmosome and that loss of SNAP29 will lead to defects in cardiac desmosome ultrastructure and stability leading to impaired heart function and arrhythmias. To test this hypothesis, our aims are as follows: i) to characterize the molecular interactions between SNAP29 and DSP as well as understand the relevance of these interactions in AC using forced yeast-2-hybrid and cardiomyocyte overexpression of mutant sequences and ii) to characterize the role of SNAP29 in the developing and adult heart by assessing the cardiac phenotype of global SNAP29 null mice and cardiac-specific SNAP29 null mice by determining a) levels and localization of desmosomal and vesicular trafficking proteins using fluorescent immunostaining and western blot, b) desmosome ultrastructure using transmission electron microscopy, c) cardiac morphology and function using histological techniques, echocardiography, MRI, and surface electrocardiography.

描述（由申请人提供）：心律失常性心肌病（AC）是一种基于遗传的心脏病，其特征是心室功能障碍，心肌的纤维曲霉替代和心室心律失常，导致心脏突然死亡。最近的人类临床数据表明，只有40％的AC患者含有脱粘组成分（即Desmoplakin（DSP），Desmoglein，desmocollin，desmocollin，plakoglobin，plakophilin）的成分中的突变，一种在相邻细胞之间建立机械链接的间结构。其余的致病突变在很大程度上是未知的，这表明新型脱发相互作用蛋白的识别和表征至关重要。为了应对这一挑战，我们着手通过在成人人体心脏cDNA文库中以DSP为诱饵来鉴定新型的脱发相关蛋白质。在此屏幕中，我们确定了突触小体相关蛋白29（SNAP29），这是一种囊泡运输蛋白，在介导膜融合中作用，尽管尚未确定其在心脏中的功能。在人类中，SNAP29突变与脱染色体疾病Cednik综合征有关，其中包括神经系统表现形式以及与脱骨体不稳定性相关的皮肤缺陷。我们的初步数据表明，SNAP29与DSP在成年小鼠和人类心脏中的脱骨体的远端共定位，这表明这种相互作用发生在完整的心脏中。在表现出AC特征的DSP心脏特异性基因敲除（DSP-CKO）的心脏中，SNAP29在连接部分中减少了SNAP29，表明SNAP29和心脏中DSP之间的功能相互作用。相反，使用我们最近产生的SNAP29全球敲除（SNAP29-GKO）小鼠的心脏，这些小鼠表现出预性致死性，增加心脏体重与体重比和皮肤缺陷，连接级分的DSP减少了DSP，从而为SNAP29，DSP和Desmosome之间的功能关系提供了惊人的证据。 SNAP29在心脏功能中的重要性得到了我们在斑马鱼中击倒SNAP29的实验进一步支持的，从而导致心脏缺陷和心律不齐。我们假设SNAP29是心脏脱骨体的新成分，而SNAP29的丧失将导致心脏脱骨体超微结构和稳定性缺陷，从而导致心脏功能和心律失常受损。要检验这一假设，我们的目的如下：i）表征SNAP29和DSP之间的分子相互作用，以及使用强制酵母2-杂交和心肌细胞过表达突变序列和II的AC中这些相互作用的相关性，以通过评估SNAP29在不断发展的Cardec 2中的作用来表征Global and card 29的作用，以评估Global and card 2 nat nat cardipy snap29 NAP的作用。 SNAP29通过a）使用荧光免疫染色和蛋白质印迹来确定a）脱骨和囊泡运输蛋白的水平和定位，b）使用透射电子显微镜，c）使用心脏形态学和功能，使用组织学技术，超声心动图，MRI，MRI，MRI和表面电子图。