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(申请人提供)：致心律失常性心肌病(AC)是一种遗传性心脏病，其特征是心功能不全、心肌纤维脂肪替代和室性心律失常导致心源性猝死。最近的人类临床数据显示，只有40%的AC患者的桥粒成分(即桥粒蛋白、桥粒芯糖蛋白、桥粒芯粒蛋白、蛋白球蛋白、蛋白亲和素)存在突变，桥粒是一种在相邻细胞之间建立机械联系的细胞间结构。其余的致病突变在很大程度上是未知的，这表明鉴定和鉴定新的桥粒相互作用蛋白是至关重要的。为了解决这一挑战，我们开始通过在成人心脏cDNA文库中进行以DSP为诱饵的酵母-2-杂交筛选来鉴定新的桥粒相关蛋白。在这个筛选中，我们发现了突触体相关蛋白29(SNAP29)，它是一种囊泡运输蛋白，具有介导膜融合的作用，尽管它在心脏中的功能尚未确定。在人类中，SNAP29突变与桥粒疾病CEDNIK综合征有关，该综合征包括神经系统表现以及与桥粒不稳定相关的皮肤缺陷。我们的初步数据显示，SNAP29与DSP共定位于成年小鼠和人类心脏中桥粒所在的细胞的远端，表明这种相互作用发生在完整的心脏中。在具有AC特征的DSP心脏特异基因敲除(DSP-CKO)小鼠的心脏中，SNAP29的连接部分减少，表明SNAP29与DSP在心脏中存在功能相互作用。相反，使用我们最近产生的SNAP29全局敲除(SNAP29-GKO)小鼠的心脏，这些小鼠表现出断奶前的致死性，心脏重量与体重的比率增加，以及皮肤缺陷，DSP的连接部分减少，为SNAP29、DSP和桥粒之间的功能关系提供了显著的证据。我们的实验进一步支持了SNAP29在心脏功能中的重要性，因为我们的实验敲除了斑马鱼中的SNAP29，这会导致心脏缺陷和心律失常。我们推测SNAP29是心脏桥粒的一个新成分，SNAP29的缺失将导致心脏桥粒超微结构和稳定性的缺陷，从而导致心功能受损和心律失常。为了验证这一假说，我们的目标如下：i)通过强制酵母2-杂交来鉴定SNAP29与DSP之间的分子相互作用，并了解这些相互作用在AC中的相关性；ii)通过检测SNAP29缺失小鼠和心脏特异SNAP29缺失小鼠的心脏表型来表征SNAP29在发育和成年心脏中的作用；a)利用荧光免疫染色和蛋白质印迹法检测桥粒和囊泡运输蛋白的水平和定位；b)利用透射电子显微镜测定桥粒和囊泡运输蛋白的超微结构；c)利用组织学技术、超声心动图、MRI和表面心电图测定心脏形态和功能。