Alternative protein isoforms in ventricular remodeling

项目摘要 许多心脏基因经历选择性剪接以产生心脏与骨骼，以及成人与胎儿同种型。 选择性剪接模式的差异调节是改变遗传程序的中心组成部分 在缺血性损伤后心脏重塑过程中发生。选择性剪接已被证明是因果调节 心脏重塑的严重程度，因此代表了阻止疾病的潜在治疗靶点 进展尽管如此，尽管现在已经有大量的替代同种型转录本， 在心脏基因组中发现，目前对蛋白质编码潜力的认识， 这些替代同种型的功能较差。大多数已发现的同种型尚未被检测到， 蛋白质水平-因此，替代亚型对蛋白质丰度，基序特征， 蛋白质-蛋白质相互作用几乎是未知的。这种知识差距阻碍了对 选择性剪接对健康和疾病心肌的影响。 近年来，蛋白质组学技术取得了进展，现在已经处于有利地位， 心血管科学，但异构体蛋白的检测仍然是一个巨大的挑战。我们和 其他人最近表明，转录组学（RNA-seq）和蛋白质组学（质谱）数据可能 结合起来更深入地探索心脏蛋白质组。在本项目中，我们将联合收割机 RNA-seq（模拟转录异构体）和蛋白质组学（提供蛋白质水平的 证据）来检查心脏可变剪接。具体来说，我们将应用这种RNA测序指导的- 蛋白质组学方法：（i）定义胎儿和成人心脏中替代蛋白质同种型的景观;（ii） 使用心肌梗塞小鼠模型鉴定重塑心脏中的疾病标记同种型， 异丙肾上腺素刺激;（iii）通过鉴定同种型- 特异性蛋白质-蛋白质相互作用和疾病中同种型表达的调节剂;和（iv）阐明 同种型特异性过表达/敲低对肌细胞功能表型影响。我们预计 这些研究的完成将为理解选择性剪接的作用开辟新的途径 在心脏病中。

PROJECT SUMMARY Many cardiac genes undergo alternative splicing to produce cardiac vs. skeletal, and adult vs. fetal isoforms. Differential regulation of alternative splicing patterns is a central component of the altered genetic program during cardiac remodeling after ischemic injuries. Alternative splicing has been shown to causally modulate the severity of cardiac remodeling, and thus represents a potential therapeutic target to halt disease progression. Nevertheless, although a large number of alternative isoform transcripts have now been discovered in the cardiac genome, current knowledge on the protein coding potential and hence molecular functions of these alternative isoform is poor. The majority of discovered isoforms were yet to be detected at the protein level -- therefore the effects of alternative isoforms on protein abundance, motif features, and protein-protein interactions are virtually unknown. This knowledge gap obstructs a fuller understanding on the impact of alternative splicing on the myocardium in health and disease. Proteomics technologies have advanced in recent years and are now well positioned to transform cardiovascular sciences, but the detection of isoform proteins remains a substantial challenge. We and others have recently shown that transcriptomics (RNA-seq) and proteomics (mass spectrometry) data may be combined to probe more deeply into the cardiac proteome. In this project, we will combine the respective strengths of RNA-seq (modeling transcript isoforms) and proteomics (providing protein-level evidence) to examine cardiac alternative splicing. Specifically, we will apply this RNA-seq-guided- proteomics approach to: (i) define the landscape of alternative protein isoforms in fetal and adult hearts; (ii) identify disease signature isoforms in remodeling hearts using mouse models of myocardial infarction and isoproterenol stimulation; (iii) examine the functional consequence of protein isoforms by identifying isoform- specific protein-protein interactions and modulators of isoform expression in disease; and (iv) elucidate the impact of isoform-specific overexpression/knockdown on myocyte functional phenotypes. We anticipate the completion of the proposed studies will open new avenues into understanding the role of alternative splicing in heart diseases.