Alternative Protein Isoforms in Ventricular Remodeling

项目摘要 心脏肥大和衰竭涉及通过选择性剪接的基因表达的重新布线，但它仍然存在。 不清楚哪些剪接变化与疾病发展相关，以及剪接变体如何影响蛋白质 功能尽管已经发现了许多替代转录物同种型，但并非所有转录物都被翻译成 蛋白质，而是可以通过无义介导的衰变或共翻译蛋白质质量控制降解。 蛋白质组学方法，可以识别和定量剪接变异蛋白质经验和大规模 为研究选择性剪接如何调节心脏基因表达提供了必要的工具。我们和其他人 显示组织特异性剪接变体蛋白可以使用组合的RNA测序来鉴定， 质谱方法。因此，我们在这里的目标是研究替代方案的机制， 剪接调节肥大和衰竭心脏的遗传程序，通过识别蛋白质， 通过剪接协调调节的途径，产生的蛋白质同种型种类的互补 （“蛋白质”）在心脏，和蛋白质序列对蛋白质结构和功能的后果。 具体而言，我们计划：（1）应用定量RNA指导的蛋白质组学框架，以确定关键异构体 在转录和蛋白质水平上的开关，重点是剪接因子和RNA结合的变化 （2）将联合收割机RNA引导的蛋白质组学和 蛋白质组范围的生物物理学方法，我们将询问剪接变异体对蛋白质结构的影响 和热稳定性，并通过选择性剪接发现重要的异构体，本质上是无序的， 调节性翻译后修饰模块。我们预计，这些目标的成功实现将 对选择性剪接调节网络如何重新编程心脏产生新的概念性见解 蛋白质组在病理性重塑和心力衰竭，更普遍地说，有助于方法和概念 阐明选择性剪接在心脏中的调节和功能。

PROJECT SUMMARY Cardiac hypertrophy and failure involve a rewiring of gene expression through alternative splicing, but it remains unclear which splicing changes are relevant to disease development, and how splice variants affect protein function. Although many alternative transcript isoforms have been discovered, not all are translated into proteins and instead may be degraded via non-sense mediated decay or co-translational protein quality control. Proteomics methods that can identify and quantitate splice variant proteins empirically and on a large scale provide essential tools to study how alternative splicing regulates cardiac gene expression. We and others showed that tissue-specific splice variant proteins may be identified using a combined RNA sequencing and mass spectrometry approach. Accordingly, our goal here is to examine the mechanisms by which alternative splicing regulates the genetic program in hypertrophic and failing hearts, by identifying the proteins and pathways that are coordinately regulated by splicing, the resulting complement of protein isoform species (`proteoforms') in the heart, and the consequences of proteoform sequences on protein structure and function. Specifically, we plan to: (1) apply a quantitative RNA-guided proteomics framework to identify key isoform switches at the transcript and the protein level, with emphasis on the changes in splice factors and RNA-binding proteins in mouse models of systolic and diastolic dysfunction; (2) combine RNA-guided proteomics and proteome-wide biophysics approaches, we will interrogate the impact of splice variants on protein structure and thermal stability, and discover significant isoforms through alternative splicing, intrinsically disordered, and regulatory post-translational modification modules. We anticipate the successful completion of these aims will generate new conceptual insights into how alternative splicing regulatory networks reprogram the cardiac proteome in pathological remodeling and heart failure, and more generally, contribute to methods and concepts to elucidate the regulation and function of alternative splicing in the heart.