Alternative Protein Isoforms in Ventricular Remodeling

项目总结 心肌肥大和衰竭涉及通过选择性剪接重新连接基因表达，但它仍然存在 不清楚哪些剪接变化与疾病的发展相关，以及剪接变体如何影响蛋白质 功能。虽然已经发现了许多可供选择的转录异构体，但并不是所有的都被翻译成 而可能通过无义介导的衰变或共翻译的蛋白质质量控制而被降解。 大规模、经验性地鉴定和定量剪接变异蛋白的蛋白质组学方法 为研究选择性剪接如何调控心脏基因表达提供必要的工具。我们和其他人 显示了组织特异性剪接变体蛋白可以使用组合的RNA测序和 质谱学方法。因此，我们在这里的目标是检查替代方案的机制 剪接通过识别蛋白质和蛋白质来调节肥厚和衰竭心脏的遗传程序 由剪接协调调节的通路，由此产生的蛋白质异构体物种的补充 (‘蛋白质形式’)，以及蛋白质形式序列对蛋白质结构和功能的影响。 具体地说，我们计划：(1)应用定量rna引导的蛋白质组学框架来鉴定关键异构体 在转录本和蛋白质水平上进行切换，重点是剪接因子和RNA结合的变化 小鼠收缩和舒张期功能障碍模型中的蛋白质；(2)将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，本质上无序，以及 监管翻译后修改模块。我们期待着这些目标的成功实现将 对选择性剪接调节网络如何对心脏重新编程产生新的概念性见解 蛋白质组在病理重塑和心力衰竭中的作用，更广泛地说，有助于方法和概念 目的：阐明心脏选择性剪接的调控和功能。

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.