Sculpting the ubiquitin status of KCNQ1 in cardiac health and disease

项目总结 QT间期延长综合征(LQTS)是一种后天性或遗传性疾病，以QT间期延长为特征， 劳累引发的心律失常和心源性猝死。最常见的遗传性LQTS亚型LQT1， 由KCNQ1功能缺失突变所致，KCNQ1是一种电压门控性钾通道， 与辅助亚单位KCNE1结合，对心脏复极起关键作用。在LQT1中有越来越多的共识，因为 在许多离子通道病中，大多数突变是由于通道表达减少所致 表膜上的亚基。相比之下，可用于调查人口贩运缺陷和 阐明机械论的洞察力仍然有限和不发达，阻碍了新潜力的发展 治疗策略。 泛素已经成为一种重要的翻译后信号，在分类、信号传递、 和退化。以前的过度表达研究强调了E3泛素连接酶NEDD4L的作用 在监管第一季度的地面贩运和稳定方面。然而，无法有选择地调查 自然细胞环境中的这些过程留下了许多与以下方面有关的基本未知因素 泛素对Q1的调节及其对LQT1的贡献。在这项提案中，我希望解决这一关键差距 两个平行、互补的目标。首先，我将开发工程E3泛素连接酶和去泛素酶 (DUBS)精确和特异地操纵细胞中KCNQ1亚基的泛素化模式 背景。利用质谱学方法，我将进一步询问负责 发散细胞生物学过程中Q1的迁移和稳定。第二，我将利用工程配音来探测 异常泛素化在LQT1运输缺陷突变不同亚群中的作用。通过服用 连接特异性DUBS和定量质谱分析的优势，我将探索不同的泛素 这些突变体的运输和稳定性的特征(即泛素链类型、修饰的赖氨酸残基) 频道。总而言之，这项重点突出、但具有创新性的建议不仅将对我的发展做出重大贡献 一位科学家，但也提供了一个通用的工具集来探索离子通道的潜在细胞生物学 心脏和阐明新的以患者为基础的遗传性心律失常的治疗策略。 好了！

PROJECT SUMMARY Long QT Syndrome (LQTS) is an acquired or inherited disorder, characterized by prolonged QT interval, exertion-triggered arrhythmias, and sudden cardiac death. The most prevalent hereditary LQTS subtype, LQT1, results from loss-of-function mutations in the KCNQ1, a voltage-gated potassium channel that, in combination with the auxiliary subunit KCNE1, is critical for cardiac repolarization. There is growing consensus in LQT1, as with many ion channelopathies, that a majority of mutations result from reduced expression of channel subunits at the surface membrane. In contrast, the tools available to probe trafficking deficiencies and elucidate mechanistic insight remain limited and underdeveloped, hindering the progress of new potential therapeutic strategies. Ubiquitin has emerged as an important post-translational signal with diverse roles in sorting, signaling, and degradation. Previous over-expression studies have highlighted the role of the E3 ubiquitin ligase NEDD4L in the regulation of Q1 surface trafficking and stability. Nevertheless, the inability to selectively investigate these processes in the native cellular environment have left behind many fundamental unknowns relating to ubiquitin regulation of Q1 and its contributions to LQT1. In this proposal, I look to address this critical gap in two parallel, complementary aims. First, I will develop engineered E3 ubiquitin ligases and deubiquitinases (DUBs) to precisely and specifically manipulate the ubiquitination pattern of KCNQ1 subunits in the cellular context. Utilizing mass spectrometric approaches, I will further interrogate the molecular code responsible for divergent cell biological process of Q1 trafficking and stability. Second, I will utilize engineered DUBs to probe the role of aberrant ubiquitination in distinct subsets of LQT1 trafficking-deficient mutations. Through taking advantage of linkage-specific DUBs and quantitative mass spectrometric analyses, I will probe distinct ubiquitin profiles (i.e. ubiquitin chain types, modified lysine residues) in the trafficking and stability of these mutant channels. In all, this focused, yet innovative proposal will not only contribute significantly to my development as a scientist, but also provide a generalizable toolset to probe the underlying cell biology of ion channels in the heart and illuminate new patient-based therapeutic strategies in inherited arrhythmic disorders. !