Sculpting the ubiquitin status of KCNQ1 in cardiac health and disease

塑造 KCNQ1 在心脏健康和疾病中的泛素状态

• 批准号: 10004713
• 负责人: Scott A Kanner
• 金额: $ 2.19万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-04 至 2021-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004713
• 关键词: Action Potentials; Address; Arrhythmia; Biochemical; Biological Assay; Biological Process; Cardiac; Cardiac health; Cells; Cellular biology; Code; Consensus; Data; Defect; Development; Disease; Electrophysiology (science); Engineering; Environment; Event; Exertion; Exhibits; Flow Cytometry; Functional disorder; Gap Junctions; Health; Heart; Heart Diseases; Hereditary Disease; Human; Impairment; Inherited; Ion Channel; Ions; Left; Ligase; Live Birth; Long QT Syndrome; Lysine; Mass Spectrum Analysis; Mediating; Membrane; Molecular; Mutation; Outcome; Pathology; Patients; Pattern; Process; Proteins; Regulation; Role; Scientist; Shapes; Signal Transduction; Sorting - Cell Movement; Specificity; Surface; Syncope; Testing; Therapeutic; Ubiquitin; Ubiquitination; Voltage-Gated Potassium Channel; Work; base; density; engineering design; functional outcomes; innovation; insight; loss of function; loss of function mutation; mutant; novel; novel therapeutics; overexpression; receptor; repaired; sudden cardiac death; tool; trafficking; ubiquitin-protein ligase

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. !

项目摘要 长QT综合征（LQTS）是一种获得性或遗传性疾病，其特征是QT间期延长， 劳累引发的心律失常和心源性猝死最普遍的遗传性LQTS亚型，LQT1， KCNQ1是一种电压门控钾通道， 与辅助亚基KCNE1一起，对心脏复极至关重要。在LQT 1中有越来越多的共识， 在许多离子通道病中，大多数突变是由通道蛋白表达减少引起的。 亚基在表面膜。相比之下，现有的调查贩运缺陷的工具， 阐明机械的洞察力仍然有限和欠发达，阻碍了新的潜力的进展 治疗策略 泛素已经成为一种重要的翻译后信号，在分选，信号传导， 和退化。先前的过表达研究强调了E3泛素连接酶NEDD4L的作用 在调控Q1的表面贩运和稳定性。然而，无法有选择地调查 这些在天然细胞环境中的过程留下了许多基本的未知， 泛素对Q1的调节及其对LQT1的贡献。在本提案中，我希望解决这一关键差距， 两个平行互补的目标首先，我将开发工程化的E3泛素连接酶和去泛素化酶 （DUBs）来精确和特异性地操纵细胞中KCNQ1亚基的泛素化模式。 上下文利用质谱方法，我将进一步询问负责 趋异细胞Q1运输和稳定的生物学过程。第二，我将利用工程DUB探测 异常泛素化在LQT1运输缺陷突变的不同亚群中的作用。通过采取 利用连接特异性DUBs和定量质谱分析，我将探测不同的泛素 这些突变体在运输和稳定性方面的特征（即泛素链类型，修饰的赖氨酸残基） 渠道总而言之，这一重点突出但富有创新精神的建议不仅将大大有助于我的发展， 一个科学家，而且还提供了一个可推广的工具集，以探测细胞中离子通道的基本细胞生物学， 心脏和照亮新的患者为基础的治疗策略在遗传性痴呆症。 !

项目成果

The mutation L69P in the PAS domain of the hERG potassium channel results in LQTS by trafficking deficiency.

hERG 钾通道 PAS 结构域中的突变 L69P 通过运输缺陷导致 LQTS。

• DOI: 10.1080/19336950.2020.1751522
• 发表时间: 2020
• 期刊: Channels (Austin, Tex.)
• 作者: Jenewein,Tina;Kanner,ScottA;Bauer,Daniel;Hertel,Brigitte;Colecraft,HenryM;Moroni,Anna;Thiel,Gerhard;Kauferstein,Silke
• 通讯作者: Kauferstein,Silke