Ubiquitin Regulation of K Channels in Health and Disease

K 通道在健康和疾病中的泛素调节

• 批准号: 10470075
• 负责人: Henry M. Colecraft
• 金额: $ 40.28万
• 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: 2018-04-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470075
• 关键词: Action Potentials; Address; Antibodies; Arrhythmia; Biochemistry; Cardiac; Cardiac Myocytes; Catalytic Domain; Code; Dangerousness; Data; Deubiquitination; Disease; Electrophysiology (science); Engineering; Enzymes; Flow Cytometry; Functional disorder; Health; Heart; Human; Inherited; Ion Channel; Life; Live Birth; Long QT Syndrome; Lysine; Mass Spectrum Analysis; Membrane Proteins; Methods; Molecular; Monoubiquitination; Muscle Cells; Mutation; Pathologic; Pathway interactions; Phenotype; Physiological; Polyubiquitin; Post-Translational Protein Processing; Potassium Channel; Process; Proteins; Proteomics; Regulation; Risk; Role; Sarcolemma; Signal Transduction; Site-Directed Mutagenesis; Sorting - Cell Movement; Substrate Interaction; Surface; Ubiquitin; Ubiquitination; Ventricular; Ventricular Arrhythmia; Western Blotting; base; delayed rectifier potassium channel; density; genetic approach; innovation; insight; loss of function mutation; mutant; novel strategies; personalized medicine; protein transport; spatiotemporal; sudden cardiac death; trafficking; ubiquitin-protein ligase

SUMMARY The functional repertoire of KCNQ1 and HERG channels on the cardiomyocyte sarcolemma is sustained by dynamic protein trafficking, sorting, and degradation processes. Reduced surface density of KCNQ1 and HERG is a major mechanism underlying LQT1 and LQT2, respectively, motivating a need to understand fundamental mechanisms regulating channel trafficking and stability. Posttranslational modifications by ubiquitin looms as a particularly powerful determinant of KCNQ1 and HERG channels as they can potentially regulate multiple aspects of protein fate including sub-cellular localization, stability, interaction partners, and function. It is known in coarse outline that ubiquitination regulates functional expression of KCNQ1 and HERG channels. However, the full scope and mechanistic bases of ubiquitin regulation of these channels, and the potential contributions of this posttranslational modification to LQTS are not known. There are several formidable obstacles to progress on these fronts owing to: diversity in the E2 ubiquitin conjugating, E3 ubiquitin ligase, and deubiquitination (DUB) enzymes; promiscuity among E3 ligase/substrate and DUB/substrate interactions; intrinsic complexity of the ubiquitin code (monoubiquitination vs polyubiquitiation; distinctive possible polyubiquitin chain linkages with different degradative and non-degradative signaling functions); and lack of spatio-temporal control over ubiquitination of specific substrates. This proposal is founded on exciting preliminary data in which we have circumvented the above complications by engineering methods to selectively target specific E3 ligases or DUBs to tagged KCNQ1 and HERG, respectively. Current dogma in the ubiquitin field holds that K48 ubiquitin chains are degradative while K63 chains have non-degradative signaling functions. Remarkably, our preliminary results enabled by the novel approaches indicate the exact opposite is true for KCNQ1 and HERG, possibly revealing a fundamental difference between cytosolic and membrane proteins. Our preliminary results further suggest that aberrant ubiquitination may underlie KCNQ1/HERG trafficking deficits in some LQT1/LQT2 mutations, and that this pathway may be targeted to rectify underlying abnormalities. Our long term objective is to elucidate molecular mechanisms controlling the surface density and functional regulation of KCNQ1 and HERG channels in heart under both physiological and pathological conditions, and to bridge the mechanistic insights to advance personalized therapy for LQTS and life- threatening cardiac arrhythmias. We combine state-of-the-art, innovative approaches: develop engineered E3 ligases/DUBs to enable unprecedented spatio-temporal control of KCNQ1/HERG ubiquitination; high- throughput flow cytometry; proteomics; biochemistry; and electrophysiology to address three specific aims: 1) Develop and utilize engineered E3 ligases to control spatiotemporal, linkage-specific ubiquitination of KCNQ1 and HERG, and to elucidate the ubiquitin code regulation of these channels. 2) Develop and utilize engineered deubiquitinases to control spatiotemporal, linkage-specific deubiquitination of KCNQ1 and HERG to elucidate ubiquitin code regulation of these channels. 3) Determine role of aberrant ubiquitination in diverse trafficking- deficient LQT1/LQT2 mutations, and assess opportunities for rescue.

概括 KCNQ1的功能曲目和心肌细胞肌膜上的HERG通道由 动态蛋白质运输，分类和降解过程。 KCNQ1的表面密度降低 HERG分别是LQT1和LQT2的主要机制 调节渠道贩运和稳定性的基本机制。翻译后修改 泛素危机是KCNQ1和HERG频道特别有力的决定因素 调节蛋白质命运的多个方面，包括亚细胞定位，稳定性，相互作用伙伴和 功能。在粗略的轮廓中已知泛素化调节KCNQ1和HERG的功能表达 频道。但是，这些通道的泛素调节的完整范围和机械基础，以及 这种翻译后修饰对LQT的潜在贡献尚不清楚。有几个 由于：E2泛素结合的多样性，E3泛素 连接酶和去泛素化（DUB）酶； E3连接酶/底物和配音/底物之间的滥交 互动；泛素代码的内在复杂性（单泛素化与多泛素化；独特的 可能具有不同的降解和非降解信号函数的多泛素链链接）；和 缺乏对特定底物泛素化的时空控制。该建议建立在令人兴奋的基础上 我们通过工程方法将上述并发症规避上述并发症的初步数据 有选择地将特定的E3连接酶或配音靶向标记的KCNQ1和HERG。当前的教条 泛素场认为K48泛素链是降解的，而K63链具有非降解信号传导 功能。值得注意的是，我们的初步结果是通过新颖的方法来实现的，表明相反的是 对于KCNQ1和HERG来说是正确的，可能揭示了胞质和膜之间的基本差异 蛋白质。我们的初步结果进一步表明，异常的泛素化可能是KCNQ1/HERG的基础 某些LQT1/LQT2突变中的贩运缺陷，并且该途径可能针对纠正基础 异常。我们的长期目标是阐明控制表面密度的分子机制 在生理和病理学下，KCNQ1和HERG通道的功能调节 条件，并弥合机械见解，以推动针对LQT和LIFE的个性化治疗 威胁心律不齐。我们结合了最先进的创新方法：开发工程E3 连接酶/配音可以实现KCNQ1/HERG泛素化的前所未有的时空控制；高的- 吞吐量流式细胞仪；蛋白质组学；生物化学；和电生理学以解决三个特定目的：1） 开发和利用工程的E3连接酶来控制KCNQ1的时空，链接特异性的泛素化 和HERG，并阐明这些渠道的泛素代码调节。 2）开发和利用工程 去泛素酶控制KCNQ1和HERG的时空，特定于连锁的去泛素化以阐明 这些渠道的泛素代码调节。 3）确定异常泛素化在各种贩运中的作用 - 缺乏LQT1/LQT2突变，并评估救援的机会。