Ubiquitin Regulation of K Channels in Health and Disease

总结 KCNQ 1和HERG通道在心肌细胞肌膜上的功能库通过以下方式维持： 动态蛋白质运输、分选和降解过程。KCNQ 1的表面密度降低， HERG分别是LQT 1和LQT 2的主要机制，激发了理解LQT 1和LQT 2的需要。 规范渠道贩运和稳定的基本机制。翻译后修饰 泛素作为KCNQ 1和HERG通道的一个特别强大的决定因素，因为它们可能 调节蛋白质命运的多个方面，包括亚细胞定位、稳定性、相互作用伴侣，以及 功能已知泛素化调节KCNQ 1和HERG的功能表达 渠道然而，这些通道的泛蛋白调节的全部范围和机制基础，以及 这种翻译后修饰对LQTS的潜在作用尚不清楚。有几 这些方面的进展面临巨大障碍，原因是：E2泛素缀合，E3泛素 连接酶和去泛素化（DUB）酶; E3连接酶/底物和DUB/底物之间的混杂 相互作用;泛素密码的内在复杂性（单泛素化与多泛素化;独特的 具有不同降解和非降解信号传导功能的可能的多聚泛素链连接）;以及 缺乏对特定底物泛素化的时空控制。这项提议是建立在令人兴奋的 我们通过工程方法规避了上述并发症的初步数据， 选择性地将特异性E3连接酶或DUB分别靶向标记的KCNQ 1和HERG。目前的教条， 泛素领域认为K48泛素链是降解性的，而K63链是非降解性的 功能协调发展的值得注意的是，我们通过新方法实现的初步结果表明， 对于KCNQ 1和HERG，这可能揭示了胞质和膜之间的根本差异 proteins.我们的初步结果进一步表明，KCNQ 1/HERG的泛素化异常可能是其基础。 一些LQT 1/LQT 2突变的运输缺陷，并且该途径可能被靶向以纠正潜在的LQT 1/LQT 2突变。 异常我们的长期目标是阐明控制表面密度的分子机制 KCNQ 1和HERG通道在生理和病理条件下的功能调节 条件，并弥合机制的见解，以推进个性化治疗LQTS和生活- 有心律失常的危险我们将联合收割机与最先进的创新方法相结合： 连接酶/DUB，以实现KCNQ 1/HERG泛素化的前所未有的时空控制; 通量流式细胞术;蛋白质组学;生物化学;和电生理学，以解决三个具体目标：1） 开发和利用工程化E3连接酶来控制KCNQ 1的时空，连接特异性泛素化 和HERG，并阐明这些通道的泛素编码调节。2)开发和利用工程 去泛素化酶来控制时空，KCNQ 1和HERG的连接特异性去泛素化，以阐明 泛素编码调控这些通道。3)确定异常泛素化在多种贩运中的作用- LQT 1/LQT 2突变缺陷，并评估挽救的机会。

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.