Ubiquitin Regulation of K Channels in Health and Disease

摘要 KCNQ1和HERG通道在心肌细胞肌膜上的功能谱系由 动态的蛋白质运输、分类和降解过程。降低KCNQ1和KCNQ1的表面密度 HERG分别是LQT1和LQT2的主要机制，激发了理解的需要 规范渠道贩运和稳定的基本机制。翻译后修饰语 泛素是KCNQ1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、相互作用伙伴和 功能。我们大致知道泛素化调节KCNQ1和HERG的功能表达 频道。然而，泛素调节这些通道的全部范围和机制基础，以及 这种翻译后修饰对LQTS的潜在贡献尚不清楚。有几个 在这些方面取得进展的巨大障碍是由于：E2泛素结合、E3泛素的多样性 连接酶和脱泛素化(DUB)酶；E3连接酶/底物和DUB/底物的混杂 相互作用；泛素编码的内在复杂性(单一泛素化与多泛素化；独特 可能的多泛素链连接，具有不同的降解和非降解信号功能)；以及 缺乏对特定底物泛素化的时空控制。这项建议是建立在激动人心的基础上的 初步数据中，我们已经通过工程方法避开了上述复杂情况 选择性地将特定的E3连接酶或DUB分别靶向标记的KCNQ1和HERG。当今世界的教条 泛素场认为，K48泛素链是降解性的，而K63链具有非降解性信号 功能。值得注意的是，我们通过新方法获得的初步结果表明，情况恰恰相反 KCNQ1和HERG为真，可能揭示了细胞质和膜之间的根本区别 蛋白质。我们的初步结果进一步表明，异常泛素化可能是KCNQ1/HERG的基础 一些LQT1/LQT2突变中的运输缺陷，这一途径可能是为了纠正潜在的 异常现象。我们的长期目标是阐明控制表面密度的分子机制 生理和病理条件下心脏KCNQ1和HERG通道的功能调节 条件，并弥合机械洞察力，以推进LQTS和LIFE的个性化治疗- 严重的心律失常。我们结合了最先进的创新方法：开发工程化的E3 连接酶/DUBS实现对KCNQ1/HERG泛素化的前所未有的时空控制； 吞吐量流式细胞术；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E3连接酶来控制KCNQ1的时空、连锁特异性泛素化 和Herg，并阐明这些通道的泛素密码调节。2)开发利用工程化 去泛素酶控制KCNQ1和HERG的时空、连锁特异性去泛素化 这些频道的泛素码调节。3)确定异常泛素化在不同贩运中的作用- 缺乏LQT1/LQT2突变，并评估拯救机会。

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.