Chip phosphorylation stimulates the degradation of mutant transthyretin to attenuate cardiac amyloidosis

芯片磷酸化刺激突变运甲状腺素蛋白的降解以减轻心脏淀粉样变性

• 批准号: 10905158
• 负责人: Mark John Ranek
• 金额: $ 66.97万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10905158
• 关键词: Address; Affect; Amyloid Fibrils; Attenuated; Autophagocytosis; Biopsy; Cardiac; Cardiac Myocytes; Cell Death; Complex; Cyclic GMP-Dependent Protein Kinases; Data; Deposition; Depressed mood; Devices; Dissociation; Excision; Excretory function; Female; Fibroblasts; Functional disorder; GCG gene; Gene Mutation; Genes; Hallervorden-Spatz Syndrome; Heart; Heart Diseases; Heat Shock 70kD Protein Binding Protein; Hepatic; Human; In Vitro; Incubated; Isoleucine; Knock-in Mouse; LIF gene; Lysosomes; Mediating; Mediator; Methods; Microfilaments; Modeling; Molecular Chaperones; Mus; Mutation; Myocardium; Organoids; Pathogenesis; Patients; Phosphorylation; Physiological; Plasma; Postmenopause; Prealbumin; Process; Proteins; Qi; RBBP9 gene; Relaxation; Reporting; Restrictive Cardiomyopathy; Serine; Signal Transduction; Testing; Valine; Woman; Work; biobank; cardiac amyloidosis; cardiac tissue engineering; cytotoxicity; heart function; human tissue; improved; in vivo; induced pluripotent stem cell derived cardiomyocytes; insight; male; men; meter; multicatalytic endopeptidase complex; mutant; new therapeutic target; novel; novel therapeutic intervention; pharmacologic; protein aggregation; protein degradation; protein protein interaction; proteostasis; therapeutic target; tool; ubiquitin ligase; uptake

PROJECT SUMMARY/ABSTRACT Cardiac amyloidosis can be caused by a mutation in transthyretin (TTR) (e.g. valine 122 to isoleucine, VI) [ATTR- CM] that will aggregate when taken up by the myocardium, resulting in cytotoxicity and ultimately dysfunction. The mechanisms underlying the pathogenesis of ATTR-CM remain unknown. Further, methods to enhance the degradation of dissociated and deposited transthyretin is a critical unmet need. We reported protein kinase G (PKG) can enhance protein degradation via the proteasome and lysosome to attenuate cardiac disease. We recently uncovered that PKG also phosphorylates a ubiquitin ligase/co-chaperone, Chip (carboxyl terminus of Hsc70-interacting protein), at serine 19 (human; S20, mouse). Chip is a primary mediator of cardiomyocyte proteostasis by ubiquitinating and shuttling proteins for degradation. With new and exciting pilot data we show PKG activity and Chip S19 phosphorylation (pS19) are uniquely depressed in ATTR-CM patients. We also reveal cardiomyocytes isolated from ATTR-CM patients have reduced myofibrillar function. The field has been stymied by lack of models, especially in vivo, and access to human tissue. We addressed these limitations by creating novel models and a biorepository of biopsies from ATTR-CM patients. In vitro, we developed engineered heart tissue (EHT) and cardiac organoids formed from human iPSC-derived cardiomyocytes and fibroblasts. To create ATTR-CM in vitro we incubate EHTs in TTRVI (5 µM, same as ATTR-CM plasma) or culture cardiac organoids with TTRVI hepatic organoids (excretes TTRVI at a similar concentration) in an interconnected microphysiological device for 14 days, resulting in cellular uptake, protein aggregation, lower PKG activity, cell death, and (in EHTs) reduced function. Our new TTRVI knock in mouse develops diastolic dysfunction, increased expression of fibrotic genes, and decreased PKG signaling. Males and ovariectomized female mice, but not intact females, develop ATTR-CM, similar to human ATTR-CM which affects men and post-menopausal women. Our pilot data shows activating PKG or expressing a Chip pS19-mimic (ChipSE) facilitates the clearance of TTRVI to enhance cardiac function (mice and EHTs) and reduce cytotoxicity (organoids). This project will provide new mechanistic insight into ATTR-CM by testing the impact of PKG activity and Chip pS19 in vitro and in vivo, tests a new therapeutic strategy, and determines the translational relevance in human patients. Aim 1 tests if PKG stimulation or ChipSE attenuates markers of ATTR-CM in vitro and if chaperone-mediated autophagy is the degradative process utilized. We also developed and will further test a novel tool (PROTAC) specifically targeting Chip for TTR to enhance TTRVI removal. In Aim 2, we test the ability of various PKG activators protect against ATTR-CM and if this occurs in a Chip pS20 dependent manner. In Aim 3, we will test the relevance of PKG signaling and Chip pS19 in human ATTR-CM. We further interrogate myofilaments isolated from ATTR-CM human myocardium and test if PKG activation can improve contraction and relaxation. The work is highly translational, as it provides key mechanistic insight into and potentially identifies a new therapeutic target for ATTR-CM patients.

项目摘要/摘要 心脏淀粉样蛋白病可能是由经腹蛋白（TTR）突变引起的（例如，valine 122 to Isoleucine，vi）[attry-） cm]将通过心肌吸收时会聚集，从而导致细胞毒性和最终功能障碍。 ATTR-CM发病机理的基础机制仍然未知。此外，增强的方法 解离和沉积的转染素的降解是至关重要的未满足需求。我们报道了蛋白激酶G （PKG）可以通过蛋白酶体和溶酶体增强蛋白质降解，从而减轻心脏病。我们 最近发现，PKG还磷酸化了泛素连接酶/co-Chaperone，Chip（Chip）（羧基末端 HSC70相互作用蛋白），在丝氨酸19（人； S20，小鼠）。芯片是心肌细胞的主要介体 通过泛素化和穿梭蛋白质降解蛋白质的蛋白质静脉曲张。我们显示的新的和令人兴奋的飞行员数据 PKG活性和CHIP S19磷酸化（PS19）在ATTR-CM患者中独特地抑郁。我们也揭示 从ATTR-CM患者中分离出的心肌细胞降低了肌原纤维功能。该领域被阻碍了 由于缺乏模型，尤其是体内，并获得人体组织。我们通过创建来解决这些限制 新型模型和来自Attr-CM患者的活检的生物遗物。在体外，我们开发了工程的心脏 由人IPSC衍生的心肌细胞和成纤维细胞形成的组织（EHT）和心脏器官。创建 ATTR-CM体外我们在TTRVI（5 µM，与Attr-CM等离子体）或培养心脏器官中孵育EHT 在互连的微生物生理学中，与TTRVI肝癌（以相似浓度的排泄TTRVI）一起使用 设备14天，导致细胞摄取，蛋白质聚集，较低的PKG活性，细胞死亡和（在EHT中） 功能降低。我们新的TTRVI敲击小鼠发育型舒张功能障碍，纤维化的表达增加 基因和改善的PKG信号传导。雄性和卵巢切除的雌性小鼠，但不是完整的女性 Attr-CM，类似于影响男性和绝经后妇女的人类Attr-CM。我们的飞行员数据显示 激活PKG或表达芯片PS19-MIMIM（芯片）促进了TTRVI的清除以增强心脏 功能（小鼠和EHT）并降低细胞毒性（器官）。该项目将提供新的机械洞察力 通过在体外和体内测试PKG活动和芯片PS19的影响进入ATTR-CM，测试一种新的疗法 策略，并确定人类患者的翻译相关性。 AIR 1测试是PKG刺激或芯片 在体外减弱ATTR-CM的标记，如果伴侣介导的自噬是降解过程 利用。我们还开发了并将进一步测试一种专门针对TTR的芯片的新颖工具（Protac） 增强TTRVI去除。在AIM 2中，我们测试了各种PKG激活剂的能力，可以防止Attr-CM以及 这是以CHIP PS20的依赖方式发生的。在AIM 3中，我们将测试PKG信号和芯片的相关性 人类attr-cm中的ps19。我们进一步询问与attr-cm人体心肌分离的肌膜和 测试PKG激活是否可以改善收缩和放松。这项工作是高度翻译的，因为它提供了关键 对ATTR-CM患者的机械洞察力并有可能确定一个新的治疗靶点。