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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要心脏淀粉样变性可由甲状腺素运载蛋白 (TTR) 突变引起（例如缬氨酸 122 变为异亮氨酸，VI）[ATTR- CM] 当被心肌吸收时会聚集，导致细胞毒性并最终导致功能障碍。此外，增强 ATTR-CM 发病机制的方法仍不清楚。我们报道了蛋白激酶 G 的关键未满足需求。 (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。最近发现 PKG 还磷酸化泛素连接酶/共伴侣 Chip（Chip 的羧基末端） Hsc70 相互作用蛋白），位于丝氨酸 19（人；S20，小鼠）。通过泛素化和穿梭蛋白质降解来实现蛋白质稳态。我们展示了新的、令人兴奋的试验数据。我们还发现，ATTR-CM 患者的 PKG 活性和 Chip S19 磷酸化 (pS19) 特别受到抑制。从 ATTR-CM 患者中分离出的心肌细胞的肌原纤维功能降低了，这一领域受到了阻碍。由于缺乏模型，尤其是体内模型，以及人体组织的获取，我们通过创建来解决这些限制。我们在体外开发了工程心脏。由人类 iPSC 衍生的心肌细胞和成纤维细胞形成的组织 (EHT) 和心脏类器官。 ATTR-CM 体外，我们在 TTRVI（5 µM，与 ATTR-CM 血浆相同）中孵育 EHT 或培养心脏类器官与 TTRVI 肝类器官（以相似浓度排泄 TTRVI）在相互关联的微生理学中设备持续 14 天，导致细胞摄取、蛋白质聚集、PKG 活性降低、细胞死亡，以及（在 EHT 中）我们新的 TTRVI 敲除小鼠会导致舒张功能障碍，纤维化表达增加。雄性和切除卵巢的雌性小鼠（但未切除卵巢的雌性小鼠）会发育。 ATTR-CM 与人类 ATTR-CM 类似，我们的试点数据显示，它会影响男性和绝经后女性。激活 PKG 或表达 Chip pS19-mimic (ChipSE) 可促进 TTRVI 的清除，从而增强心脏功能该项目将提供新的机制见解。通过在体外和体内测试 PKG 活性和芯片 pS19 的影响，进入 ATTR-CM，测试一种新的治疗方法策略，并确定人类患者的翻译相关性，目标 1 测试 PKG 刺激或 ChipSE。体外减弱 ATTR-CM 标记物，并且如果伴侣介导的自噬是降解过程我们还开发了并将进一步测试一种专门针对 TTR 芯片的新工具 (PROTAC)。在目标 2 中，我们测试了各种 PKG 激活剂抵御 ATTR-CM 的能力。这以芯片 pS20 相关的方式发生。在目标 3 中，我们将测试 PKG 信号和芯片的相关性。我们进一步研究了从 ATTR-CM 人类心肌中分离出的肌丝和测试 PKG 激活是否可以改善收缩和松弛这项工作具有高度转化性，因为它提供了关键。深入了解 ATTR-CM 患者的机制并可能确定新的治疗靶点。