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:
- 金额:$ 66.97万
- 依托单位:
- 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:
- 关键词:AddressAffectAmyloid FibrilsAttenuatedAutophagocytosisBiopsyCardiacCardiac MyocytesCell DeathComplexCyclic GMP-Dependent Protein KinasesDataDepositionDepressed moodDevicesDissociationExcisionExcretory functionFemaleFibroblastsFunctional disorderGCG geneGene MutationGenesHallervorden-Spatz SyndromeHeartHeart DiseasesHeat Shock 70kD Protein Binding ProteinHepaticHumanIn VitroIncubatedIsoleucineKnock-in MouseLIF geneLysosomesMediatingMediatorMethodsMicrofilamentsModelingMolecular ChaperonesMusMutationMyocardiumOrganoidsPathogenesisPatientsPhosphorylationPhysiologicalPlasmaPostmenopausePrealbuminProcessProteinsQiRBBP9 geneRelaxationReportingRestrictive CardiomyopathySerineSignal TransductionTestingValineWomanWorkbiobankcardiac amyloidosiscardiac tissue engineeringcytotoxicityheart functionhuman tissueimprovedin vivoinduced pluripotent stem cell derived cardiomyocytesinsightmalemenmetermulticatalytic endopeptidase complexmutantnew therapeutic targetnovelnovel therapeutic interventionpharmacologicprotein aggregationprotein degradationprotein protein interactionproteostasistherapeutic targettoolubiquitin ligaseuptake
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.
项目总结/摘要
心脏淀粉样变性可由甲状腺素运载蛋白(TTR)突变(例如缬氨酸122突变为异亮氨酸,VI)引起[ATTR-122]。
CM],当被心肌吸收时将聚集,导致细胞毒性和最终功能障碍。
ATTR-CM的发病机制尚不清楚。此外,还提供了增强免疫应答的方法。
解离和沉积的甲状腺素运载蛋白的降解是一个关键的未满足的需求。我们报道了蛋白激酶G
(PKG)可通过蛋白酶体和溶酶体促进蛋白质降解,从而减轻心脏疾病。我们
最近发现PKG还磷酸化泛素连接酶/共伴侣,Chip(其羧基末端),
Hsc 70相互作用蛋白),在丝氨酸19处(人; S20,小鼠)。Chip是心肌细胞的主要介质
通过遍在化和穿梭蛋白质进行降解来实现蛋白质稳定。通过新的令人兴奋的试验数据,我们展示了
PKG活性和芯片S19磷酸化(pS19)在ATTR-CM患者中被独特地抑制。我们还揭示了
从ATTR-CM患者分离的心肌细胞具有降低的肌原纤维功能。该领域已被阻碍
缺乏模型,特别是体内模型,以及无法获得人体组织。我们通过创建
新的模型和ATTR-CM患者活检的生物储存库。在体外,我们开发了工程心脏
组织(EHT)和由人iPSC衍生的心肌细胞和成纤维细胞形成的心脏类器官。创建
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
器械14天,导致细胞摄取、蛋白质聚集、PKG活性降低、细胞死亡和(EHT)
简化功能。我们的新TTRVI敲入小鼠发展舒张功能障碍,增加纤维化的表达,
基因,并降低PKG信号传导。雄性和卵巢切除的雌性小鼠,但不是完整的雌性,
ATTR-CM,类似于影响男性和绝经后女性的人ATTR-CM。我们的试点数据显示
激活PKG或表达Chip pS19模拟物(ChipSE)促进TTRVI的清除,以增强心脏功能。
功能(小鼠和EHT)和减少细胞毒性(类器官)。该项目将提供新的机械见解
通过测试PKG活性和芯片pS19在体外和体内的影响,将其转化为ATTR-CM,测试了一种新的治疗方法,
策略,并确定人类患者中的翻译相关性。目标1测试PKG刺激或ChipSE
在体外减弱ATTR-CM的标记物,并且如果分子伴侣介导的自噬是降解过程,
利用。我们还开发并将进一步测试一种专门针对TTR芯片的新工具(PROTAC),
增强TTRVI去除。在目的2中,我们测试了各种PKG激活剂保护ATTR-CM的能力,
这以芯片pS20依赖的方式发生。在目标3中,我们将测试PKG信号传导和芯片的相关性。
人ATTR-CM中的pS19。我们进一步询问从ATTR-CM人心肌分离的肌丝,
测试PKG激活是否可以改善收缩和舒张。这项工作是高度翻译,因为它提供了关键
这是对ATTR-CM患者的一种机制性洞察,并可能识别出ATTR-CM患者的新治疗靶标。
项目成果
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