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心肌细胞中Junctophilin-2蛋白钙蛋白酶依赖裂解的分子机理

来源:生物谷 2018-11-14 14:52

心肌细胞的兴奋收缩偶联是控制心肌收缩的中心机制。在这一过程中,细胞膜去极化引起的钙离子内流诱导肌浆网的钙离子释放,进而激动肌丝,诱发收缩。心肌的舒张则伴随着钙离子回收入肌浆网。这一称为钙离子瞬变的的动态平衡控制着心肌细胞的搏动。在细胞水平,兴奋收缩偶联依赖于称为dyad 的特殊微结构。这一结构中,内陷的细胞膜与肌浆网紧密连接,为两个膜结构上离子通道的交互作用提供物理保障,确保了正常的钙离子瞬变。在多种心脏疾病的早期,心肌细胞的dyad即发生变构,细胞膜与肌浆网的联系减弱,破坏了兴奋收缩偶联和钙离子稳态平衡,是造成心功能异常的原因之一。但是,长久以来,人们并不清楚病理刺激引起的dyad变构和转录调控之间是否有内在的交互作用,以及心肌细胞在面临兴奋收缩解偶联的压力时是否有自我保护的机制。

近期,美国爱荷华大学的华人科学家团队发现,维持兴奋收缩偶联的结构蛋白Junctophilin2经蛋白酶切割后获得转录调控功能,参与心肌保护机制。 本文的主要完成人和第一作者是爱荷华大学的郭昂助理教授,通讯作者是爱荷华大学的宋龙生教授。

Junctophilin2 (JP2)一直以来被认为是维持dyad的结构蛋白之一。该蛋白的N端通过MORN结构域附着在细胞膜上,而C端的跨膜结构域锚定在肌浆网膜上,这一结构赋予了该蛋白如桥梁一般连接细胞膜和肌浆网膜,保障了两个膜结构间的紧密联系。心脏组织中的JP2的蛋白水平在多种心脏病模型和心脏病人组织中有明显减少,这与病理状态下兴奋收缩偶联的减弱一致。郭昂博士在宋龙生实验室的工作发现,心肌病理刺激下,钙离子依赖的蛋白酶calpain介导的水解将JP2首尾结构域分离,去除了JP2在维持dyad结构和兴奋收缩偶联中的作用。然而,郭昂博士并未止步于此。通过分析JP2的序列,他发现了JP2的中间区域含有进化保守的细胞核定位信号(NLS)和类似helix-turn-helix的alanine rich region(ARR),JP2 C端的跨膜区间含有出核信号(NES),而主要的calpain酶切位点恰巧位于NLS和NES之间,这一发现促使郭昂博士做出更进一步的推测:calpain介导的酶切在去除全长JP2作为结构蛋白功能的同时,可能释放其NLS活性,诱导其转化为核蛋白发挥新功能。他设计了一个巧妙的实验,证实了通过酶切去除JP2 C端,足以使已经定位在细胞膜上的JP2 N端 (JP2NT)转移到细胞核中,并验证了JP2NT的核定位是由其保守NLS决定的, 而且JPNT通过一个进化保守的ARR与DNA结合。因而,在全长JP2中,NES和NLS作用互相中和,当C端的NES被calpain切除后,NLS发挥作用,引起JP2NT入核, 并与DNA结合。染色质免疫共沉淀实验表明, JP2NT 广泛富集在很多基因的转录起始区域,并影响启动子活性。一系列实验证明,JP2NT识别MEF2 结合元件(MRE)和TATA  box,与MEF2C 和TATA box binding protein (TBP)结合,并抑制MEF2 介导的转录。提升心肌细胞内JP2NT水平改变转录因子MEF2C 和TATA box binding protein (TBP)的DNA结合位点。郭昂博士与宋龙生教授研究团队进一步发现,细胞核内的JP2NT水平在病理刺激下显着升高。心肌特异过表达JP2NT改变了病理刺激引起的心肌转录重构,抑制了心室高压引起的小鼠心肌肥大和心衰的发生。 而缺失JP2 中NLS的突变小鼠则更易发生心肌肥大和心衰,揭示了JP2NT作为转录调控因子的心肌保护作用。

这一发现揭示了一个心肌细胞中兴奋收缩解偶联诱导转录重构的机制,在病理刺激破坏兴奋收缩偶联蛋白的同时,由降解的蛋白片段启动了心肌保护性的转录改变。哺乳动物成年心肌细胞缺乏再生能力,为了长久维持心功能,机体不可以轻易放弃受损的心肌细胞,因而需要自我保护的机制,而JP2蛋白的特性提供了一个这样的机制。作为一个古老的保守蛋白,JP2 的核定位序列和DNA结合区域的保守进化应该是在重要的进化压力下实现的,这些进化压力是否部分来自除心脏以外的组织生理需要,其核蛋白属性是否在其他的组织和生理系统中发挥作用,这些问题值得进一步研究。

Cardiac excitation-contraction (E-C) coupling refers to a cascade of Ca2+-mediated events whereby membrane depolarization leads to cell contraction, and it is central to contractile function of the heart. At the subcellular level, E-C coupling occurs within a specialized ultrastructural microdomain of the cardiomyocyte termed the cardiac dyad. In various forms of heart disease, such as pathological hypertrophy and heart failure, the E-C coupling process is abnormal due, in part, to ultrastructural remodeling. Abnormal Ca2+ homeostasis (as a result of failed E-C coupling) triggers maladaptive remodeling at the transcriptional level, contributing to pathological myocardial remodeling, hypertrophy and heart failure. However, it remains unclear whether cardiomyocytes possess a self-protective or homeostatic mechanism that mitigates adverse myocardial remodeling. It is also unknown whether there is an intrinsic connection between cardiac ultrastructural remodeling at E-C coupling sites and transcriptional reprogramming in stressed hearts.

Junctophilin-2 (JP2) is a structural protein that organizes the E-C coupling ultrastructural machinery in cardiomyocytes. We previously demonstrated that calpain-mediated proteolytic cleavage of JP2 is key to its downregulation in the diseased heart following cardiac stress, and that this cleavage contributes to the observed: loss of ultrastructural integrity at cardiac dyads; E-C uncoupling and disruption of Ca2+ handling that results in heart failure. Computational analyses predicted that JP2 contains a nuclear localization signal (NLS), as well as an alanine-rich region (ARR) with characteristics of a helix-turn-helix structure, a structure motif capable of DNA binding. Herein we tested the hypothesis that JP2 encodes a stress-adaptive transcriptional regulator, which transduces mechanical information (E-C uncoupling) into salutary transcriptional reprogramming in the myocardium in the setting of cardiac stress.

RESULTS: Biochemical, mutagenesis and confocal imaging analyses revealed that stress-induced proteolysis of JP2 liberated an N-terminal fragment (JP2NT) that was imported into the nucleus through its NLS. Further biochemical and microarray assays showed that in the nucleus JP2NT associated with chromatin and regulated transcription of a wide spectrum of genes via an evolutionarily conserved ARR located in the α-helix region of JP2. Chromatin immunoprecipitation sequencing (ChIP-seq) of JP2NT-overexpressing hearts revealed that it bound preferentially to the transcription start sites (TSSs) of genes, and gel shift studies defined the DNA binding motifs of JP2NT as the TATA box and a MEF2-response element (MRE). Elevation of JP2NT levels by JP2NT overexpression altered the in vivo genomic binding profile of TATA-box binding protein (TBP) and MEF2C. In addition, JP2NT suppressed the transcriptional activity of MEF2C by competing for MRE. Importantly, overexpression of JP2NT in mice led to profound reprogramming of the transcriptome in the setting of stress and attenuated hypertrophic remodeling and the progression of heart failure. Conversely, loss of JP2NT function by deletion of the JP2 NLS in mice accelerated the development of hypertrophy and heart failure following cardiac stress.

CONCLUSION: Our data reveal that calpain-mediated cleavage of JP2 transforms this E-C coupling structural protein into a transcriptional regulator that is shuttled into the nucleus and binds to promoters of target genes, inducing cardioprotective transcriptional reprogramming. These data reveal that cardiomyocytes possess a self-protective mechanism that counters pathological transcriptional remodeling following cardiac stress. Our findings also identify an intrinsic direct connection between ultrastructural remodeling and transcriptional reprogramming in the stressed heart.(生物谷Bioon.com)
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