In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.
In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.
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基于 FRET 的原位心肌细胞中肌球蛋白结合蛋白 C 的 N 末端定位。
DOI：
10.1073/pnas.2222005120
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发表时间：
2023-03-21
影响因子：
11.1
通讯作者：
Kampourakis, Thomas
中科院分区：
文献类型：
--
作者：
Chandler, Jessica;Treacy, Conor;Ameer-Beg, Simon;Ehler, Elisabeth;Irving, Malcolm;Kampourakis, Thomas
关键词：
心肌肌球蛋白结合蛋白-C(cMyBP-C)是一组与心脏疾病和心力衰竭的发生密切相关的肌瘤蛋白之一。然而，cMyBP-C调节收缩的分子机制却知之甚少。在这项研究中，我们使用了一种新开发的基于荧光寿命的Foerster共振能量转移分析(FRET)方法来监测cMyBP-C在天然环境中的调节作用。我们发现，在心肌的松弛状态下，cMyBP-C存在多个群体，反映了其N-末端结构域的细丝结合和粗丝结合状态，这些状态进一步受到磷酸化的调节。我们的发现对健康和疾病中的心肌功能具有重要的意义。心肌肌球蛋白结合蛋白-C(cMyBP-C)是一种厚丝相关调节蛋白，在肥厚型心肌病(HCM)患者中经常发现突变。最近的体外实验强调了其N-末端区域(NcMyBP-C)对心肌收缩的功能意义，报道了与粗丝和细丝的调节相互作用。为了更好地了解cMyBP-C在其天然肌节环境中的相互作用，建立了原位Foerster共振能量转移-荧光寿命成像技术(FRET-FLIM)来确定NcMyBP-C与分离的新生大鼠心肌细胞(NRCs)粗丝和细丝之间的空间关系。体外研究表明，将基因编码的荧光团与NcMyBP-C连接对其与粗丝蛋白和细丝蛋白的结合没有或几乎没有影响。用该方法检测了NcMyBP-C标记的MTFP与Phalloidin-iFluor 514标记NRCS中肌动蛋白细丝之间的FRET。测得的FRET效率介于供体连接到心肌肌球蛋白调节轻链(粗纤维)和肌钙蛋白T(细纤维)之间。这些结果与cMyBP-C的多种构象共存一致，其中一些构象的N-末端结构域与细丝结合，另一些构象与粗丝结合，支持了这些构象之间的动态交换在收缩调节中介导了丝间信号的假设。此外，用β肾上腺素能激动剂刺激NRCs可以减少NcMyBP-C和肌动蛋白结合的Phalloidin之间的FRET，这表明cMyBP-C的磷酸化减少了它与细丝的相互作用。
Cardiac myosin binding protein-C (cMyBP-C) is one of a group of sarcomeric proteins that have been frequently implicated in the development of heart disease and heart failure. However, the molecular mechanisms underlying contractile regulation by cMyBP-C are poorly understood. In this study, we used a newly developed fluorescence lifetime-based Foerster resonance energy transfer assay (FRET) approach to monitor cMyBP-C’s regulatory interactions in the native environment of the intact sarcomere lattice of isolated cardiac muscle cells. We show that multiple populations of cMyBP-C exist during the relaxed state of cardiac muscle, reflecting thin and thick filament–bound states of its N-terminal domains, which are further modulated by phosphorylation. Our findings have important implications for heart muscle function in health and disease. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament–associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer–fluorescence lifetime imaging (FRET–FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with β-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.
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DOI：
10.1074/jbc.ac118.004816
发表时间：
2018-09-14
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Kampourakis T;Ponnam S;Sun YB;Sevrieva I;Irving M
通讯作者：
Irving M
影响因子：
2.9
作者：
Belknap, Betty;Harris, Samantha P.;White, Howard D.
通讯作者：
White, Howard D.
影响因子：
3.5
作者：
Gruen, M;Prinz, H;Gautel, M
通讯作者：
Gautel, M
DOI：
10.1073/pnas.1521281113
发表时间：
2016-03-22
影响因子：
11.1
作者：
Colson, Brett A.;Thompson, Andrew R.;Thomas, David D.
通讯作者：
Thomas, David D.
影响因子：
5.6
作者：
Gollapudi, Sampath K.;Gallon, Clare E.;Chandra, Murali
通讯作者：
Chandra, Murali