Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling.
Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling.
复制标题
扩张型心肌病变异体 R14del 增加了受磷蛋白五聚体的稳定性，削弱了心脏钙处理的动态调节。
DOI：
10.1101/2023.05.26.542463
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Robia,SethL
中科院分区：
文献类型：
--
作者：
Cleary,SeanR;Teng,AllenCT;Kongmeneck,AudreyDeyawe;Fang,Xuan;Phillips,TaylorA;Cho,EllenE;Kekenes-Huskey,Peter;Gramolini,AnthonyO;Robia,SethL
肌浆网Ca ~（2+）ATP酶（SERCA）是一种膜转运蛋白，能产生和维持细胞内Ca ~（2+）的储存。在心脏中，SERCA通过与跨膜微肽受磷蛋白（PLB）的单体形式的抑制性相互作用来调节。PLB还形成了贪婪的同源五聚体，并且五聚体和SERCA之间PLB的动态交换是心脏对运动的反应性的重要决定因素。在这里，我们研究了两种自然发生的致病性PLB变体：Arg 9的半胱氨酸取代（R9 C）和Arg 14的框内缺失（R14 del）。这两种变异都与扩张型心肌病有关。我们以前表明，R9 C突变导致二硫键交联和五聚体的超稳定。虽然R14 del的致病机制尚不清楚，但我们假设该突变也可能改变五聚体的稳定性。免疫印迹显示，与WT-PLB相比，R14 del-PLB的五聚体：单体比率显著增加。我们使用荧光共振能量转移（FRET）显微镜定量活细胞中的同源寡聚化和SERCA结合。与WT相比，R14 del-PLB显示出对同源寡聚化的增加的亲和力和对SERCA的降低的结合亲和力。这些数据表明，与R9 C一样，R14 del突变使PLB稳定在五聚体中，降低了其调节SERCA的能力。R14 del突变降低了钙离子瞬时升高后PLB从五聚体中解结合的速率，限制了PLB-SERCA复合物的恢复。一个计算模型预测，R14 del对PLB五聚体的超稳定性损害了心脏Ca 2+处理对休息和运动之间心率变化的反应能力。我们推测，受损的生理应激反应有助于在人类携带者的R14 del突变的mammogenesis。
The sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and the dynamic exchange of PLB between pentamers and SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic variants of PLB: a cysteine substitution of Arg9 (R9C) and an in-frame deletion of Arg14 (R14del). Both variants are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized this mutation may also alter pentamer stability. Immunoblots revealed a significantly increased pentamer: monomer ratio for R14del-PLB compared to WT-PLB. We quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT. The data suggest that, like R9C, the R14del mutation stabilizes PLB in pentamers, decreasing its ability to regulate SERCA. The R14del mutation reduced the rate of PLB unbinding from pentamers after transient elevations of Ca2+, limiting the recovery of PLB-SERCA complexes. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca2+handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.