The Ca-ATPase isoform 2a (SERCA2a) re-sequesters cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. A central factor in heart failure is abnormally high cytosolic [Ca2+], resulting in pathophysiology and decreased cardiac performance. Therefore, augmentation of the SERCA2a Ca2+ transport activity is a promising therapeutic approach. A novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing the protein phospholamban (PLB) from its inhibitory site on SERCA2a. In the present study, we have developed several cell-based FRET biosensor systems for time-resolved FRET (TR-FRET) measurements of the protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the putative SERCA2a binding site, we transiently transfected DWORF into a stable cell line expressing SERCA2a labeled with green fluorescent protein (GFP, the FRET donor) and PLB labeled with red fluorescent protein (RFP, the FRET acceptor). We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Functional analysis demonstrates that DWORF activates SERCA in both the presence and absence of PLB. Furthermore, using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues that are necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

中文翻译：

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DWORF的跨膜结构域直接激活SERCA。P15和W22残基至关重要。

Ca-ATPase同工型2a（SERCA2a）将胞质Ca2 +重新隔离到心肌细胞的肌质网（SR）中，从而在舒张期使肌肉松弛。心力衰竭的中心因素是异常高的胞浆[Ca2 +]，导致病理生理和心脏功能下降。因此，增强SERCA2a Ca2 +转运活性是一种有前途的治疗方法。提出了一种新的跨膜肽，矮开放阅读框（DWORF），通过从其在SERCA2a的抑制位点置换蛋白磷脂酰磷脂（PLB）来增强SR Ca2 +的摄取和心肌细胞的收缩性。在本研究中，我们已经开发了几种基于细胞的FRET生物传感器系统，用于时间分辨FRET（TR-FRET）测量SERCA2a与PLB和/或DWORF的蛋白质-蛋白质相互作用和结构变化。为了测试DWORF与PLB竞争以占据假定的SERCA2a结合位点的假设，我们将DWORF瞬时转染到稳定的细胞系中，该细胞系表达用绿色荧光蛋白（GFP，FRET供体）标记的SERCA2a和用红色荧光蛋白（RFP， FRET受体）。我们观察到FRET效率的显着降低，与SERCA2a与PLB结合的比例的降低相一致。功能分析表明，无论有无PLB，DWORF均可激活SERCA。此外，使用定点诱变，我们生成了不激活SERCA的DWORF变体，从而鉴定了功能性SERCA2a-DWORF相互作用所必需的残基。