Phospholamban mutants compete with wild type for SERCA binding in living cells.
Phospholamban mutants compete with wild type for SERCA binding in living cells.
复制标题
Phospholamban 突变体与野生型竞争活细胞中的 SERCA 结合。
DOI：
10.1016/j.bbrc.2012.02.125
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发表时间：
2012-04-06
影响因子：
3.1
通讯作者：
Thomas DD
中科院分区：
文献类型：
--
作者：
Gruber SJ;Haydon S;Thomas DD
我们利用在HEK细胞中稳定表达的荧光融合蛋白直接检测活细胞中肌浆网Ca-ATP酶（SERCA）和受磷蛋白（PLB）之间的相互作用，以设计用于基因治疗的PLB突变体。肌细胞中的Ca 2+循环强烈依赖于SERCA。心力衰竭（HF）占美国死亡人数的12%，通常表现出SERCA活性降低，HF的几种潜在疗法旨在增加SERCA活性。我们正在研究使用LOF-PLB突变体（PLBM）作为基因治疗载体，以增加SERCA活性。活性SERCA 1a和WT-PLB，在其N末端标记有荧光蛋白（CFP和YFP），在稳定的HEK细胞系中共表达，并使用荧光共振能量转移（FRET）直接检测它们的相互作用。由毛喉素诱导的PLB磷酸化导致FRET从CFP-SERCA增加到YFP-PLB，表明SERCA抑制可以在不解离复合物的情况下减轻。这表明LOF突变体可能以足够的亲和力与SERCA结合，以有效地完成WT-PLB，从而减轻SERCA抑制。因此，我们在CFP-SERCA/YFP-PLB细胞系中瞬时表达了一系列PLBM，并发现FRET降低，这意味着PLBM和WT-PLB之间竞争结合SERCA。这些结果确立了这种FRET测定法作为筛选PLBM的快速定量手段，以优化基因治疗以激活SERCA，这是HF基因治疗所需的。
We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca2+ cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLBM) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLBM in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLBM and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLBM for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.