细胞穿透肽(CPPs)是短肽序列,其具有穿过细胞膜并递送货物的能力。尽管CPP以最小的脱靶效应完成这项任务至关重要,在许多情况下,这种行动没有得到强有力的筛选。我们目前研究了常用的CPPsTAT和聚精氨酸Arg9和Arg11是否对细胞Ca2稳态产生脱靶作用。在使用心脏左心室或比目鱼肌的肌细胞和匀浆的实验中,我们观察到与聚精氨酸CPPs孵育后,Ca2再循环到肌浆网(SR)的明显抑制作用。在这两个组织中,SRCa2+泄漏率保持不变,表明从细胞溶质中长期去除Ca2+源于对SRCa2+ATP酶2(SERCA2)的抑制。用TAT处理后没有发生这种抑制作用,或在表达SERCA1的趾长伸肌的制剂中。在过表达单个SERCA同工型的HEK细胞中的实验证实,聚精氨酸孵育特异性抑制SERCA2a和2b的活性,但不是SERCA1或3。SERCA2活性的衰减不依赖于磷脂的存在,基于ELISA的分析揭示了多聚精氨酸与蛋白质的致动器结构域之间的直接相互作用。表面等离子体共振实验证实了SERCA2的该区域内的强结合,以及两种物质之间的缓慢解离。基于这些观察,我们敦促谨慎使用聚精氨酸CPPs。的确,SERCA2在不同的细胞类型中表达,在实验和治疗环境中,应预期SERCA2结合和抑制的广泛后果.
Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg9 and Arg11 exert off-target effects on cellular Ca2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (
SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of
SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of
SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as
SERCA2 is expressed in diverse cell types, the wide-ranging consequences of
SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.