Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Iα inhibit NO-induced cerebral dilation
Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Iα inhibit NO-induced cerebral dilation
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DOI：
10.1073/pnas.97.26.14772
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发表时间：
2000-12-19
影响因子：
11.1
通讯作者：
Tegge, WJ
中科院分区：
文献类型：
--
作者：
Dostmann, WRG;Taylor, MS;Tegge, WJ
通过使用P-32-自磷酸化cGMP依赖性蛋白激酶1 α（cGPK）筛选纤维素纸上的八聚体肽文库阵列，以鉴定对cGPK具有高结合亲和力的肽序列。肽中每个氨基酸位置的迭代去卷积鉴定序列LRK 5 H（W 45）具有最高的结合亲和力。W 45与cGPK的结合导致对cGPK和cAMP依赖性蛋白激酶（cAPK）的选择性抑制，Ki值分别为0.8 μ M和560 μ M。将W 45融合到来自HIV-1达特蛋白（YGRKKRRQRRRPP-LRK 5 H，DT-2）或果蝇足同源域（RQIKIWIPNRRMKWKK-LRK 5 H，DT-3）的膜转位信号被证明是用于细胞内递送这些高电荷肽的有效方法。通过使用荧光素标记的DT-2和DT-3证明了肽快速易位到完整的脑动脉中。融合肽的抑制效力甚至大于W 45的抑制效力，DT-2和DT-3的Ki值分别为12.5 nM和25 nM。两种肽仍然是cAPK的不良抑制剂。在体外证明了DT-2或DT-3在cAPK存在下对cGPK的选择性抑制。在加压脑动脉中，DT-2和DT-3显著降低NO诱导的扩张。本研究提供了一类选择性cGPK抑制剂肽在血管平滑肌中的功能表征，并揭示了cGPK在调节血管收缩性中的核心作用。
Arrays of octameric peptide libraries on cellulose paper were screened by using P-32-autophosphorylated cGMP-dependent protein kinase 1 alpha (cGPK) to identify peptide sequences with high binding affinity for cGPK. Iterative deconvolution of every amino acid position in the peptides identified the sequence LRK5H (W45) as having the highest binding affinity. Binding of W45 to cGPK resulted in selective inhibition of the kinase with K-i values of 0.8 muM and 560 muM for cGPK and cAMP-dependent protein kinase (cAPK), respectively. Fusion of W45 to membrane translocation signals from HIV-1 tat protein (YGRKKRRQRRRPP-LRK5H, DT-2) or Drosophila Antennapedia homeo-domain (RQIKIWFQNRRMKWKK-LRK5H, DT-3) proved to be an efficient method for intracellular delivery of these highly charged peptides. Rapid translocation of the peptides into intact cerebral arteries was demonstrated by using fluorescein-labeled DT-2 and DT-3. The inhibitory potency of the fusion peptides was even greater than that for W45, with K-i values of 12.5 nM and 25 nM for DT-2 and DT-3, respectively. Both peptides were still poor inhibitors of cAPK. Selective inhibition of cGPK by DT-2 or DT-3 in the presence of cAPK was demonstrated in vitro. In pressurized cerebral arteries, DT-2 and DT-3 substantially decreased NO-induced dilation. This study provides functional characterization of a class of selective cGPK inhibitor peptides in vascular smooth muscle and reveals a central role for cGPK in the modulation of vascular contractility.