Inhibition of NF-κB activation by peptides targeting NF-κB essential modulator (NEMO) oligomerization
Inhibition of NF-κB activation by peptides targeting NF-κB essential modulator (NEMO) oligomerization
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DOI：
10.1074/jbc.m406423200
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发表时间：
2004-12-24
影响因子：
4.8
通讯作者：
Véron, M
中科院分区：
文献类型：
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作者：
Agou, F;Courtois, G;Véron, M
NF-kappaB必需调节剂/IKK-gamma （NEMO/IKK-gamma）在NF-kappaB通路响应促炎刺激的激活中起关键作用。先前的研究表明IKK复合物的信号依赖性激活涉及NEMO的三聚化。该蛋白的最小寡聚化结构域由两个名为coil -coil 2 （CC2）和leucine zipper （LZ）的coil -coil亚结构域组成(Agou， F., Traincard， F., Vinolo， E., Courtois， G., Yamaoka， S., Israel， A.和Veron， M. (2004) J. Biol。化学，279,27861-27869)。为了寻找抑制NF-kappaB活化的药物，我们合理设计了对应于CC2和LZ亚结构域的细胞渗透肽，模拟NEMO亚基之间的接触区域。这些肽被标记为Antennapedia/Penetratin基序，并在脂多糖刺激之前传递给细胞。用荧光活化细胞分选仪监测多肽转导，在稳定转染的pre- b70z /3淋巴细胞中，用NF-kappaB依赖的β -半乳苷酶测定定量它们对脂多糖诱导的NF-kappaB活化的影响。我们发现LZ和CC2亚结构域对应的肽抑制NF-kappaB的激活，IC50在muM范围内。控制肽，包括突变的CC2和LZ肽和异源卷曲肽，没有抑制作用。设计的肽能够诱导具有组成性NF-kappaB活性的人视网膜母细胞瘤Y79细胞的细胞死亡。我们的研究结果为通过靶向NEMO蛋白的寡聚化状态来抑制NF-kappaB通路激活的新策略提供了“概念证明”。
NF-kappaB essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappaB pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappaB activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappaB activation was quantified using an NF-kappaB-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappaB activation with an IC50 in the muM range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappaB activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappaB pathway activation through targeting the oligomerization state of the NEMO protein.