Recognition mechanism of p63 by the E3 ligase Itch Novel strategy in the study and inhibition of this interaction
Recognition mechanism of p63 by the E3 ligase Itch Novel strategy in the study and inhibition of this interaction
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DOI：
10.4161/cc.21918
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发表时间：
2012-10-01
期刊：
影响因子：
4.3
通讯作者：
Melino, Sonia
中科院分区：
文献类型：
--
作者：
Bellomaria, Alessia;Barbato, Gaetano;Melino, Sonia
含HECT的E3泛素连接酶Itch介导参与细胞特化和命运的几种蛋白质的降解，包括p63和p73。痒包含四个WW结构域，其对于在靶底物上的识别是必需的，所述靶底物包含短的富含脯氨酸的序列。含有这些结构域的几种信号传导复合物与人类疾病如肌营养不良、阿尔茨海默病或亨廷顿病有关。为了进一步了解Itch-WW 2结构域的结构决定因素，我们研究了它与p63的相互作用。我们通过3D异源NMR实验，分配了均匀C-13-N-15-标记的Itch-WW 2的主链和侧链。使用其相互作用p63肽pep 63研究了Itch-WW 2结构域与p63的体外相互作用。Pep 63是一个18-mer肽，对应于p63的534-551残基的区域，包含与Itch-WW结构域相互作用的PPxY基序，并且我们鉴定了参与这种分子识别的残基。此外，在这里，采用了PPxY肽构象稳定化的策略，增加了WW-ligand结合。我们证明，环化的pep 63导致增加的肽的生物稳定性和的WW-ligand复合物。还获得了pep 63的稳定的金属结合复合物，并诱导了Itch-WW 2结构域的局部氧化损伤，证明了使用金属-pep 63复合物作为设计金属药物以抑制Itch-WW-p63识别的模型的可能性。因此，我们的数据表明，一种新的策略来研究和抑制痒E3-连接酶的识别机制。
The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer's or Huntington's diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly C-13-N-15-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534-551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase.