Molecular interaction between Smurf1 WW2 domain and PPXY motifs of Smad1, Smad5, and Smad6 - Modeling and analysis
Molecular interaction between Smurf1 WW2 domain and PPXY motifs of Smad1, Smad5, and Smad6 - Modeling and analysis
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DOI：
10.1080/07391102.2007.10507151
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发表时间：
2007-08-01
影响因子：
4.4
通讯作者：
Reddy, Boojala Vijay B.
中科院分区：
文献类型：
--
作者：
Sangadala, Sreedhara;Metpally, Raghu Prasad Rao;Reddy, Boojala Vijay B.
泛素-蛋白酶体蛋白水解途径在细胞周期、基因转录和信号转导等重要生物学过程中发挥重要作用。其中一个重要的调控机制，通过它的骨诱导活性的骨形态发生蛋白（BMP）信号被调制涉及泛素介导的蛋白酶体降解。BMP诱导的受体信号通过活化的受体I磷酸化Smad蛋白而在细胞内传递。磷酸化的Smads 1、5和8（R-Smads）与co-Smad（Smad 4）寡聚化。因此，形成的复合物易位到细胞核并与其他辅因子相互作用以调节下游靶基因的表达。R-Smads在接头区域中含有PPXY基序，其与Smad泛素调节因子I（Smurf 1）相互作用，Smurf 1是一种E3泛素连接酶，其催化靶蛋白的泛素化以用于蛋白酶体降解。Smurf I包含HECT结构域、C2结构域和2个WW结构域（WW 1、WW 2）。靶蛋白中的PPXY基序及其与Smurf I的相互作用可能构成在控制细胞的BMP反应性中调节Smads稳态水平的基础。在这里，我们提出了一个同源性为基础的模型的Smurf 1 WW 2结构域和目标八肽含有PPXY基序的Smurf 1相互作用Smads。我们进行了Smurf I WW 2结构域与Smad 1，Smad 5和Smad 6的PPXY基序的对接，并确定了参与相互作用的关键氨基酸残基。此外，我们提出的实验证据表明，WW 2结构域的Smurf I确实与Smad蛋白的相互作用，并删除WW 2结构域的Smurf I的结果在使用纯化的重组蛋白的结合损失。最后，我们还提出的数据证实，WW 2结构域的Smurf I的删除废除其在体外泛素化测定Smad 1的泛素化活性。这表明WW结构域和Smad PPXY基序之间的相互作用是Smurf 1介导的其天然靶标如Smad 1，Smad 5和Smad 6的泛素化的关键步骤。这项工作有利于进一步的策略，以解开这种相互作用的生物学功能，并帮助设计有效的模拟化合物，模拟或破坏特定的相互作用。
The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction. One of the important regulatory mechanisms by which the bone-inducing activity of the bone morphogenetic protein (BMP) signaling is modulated involves ubiquitin-mediated proteasomal degradation. The BMP induced receptor signal is transmitted intracellularly by phosphorylation of Smad proteins by the activated receptor I. The phosphorylated Smads 1, 5, and 8 (R-Smads) oligomerize with the co-Smad (Smad4). The complex, thus, formed translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. R-Smads contain PPXY motif in the linker region that interacts with Smad ubiquitin regulatory factor I (Smurf 1), an E3 ubiquitin ligase that catalyzes ubiquitination of target proteins for proteasomal degradation. Smurf I contains a HECT domain, a C2 domain, and 2 WW domains (WW1, WW2). The PPXY motif in target proteins and its interaction with Smurf I may form the basis for regulation of steady-state levels of Smads in controlling BMP-responsiveness of cells. Here, we present a homology-based model of the Smurf1 WW2 domain and the target octa-peptides containing PPXY motif of Smurf1-interacting Smads. We carried out docking of Smurf I WW2 domain with the PPXY motifs of Smad1, Smad5, and Smad6 and identified the key amino acid residues involved in interaction. Furthermore, we present experimental evidence that WW2 domain of Smurf I does indeed interact with the Smad proteins and that the deletion of WW2 domain of Smurf I results in loss of its binding to Smads using the purified recombinant proteins. Finally, we also present data confirming that the deletion of WW2 domain in Smurf I abolishes its ubiquitination activity on Smad1 in an in vitro ubiquitination assay. It shows that the interaction between the WW domain and Smad PPXY motif is a key step in Smurf1-mediated ubiquitination of its natural targets such as Smad1, Smad5, and Smad6. This work facilitates further strategies to unravel the biological function of such interactions and help in designing effective mimetic compounds that either mimic or disrupt the specific interaction.