Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2).
Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2).
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DOI：
10.1074/jbc.m114.616664
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发表时间：
2015-02-13
期刊：
影响因子：
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通讯作者：
Ciulli A
中科院分区：
文献类型：
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作者：
Bulatov E;Martin EM;Chatterjee S;Knebel A;Shimamura S;Konijnenberg A;Johnson C;Zinn N;Grandi P;Sobott F;Ciulli A
背景：CRL E3连接酶的组成亚基组装成特定的复合体。结果：从人细胞裂解液中鉴定了CRL5SOCS2的组成成分，体外重组了完整的复合体，并对蛋白质-蛋白质相互作用进行了生物物理特征分析。结论：CRL5SOCS2组分以单体形式存在，提出的结构模型得到了离子迁移质谱图的支持。意义：我们提供了对全尺寸CRL5SOCS2组装的结构见解，可以帮助开发靶向CRL复合体的小分子。多亚单位cullin环E3泛素连接酶(CRL)针对翻译后修饰的底物进行泛素化和蛋白酶体降解。细胞因子信号转导抑制因子(SOCs)蛋白在炎症过程、糖尿病和癌症中发挥重要作用，因此是治疗干预的有吸引力的靶点。在其他功能中，SOCS蛋白是CRL5复合体的底物受体。CRL家族成员SOCS2-EloBC-Cul5-Rbx2(CRL5SOCS2)与磷酸化生长激素受体结合为其主要底物。在这里，我们证明了CRL5SOCS2的成分可以使用磷酸化生长激素受体多肽装饰的珠子从K562细胞裂解物中特异性地提取出来。随后，分别在大肠杆菌和Sf21昆虫细胞中重组表达的SOCS2-EloBC和全长Cul5-Rbx2用于体外重组CRL5SOCS2复合物。最后，用不同的生物物理方法研究了配合物的组装和相互作用。与其他E3连接酶不同的是，CRL5SOCS2以单体形式存在，这一结果通过多角度静态光散射和天然MS的大小排除层析得到证实。行波离子迁移率质谱仪数据支持全尺寸CRL5SOCS2络合物的结构模型。
Background: The component subunits of CRL E3 ligases assemble into specific complexes. Results: Components of CRL5SOCS2 were identified from human cell lysate, the full-size complex was reconstituted in vitro, and protein-protein interactions were biophysically characterized. Conclusion: CRL5SOCS2 components exist in a monomeric state, and proposed structural models are supported by ion mobility mass spectrometry. Significance: We provide structural insights into the assembly of full-size CRL5SOCS2 that can aid development of small molecules targeting CRL complexes. The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes, and cancer and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5SOCS2 complexes is supported by traveling wave ion mobility mass spectrometry data.