Expansion of protein interaction maps by phage peptide display using MDM2 as a prototypical conformationally flexible target protein
Expansion of protein interaction maps by phage peptide display using MDM2 as a prototypical conformationally flexible target protein
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DOI：
10.1016/j.jmb.2004.01.017
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发表时间：
2004-03-12
影响因子：
5.6
通讯作者：
Hupp, TR
中科院分区：
文献类型：
--
作者：
Burch, L;Shimizu, H;Hupp, TR
在生物化学途径中扩展可能的蛋白质相互作用伙伴是后基因组时代的一个关键分子目标。噬菌体肽展示是一种多功能的体外工具，用于绘制新的蛋白质-蛋白质界面，并且这种技术在扩展蛋白质相互作用图中的优点是可以小心地控制诱饵蛋白构象完整性的体外操纵。噬菌体肽展示用于扩展构象响应蛋白MDM 2的结合蛋白的可能类型。富集的肽取决于MDM 2是无配体的、锌结合的还是RNA结合的，这表明MDM 2构象变化改变了富集的肽配体的类型。通过该技术鉴定的推定/确定的MDM 2结合蛋白的类别包括泛素修饰酶（F-box蛋白、UB连接酶、UBC-E1）和凋亡修饰剂（HSP 90、GAS 1、APAF 1、p53）。在可以检测的许多推定的MDM 2蛋白中，研究了HSP 90对MDM 2活性的影响，因为HSP 90与人类癌症中的p53蛋白解折叠有关。需要锌离子来重建稳定的MDM 2-HSP 90蛋白复合物。锌结合将MDM 2从单体转化为寡聚体，并激活MDM 2与其内部RING指结构域的结合，为MDM 2蛋白结合锌时的构象变化提供了证据。重组的HSP 90-MDM 2蛋白复合物在体外刺激p53四聚体的解折叠。从人细胞中纯化的能够在体外环境温度下解折叠p53的p53 DNA结合抑制剂含有HSP 90和MDM 2的共纯化池。这些数据突出了噬菌体肽展示作为一种强有力的体外方法来鉴定与构象柔性蛋白质如MDM 2结合的调节蛋白的实用性。(C)2004爱思唯尔有限公司保留所有权利。
Expanding on the possible protein interaction partners in a biochemical pathway is one key molecular goal in the post-genomic era. Phage peptide display is a versatile in vitro tool for mapping novel protein-protein interfaces and the advantage of this technique in expanding protein interaction maps is that in vitro manipulation of the bait protein conformational integrity can be controlled carefully. Phage peptide display was used to expand on the possible types of binding proteins for the conformationally responsive protein MDM2. Peptides enriched differ depending upon whether MDM2 is ligand-free, zinc-bound, or RNA-bound, suggesting that MDM2 conformational changes alter the type of peptide ligands enriched. Classes of putative/established MDM2-binding proteins identified by this technique included ubiquitin-modifying enzymes (F-box proteins, UB-ligases, UBC-E1) and apoptotic modifiers (HSP90, GAS1, APAF1, p53). Of the many putative MDM2 proteins that could be examined, the impact of HSP90 on MDM2 activity was studied, since HSP90 has been linked with p53 protein unfolding in human cancers. Zinc ions were required to reconstitute a stable MDM2-HSP90 protein complex. Zinc binding converted MDM2 from a monomer to an oligomer, and activated MDM2 binding to its internal RING finger domain, providing evidence for a conformational change in MDM2 protein when it binds zinc. Reconstitution of an HSP90-MDM2 protein complex in vitro stimulated the unfolding of the p53 tetramer. A p53 DNA-binding inhibitor purified from human cells that is capable of unfolding p53 at ambient temperature in vitro contains co-purifying pools of HSP90 and MDM2. These data highlight the utility of phage peptide display as a powerful in vitro method to identify regulatory proteins that bind to a conformationally flexible protein like MDM2. (C) 2004 Elsevier Ltd. All rights reserved.