Characterization of the function of iASPP as a scaffolding protein for selected proteins involved in inflammation

ASPP （p53的凋亡刺激蛋白）家族蛋白参与许多细胞相互作用，并开始成为许多涉及癌症生物学和炎症的蛋白质的主要支架中心。它由三个成员ASPP1， ASPP2和iASPP组成，其中最著名的是调节p53的凋亡功能，从而指导细胞命运的决定。ASPP1和ASPP2被认为是刺激p53的促凋亡功能，而iASPP则相反，抑制p53介导的促凋亡基因的转录。所有ASPP家族成员由三个结构域组成：脯氨酸富集区、锚蛋白重复序列（ARs）和SH3结构域。其中ARs和SH3结构域在c端构成一个结构实体（CTD； c端结构域）。在这项拨款申请中，我们描述了通过结合体外相互作用研究（等温滴定量热法，下拉实验），结构测定和基于细胞的功能测定（转激活测定，质谱测定）来表征iASPP与进一步相互作用伙伴的相互作用的实验。除p53外，iASPP的另外两个相互作用伙伴已部分确定：NF-kB和PP1。与p53通过锚蛋白重复序列和SH3结构域与iASPP相互作用不同，NF-kB和PP1通过聚脯氨酸结构域与SH3结构域结合。我们已经确定了与iASPP SH3结构域相互作用的一致的肽序列，并扫描了KEGG基因数据库。这已经确定了许多不同蛋白的潜在结合位点，包括AP1家族、E3连接酶Itch、MAPK激酶MKK7、非典型MAP激酶ERK8和RASSF家族的RASSF5。我们已经开始表征AP1家族的结合和相互作用的功能后果，表明iASPP结合对转录活性有抑制作用。在这里，我们建议扩展这些研究，并将其扩展到对NF-kB抑制作用的机制研究和与瘙痒相互作用的表征。

The ASPP (apoptosis-stimulating protein of p53) family of proteins is involved in many cellular interactions and is starting to emerge as a major scaffolding hub for numerous proteins involved in cancer biology and inflammation. It consists of the three members ASPP1, ASPP2 and iASPP which are best known for modulating the apoptotic function of p53, thereby directing cell fate decision. While ASPP1 and ASPP2 are supposed to stimulate the pro-apoptotic function of p53, iASPP has the opposing effect and inhibits the p53 mediated transcription of pro-apoptotic genes. All ASPP family members consist of three domains: the proline-rich region, the ankyrin repeats (ARs) and the SH3 domain. Of these the ARs and SH3 domain constitute one structural entity at the very C-terminus (CTD; C-terminal domain). In this grant application we describe experiments to characterize the interaction of iASPP with further interaction partners by a combination of in vitro interaction studies (isothermal titration calorimetry, pull down experiments), structure determination and cell based functional assays (transactivation assays, mass spectrometry). In addition to p53, two more interaction partners of iASPP are partially characterized: NF-kB and PP1. In contrast to p53 which interacts via its Ankyrin repeat and SH3 domains with iASPP, NF-kB and PP1 bind via a poly-proline domain to the SH3 domain. We have determined a consensus peptide sequence for interaction with the iASPP SH3 domain and scanned the KEGG GENES database. This has identified potential binding sites in many different proteins, including the AP1 family, the E3 ligase Itch, the MAPK kinase MKK7, the atypical MAP kinase ERK8 and RASSF5 of the RASSF family. We have started to characterize the binding and the functional consequences of the interaction for the AP1 family showing that iASPP binding has inhibitory effects on the transcriptional activity. Here we propose to extend these investigations and to extend them to mechanistic investigations of the inhibitory effect on NF-kB and characterization of the interaction with Itch.