Selective degradation of p53 mutant by an engineered ScFv-linked ubiquitin ligase

我们试图创建一种工程泛素连接酶，以ScFv作为底物识别位点，仅靶向和降解p53突变型，而不是野生型。该ScFv从表达Pab240的杂交瘤细胞cDNA文库中克隆而来，Pab240是一种识别突变p53如R273P、R175H或V143A的抗体。我们首先测试了双环指泛素连接酶是否真的针对预期的特定底物。工程连接酶含有BRCA1和BARD1的环指结构域，与底物识别位点PCNA连接，已知PCNA与细胞周期蛋白依赖性激酶抑制剂p57相互作用。双环指泛素连接酶形成了一个同源低聚物复合物，并表现出显著的连接酶活性。连接酶的共转染以蛋白酶体依赖的方式将转染的p57的表达降低到背景水平，恢复了U2OS细胞的集落形成能力，否则p57的过表达会抑制这种能力。结果表明，工程双环泛素连接酶能够靶向预期的底物。接下来，我们测试了连接到从Pab240克隆的ScFv的双环泛素连接酶。然而，到目前为止，它还没有成功，很可能是因为ScFv和p53突变体之间的亲和力问题。为了克服这一问题，采用DNA合成的方法制备了一种新的单链抗体。

We have attempted to create an engineered ubiquitin ligase with ScFv as a substrate recognition site that target and degrade only p53 mutant but not the wild type. The ScFv was cloned from cDNA library of a hybridoma cells that express Pab240, an antibody recognizes mutant p53 such as R273P, R175H, or V143A. We first tested a double RING finger ubiquitin ligase whether it actually targets the intended specific substrates. The engineered ligase contains the RING finger domains of both BRCA1 and BARD1 linked to a substrate recognition site PCNA, which is known to interact with cyclin dependent kinase inhibitor p57. The double RING finger ubiquitin ligase formed a homo-oligomer complex and exhibited significant ligase activity. Co-transfection of the ligase reduced the expression of transfected p57 to the back ground level in proteasome dependent manner, and restored the colony formation ability of U2OS cells that is otherwise inhibited by overexpressed p57. The results indicate the ability of the engineered double RING ubiquitin ligase to target the intended substrate. Next we tested the double RING ubiquitin ligase linked to the ScFv cloned from Pab240. However so far it has not been successful most likely because of the affinity problem between the ScFv and p53 mutants. To overcome the problem a new ScFv has been made by DNA synthesize method.