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

我们试图创建一种以单链抗体为底物识别位点的工程化泛素连接酶，它只针对和降解突变型p53，而不是野生型。单链抗体是从表达Pab240的杂交瘤细胞的cDNA文库中克隆出来的，Pab240是一种识别突变型P53的抗体，如R273P、R175H或V143A。我们首先测试了一种双环指泛素连接酶，看看它是否真的针对预期的特定底物。该工程连接酶含有BRCA1和BARD1的环指结构域，连接到底物识别位点PCNA，已知该位点与细胞周期蛋白依赖的激酶抑制物p57相互作用。双环指泛素连接酶形成均一低聚体复合体，并显示出显著的连接酶活性。共转染连接酶使p57的表达以蛋白酶体依赖的方式降低到基础水平，并恢复了p57过表达抑制的U2OS细胞的集落形成能力。结果表明，工程双环泛素连接酶具有靶向目标底物的能力。接下来，我们测试了与从Pab240克隆的单链抗体相连的双环泛素连接酶。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。为了解决这一问题，我们用DNA合成法合成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.