Discovery and characterisation of recifin

酪氨酸- dna磷酸二酯酶1 （TDP1）是癌细胞对拓扑异构酶抑制剂喜树碱及其衍生物致敏的分子靶点。高通量筛选TDP1活性抑制剂的天然产物提取物文库导致发现了一种具有生物活性的海绵水提取物，Axinella sp.。生物测定引导分离的源提取物导致活性成分被确定为一种新的，42个残基半胱氨酸丰富的肽，我们命名为recifin。采用Edman降解和串联质谱从头蛋白测序相结合的方法确定了recifin的一级序列和二硫键模式。核磁共振结构揭示了一种新的褶皱，包括四股反平行β -片和两个螺旋旋转，由复杂的二硫键网络稳定，在其中一条链周围形成嵌入环。在生化实验中，累西芬抑制全长TDP1，但不抑制n端截断的TDP1。酶动力学研究表明，累西芬可以特异性调节全长TDP1的酶活性，而不影响截断形式的TDP1，这表明累西芬在TDP1上有一个变构结合位点。

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the topoisomerase inhibitor camptothecin and its derivatives. High-throughput screening of natural product extract libraries for inhibitors of TDP1 activity resulted in the discovery of a bioactive aqueous extract of the marine sponge, Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be a novel, 42-residue cysteine-rich peptide we named recifin. The primary sequence and disulfide bonding pattern of recifin was determined using a combination of Edman degradation and tandem mass spectroscopy de novo protein sequencing. The NMR structure revealed a novel fold comprising a four strand anti-parallel beta-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. Recifin inhibited full-length TDP1 but not N-terminally truncated TDP1 in biochemical assays. Enzyme kinetics studies revealed that recifin can specifically modulate the enzymatic activity of full-length TDP1 while not affecting a truncated form of TDP1, suggesting an allosteric binding site for recifin on TDP1.