Discovery and characterisation of recifin

酪氨酸二酯酶1(Tdp1)是肿瘤细胞对拓扑异构酶抑制剂喜树碱及其衍生物增敏的分子靶点。对Tdp1活性抑制剂的天然产物提取物库进行高通量筛选后，发现了一种具有生物活性的海绵水提物，Axinella sp。生物测定引导的源提取物的分级导致了活性成分的分离，它被确定为一种新的，42个残基的富含半胱氨酸的多肽，我们将其命名为雷吉芬。采用Edman降解和串联质谱仪从头测序相结合的方法确定了雷吉芬的一级序列和二硫键模式。核磁共振结构揭示了一个新颖的折叠结构，它包括一个四链反平行的β-折叠和两个由复杂的二硫键网络稳定的螺旋转弯，该网络在其中一条链周围创建了一个嵌入的环。在生化分析中，Recifin抑制全长Tdp1，但不抑制N末端截短的Tdp1。酶动力学研究表明，雷吉芬可以特异性地调节全长Tdp1的酶活性，而不影响截短形式的Tdp1，这表明雷吉芬在Tdp1上存在变构结合部位。

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.