Wünsch Désirée, Hahlbrock Angelina, Heiselmayer Christina, Bäcker Sandra, Schrenk Christian, Benne Franziska, Schilling Oliver, Knauer Shirley K
Biol Chem. 2015 Apr;396(4):367-76. doi: 10.1515/hsz-2014-0318.
Proteases are key regulators of life. Human Threonine Aspartase1 processes substrates, such as the mixed-lineage leukemia (MLL) protein, containing two cleavage sites, CS1 and CS2. Likewise, MLL's Drosophila ortholog trithorax is cleaved by Drosophila Threonine Aspartase1 (dTasp), suggesting a mechanistic coevolution. However, a detailed analysis of dTasp's function was missing so far. Here, active and inactive dTasp mutants allowed to compare substrate recognition and cleavage site selectivity of human and Drosophila enzymes. In contrast to the human protease, our cell-based assay revealed a preferential processing of CS2-like (QLD↓Gx[xD/Dx]) targets for dTasp, whereas cleavage of CS1-like targets (QVD↓Gx[xD/Dx]) was significantly impaired. Systematic mutagenesis of the CS2 sequence defined the motif x[FILMW]D↓Gx[xD/Dx] as the consensus cleavage sequence for dTasp. Substrate species selectivity of the enzymes was uncovered by demonstrating that dTasp cleaves Drosophila TFIIA, but not the human ortholog, suggesting evolutionary divergence of TFIIA downstream networks. Also, Drosophila USF2 was neither predicted nor cleaved by dTasp. Moreover, we found that dTasp cleavage site selectivity is independent of heterocomplex formation, as dTasp exists predominantly as an αβ-monomer. Collectively, we provide novel insights into evolutionary similarities and divergence concerning Threonine Aspartase1 function in different species, which may aid to dissect and better target human Threonine Aspartase1 in malignancies.
蛋白酶是生命的关键调节因子。人类苏氨酸天冬氨酸酶1可处理含有两个切割位点CS1和CS2的底物，如混合谱系白血病（MLL）蛋白。同样，MLL在果蝇中的直系同源物三体胸节蛋白也被果蝇苏氨酸天冬氨酸酶1（dTasp）切割，这表明存在机制上的共同进化。然而，到目前为止，对dTasp功能的详细分析还缺失。在这里，活性和非活性dTasp突变体可用于比较人类和果蝇酶的底物识别和切割位点选择性。与人类蛋白酶不同，我们基于细胞的分析表明，dTasp优先处理类似CS2的（QLD↓Gx[xD/Dx]）靶点，而类似CS1的靶点（QVD↓Gx[xD/Dx]）的切割则明显受损。对CS2序列进行系统诱变确定了基序x[FILMW]D↓Gx[xD/Dx]为dTasp的共有切割序列。通过证明dTasp可切割果蝇TFIIA，但不能切割其人类同源物，揭示了这些酶的底物物种选择性，这表明TFIIA下游网络存在进化差异。此外，果蝇USF2既未被预测会被dTasp切割，也未被其切割。而且，我们发现dTasp的切割位点选择性与异源复合物的形成无关，因为dTasp主要以αβ单体形式存在。总的来说，我们对不同物种中苏氨酸天冬氨酸酶1功能的进化相似性和差异提供了新的见解，这可能有助于剖析并更好地靶向人类恶性肿瘤中的苏氨酸天冬氨酸酶1。
