Departmento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, 669 - 7° andar, São Paulo, Brazil.
Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes, Av. Cândido Xavier de Almeida e Souza, 200, Mogi das Cruzes, SP, Brazil.
Biochim Biophys Acta Proteins Proteom. 2017 Apr;1865(4):388-394. doi: 10.1016/j.bbapap.2017.01.002. Epub 2017 Jan 9.
Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys and Lys increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys and Lys with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
天冬氨酸蛋白酶样半胱氨酸蛋白酶（metacaspases）是半胱氨酸蛋白酶家族的成员，可能与植物和低等真核生物中的程序性细胞死亡有关。这些蛋白酶表现出依赖钙的活性和对 P1 位精氨酸残基的特异性。与半胱天冬酶不同，它们不需要加工或二聚化即可发挥活性。事实上，未加工的布氏锥虫（Trypanosoma brucei）metacaspase-2（TbMCA2）是有活性的；然而，已经表明裂解赖氨酸和赖氨酸可以增加 TbMCA2 在合成底物上的水解活性。加工后的 TbMCA2 由 3 条多肽链组成，它们通过非共价键连接在一起。通过定点突变将赖氨酸和赖氨酸替换为甘氨酸会导致未加工但具有酶活性的突变体 TbMCA2 K55/268G。为了研究这种加工对 TbMCA2 的活性和特异性的重要性，我们进行了活性测定，比较了未加工的突变体（TbMCA2 K55/268G）和加工的 TbMCA2 形式。在 TbMCA2 WT（加工形式）和 TbMCA2 K55/268G（未加工形式）之间观察到显著差异。具体而言，我们验证了尽管在用小的合成底物进行测定时，未加工的 TbMCA2 是有活性的，但 TbMCA2 形式在大底物（如偶氮酪蛋白）上不表现出水解活性，而加工的 TbMCA2 能够轻易消化这种蛋白质。这些差异对于理解 TbMCA2 的生理调节和功能可能是相关的。
