National Institute of Malaria Research, New Delhi, 110077, India; Department of Bioscience, Jamia Millia Islamia, New Delhi 110025, India.
National Institute of Malaria Research, New Delhi, 110077, India.
Int J Biol Macromol. 2019 Oct 1;138:309-320. doi: 10.1016/j.ijbiomac.2019.07.067. Epub 2019 Jul 10.
Metacaspases are clan CD cysteine peptidases found in plants, fungi and protozoa that possess a conserved Peptidase_C14 domain, homologous to the human caspases and a catalytic His/Cys dyad. Earlier reports have indicated the role of metacaspases in cell death; however, metacaspases of human malaria parasite remains poorly understood. In this study, we aimed to functionally characterize a novel malarial protease, P. falciparum metacaspase-3 (PfMCA3). Unlike other clan CD peptidases, PfMCA3 has an atypical active site serine (Ser1865) residue in place of canonical cysteine and it phylogenetically forms a distinct branch across the species. To investigate whether this domain retains catalytic activity, we expressed, purified and refolded the Peptidase_C14 domain of PfMCA3 which was found to express in all asexual stages. PfMCA3 exhibited trypsin-like serine protease activity with ser1865 acting as catalytic residue to cleave trypsin oligopeptide substrate. PfMCA3 is inhibited by trypsin-like serine protease inhibitors. Our study found that PfMCA3 enzymatic activity was abrogated when catalytic serine1865 (S1865A) was mutated. Moreover, PfMCA3 was found to be inactive against caspase substrate. Overall, our study characterizes a novel metacaspase of P. falciparum, different from human caspases and not responsible for the caspase-like activity, therefore, could be considered as a potential chemotherapeutic target.
植物、真菌和原生动物中的 metacaspases 是具有保守的 Peptidase_C14 结构域的 clan CD 半胱氨酸肽酶，与人类 caspase 同源，并且具有催化 His/Cys 二联体。早期的报道表明 metacaspases 在细胞死亡中发挥作用；然而，人类疟原虫的 metacaspases 仍然知之甚少。在这项研究中，我们旨在对一种新型疟原虫蛋白酶 PfMCA3 进行功能表征。与其他 clan CD 肽酶不同，PfMCA3 的活性位点丝氨酸（Ser1865）残基取代了典型的半胱氨酸，并且在种间形成了一个独特的分支。为了研究该结构域是否保留催化活性，我们表达、纯化和复性了 PfMCA3 的 Peptidase_C14 结构域，发现该结构域在所有无性阶段都有表达。PfMCA3 表现出胰凝乳蛋白酶样丝氨酸蛋白酶活性，其中 Ser1865 作为催化残基切割胰凝乳蛋白酶寡肽底物。PfMCA3 被胰凝乳蛋白酶样丝氨酸蛋白酶抑制剂抑制。我们的研究发现，当催化丝氨酸 1865（S1865A）发生突变时，PfMCA3 的酶活性被阻断。此外，PfMCA3 对 caspase 底物无活性。总的来说，我们的研究描述了一种新型疟原虫的 metacaspase，与人类 caspase 不同，不负责 caspase 样活性，因此可以被认为是一个潜在的化疗靶点。
