School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India.
FEBS J. 2019 Jun;286(11):2216-2234. doi: 10.1111/febs.14804. Epub 2019 Mar 25.
Entamoeba histolytica is an intestinal protist parasite that causes amoebiasis, a major source of morbidity and mortality in developing countries. Phosphoinositides are involved in signalling systems that have a role in invasion and pathogenesis of this parasite. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyses the generation of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P ), a key species of phosphoinositide that regulates various cellular processes. However, phosphatidylinositol phosphate kinase (PIPK) family of enzymes have not been characterized in E. histolytica. Here, we report the identification and characterization of type I PIPK (EhPIPKI) of E. histolytica. Computational analysis revealed homologs of type I and III PIPK family in E. histolytica and the absence of type II PIPK. In spite of low overall sequence identity, the kinase domain was found to be highly conserved. Interestingly, a unique insertion of a tandem repeat motif was observed in EhPIPKI distinguishing it from existing PIPKs of other organisms. Substrate profiling showed that EhPIPKI could phosphorylate at third and fifth hydroxyl positions of phosphatidylinositols, though the predominant substrate was phosphatidylinositol 4-phosphate (PtdIns(4)P). Furthermore, EhPIPKI underwent intracellular cleavage close to the amino-terminal, generating two distinct fragments Nter-EhPIPKI (27p) and Cter-EhPIPKI (47p). Immunofluorescence and cellular fractionation revealed that the full-length EhPIPKI and the Cter-EhPIPKI containing carboxyl-terminal activation loop were present in the plasma membrane while the Nter-EhPIPKI was observed in the cytosolic region. In conclusion, E. histolytica has a single EhPIPKI gene that displays novel properties of post-translational processing, the presence of a repeat domain and substrate specificity not observed in any PIPK enzyme so far.
溶组织内阿米巴是一种肠道原生动物寄生虫，可引起阿米巴病，这是发展中国家发病率和死亡率的主要原因。磷酸肌醇在信号系统中发挥作用，该信号系统在寄生虫的侵袭和发病机制中起作用。磷脂酰肌醇 4-磷酸 5-激酶（PIP5K）催化磷脂酰肌醇（4,5）双磷酸（PtdIns（4,5）P）的生成，该磷酸肌醇是调节各种细胞过程的关键物种。然而，磷酸肌醇磷酸激酶（PIPK）家族的酶在溶组织内阿米巴中尚未得到表征。在这里，我们报道了溶组织内阿米巴中 I 型 PIPK（EhPIPKI）的鉴定和特征。计算分析显示溶组织内阿米巴中存在 I 型和 III 型 PIPK 家族的同源物，而不存在 II 型 PIPK。尽管整体序列同一性较低，但激酶结构域被发现高度保守。有趣的是，在 EhPIPKI 中观察到串联重复基序的独特插入，将其与其他生物体的现有 PIPKs 区分开来。底物谱分析表明，EhPIPKI 可以在磷脂酰肌醇的第三和第五羟基位置磷酸化，尽管主要底物是磷脂酰肌醇 4-磷酸（PtdIns（4）P）。此外，EhPIPKI 在靠近氨基末端处发生细胞内切割，生成两个不同的片段 Nter-EhPIPKI（27p）和 Cter-EhPIPKI（47p）。免疫荧光和细胞分级分离显示全长 EhPIPKI 和含有羧基末端激活环的 Cter-EhPIPKI 存在于质膜中，而 Nter-EhPIPKI 存在于细胞质区域。总之，溶组织内阿米巴只有一个 EhPIPKI 基因，它具有独特的翻译后加工特性、重复结构域和迄今在任何 PIPK 酶中都未观察到的底物特异性。
