School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India.
PLoS Negl Trop Dis. 2018 Jun 13;12(6):e0006575. doi: 10.1371/journal.pntd.0006575. eCollection 2018 Jun.
Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis.
Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human.
Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 μM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 μM). Borrelidin inhibited the promastigotes (IC50: 21 μM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts.
氨酰-tRNA 合成酶是蛋白质合成所必需的核心酶。这些酶是细菌和真菌中已知的药物靶点。在这里，我们首次报道了利什曼原虫（一种原生动物寄生虫，内脏利什曼病的主要病原体）的苏氨酰-tRNA 合成酶（LdThrRS）的功能特征。
在大肠杆菌中表达重组 LdThrRS（rLdThrRS）并进行纯化。测定 rLdThrRS 的动力学参数。通过免疫荧光分析进行 LdThrRS 的亚细胞定位。在利什曼前鞭毛体中生成 LdThrRS 的杂合突变体。检查这些遗传操作的寄生虫的增殖、毒力、氨酰化活性以及对已知 ThrRS 抑制剂博莱霉素的敏感性。利什曼原虫 ThrRS 的计算生成结构模型与人类进行比较。
重组 LdThrRS 显示氨酰化活性，并且该蛋白可能定位于细胞质和线粒体。LdThrRS 的 3D 模型与人类 ThrRS 的比较显示出相当大的相似性。杂合寄生虫表现出限制生长表型，并且感染力减弱。这些杂合寄生虫对博莱霉素的抑制更为敏感。多次尝试获得 ThrRS 纯合缺失突变体均未成功，表明其对利什曼原虫寄生虫是必需的。博莱霉素对 LdThrRS 具有很强的亲和力（KD：0.04 μM），并且有效地抑制了 rLdThrRS 的氨酰化活性（IC50：0.06 μM）。博莱霉素抑制前鞭毛体（IC50：21 μM）阶段的寄生虫。我们的数据表明，LdThrRS 对 L. donovani 的存活至关重要，并且可能与具有强亲和力的小分子药物结合，因此使其成为药物发现努力的潜在靶标。
