Division of Biochemistry, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India.
Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India.
J Proteomics. 2021 May 30;240:104189. doi: 10.1016/j.jprot.2021.104189. Epub 2021 Mar 20.
Mitogen Activated Protein Kinase1 (MAPK1) of Leishmania donovani functions as key regulators of various cellular activities, which seem to be imperative for parasite survival, infectivity, drug resistance and post-translational modification of chaperones/co-chaperones. However, very less is known about LdMAPK1 target proteins. With recent advancements in proteomics, we aimed to identify phosphoproteins which were differentially expressed in LdMAPK1 overexpressing (Dd8++/++) and single replacement mutants (Dd8+/) as compared to wild type (Dd8+/+) parasites, utilizing LC-MS/MS approach. An in-depth label-free phospoproteomic analysis revealed that modulation of LdMAPK1 expression significantly modulates expression levels of miscellaneous phosphoproteins which may act as its targets/substrates. Out of 1974 quantified phosphoproteins in parasite, 140 were significantly differentially expressed in MAPK1 overexpressing and single replacement mutants. These differentially expressed phosphoproteins are majorly associated with metabolism, signal transduction, replication, transcription, translation, transporters and cytoskeleton/motor proteins, hence suggested that MAPK1 may act in concert to modulate global biological processes. The study further implicated possible role of LdMAPK1 in regulation and management of stress machinery in parasite through post translational modifications. Precisely, comparative phosphoproteomics study has elucidated significant role of LdMAPK1 in regulating various pathways contributing in parasite biology with relevance to future drug development. SIGNIFICANCE: MAPKinase1, the downstream kinase of MAPK signal transduction pathway, has drawn much attention as potential therapeutic drug target due to their indispensable role in survival and infectivity of Leishmania donovani. However, limited information is available about its downstream effector proteins/signaling networks. Utilizing label free LC-MS/MS analysis, phosphoproteome of LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single replacement mutants (Dd8+/-) with wild type (Dd8+/+) parasites was compared and identified 140 LdMAPK1 modulated phosphoproteins, mainly involved in pathways like signal transduction, metabolism, transcriptional, translational, post-translational modification and regulation of heat shock proteins. Interestingly, LdMAPK1 interacts directly with only six phosphoproteins i.e. casein kinase, casein kinase II, HSP83/HSP90, LACK, protein kinase and serine/threonine protein kinase. Thus, the study elucidates significant role of LdMAPK1 in Leishmania biology which may drive drug-discovery efforts against visceral leishmaniasis.
杜氏利什曼原虫有丝分裂原激活蛋白激酶 1（MAPK1）作为各种细胞活动的关键调节剂，似乎对寄生虫的生存、感染力、耐药性和伴侣蛋白/共伴侣的翻译后修饰至关重要。然而，关于 LdMAPK1 靶蛋白的了解甚少。利用蛋白质组学的最新进展，我们旨在利用 LC-MS/MS 方法，鉴定 LdMAPK1 过表达（Dd8++/++）和单替换突变体（Dd8+/-）与野生型（Dd8+/+）寄生虫相比差异表达的磷酸蛋白。深入的无标记磷酸蛋白质组学分析表明，LdMAPK1 表达的调节显著调节了多种磷酸蛋白的表达水平，这些磷酸蛋白可能作为其靶标/底物。在寄生虫中定量的 1974 种磷酸蛋白中，有 140 种在 MAPK1 过表达和单替换突变体中差异表达。这些差异表达的磷酸蛋白主要与代谢、信号转导、复制、转录、翻译、转运体和细胞骨架/运动蛋白有关，因此表明 MAPK1 可能协同作用以调节全局生物学过程。该研究进一步表明 LdMAPK1 可能通过翻译后修饰在寄生虫应激机制的调节和管理中发挥作用。具体来说，比较磷酸蛋白质组学研究阐明了 LdMAPK1 在调节参与寄生虫生物学的各种途径中的重要作用，这与未来的药物开发有关。
丝裂原激活蛋白激酶 1（MAPK1）是 MAPK 信号转导途径的下游激酶，由于其在杜氏利什曼原虫的生存和感染力中的不可或缺作用，已引起人们对其作为潜在治疗药物靶点的极大关注。然而，关于其下游效应蛋白/信号网络的信息有限。利用无标记 LC-MS/MS 分析，比较了 LdMAPK1 过表达（Dd8++/++）和 LdMAPK1 单替换突变体（Dd8+/-）与野生型（Dd8+/+）寄生虫的磷酸蛋白质组，并鉴定了 140 种 LdMAPK1 调节的磷酸蛋白，主要涉及信号转导、代谢、转录、翻译、翻译后修饰和热休克蛋白的调节等途径。有趣的是，LdMAPK1 仅与六种磷酸蛋白直接相互作用，即酪蛋白激酶、酪蛋白激酶 II、HSP83/HSP90、LACK、蛋白激酶和丝氨酸/苏氨酸蛋白激酶。因此，该研究阐明了 LdMAPK1 在利什曼原虫生物学中的重要作用，这可能推动针对内脏利什曼病的药物发现工作。
