Dataset Information

MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex.

ABSTRACT: MAP kinases (MAPK) are the most downstream kinases in signal transduction cascades and regulate critical cellular activities such as cell proliferation, differentiation, mortality, stress response, and apoptosis. The Leishmania donovani MAPK1 (LdMAPK1) is involved in parasite viability and drug resistance, but its substrates have not been identified yet. Aiming to identify the possible targets(s) of LdMAPK1, we sought to isolate interacting partners by co-immunoprecipitation, gel electrophoresis and mass spectrometry. Out of fifteen analyzed protein bands, four were identified as subunits of the HSP90 foldosome complex, namely HSP 90, HSP70, STI and SGT. Western blot analysis not only confirmed that LdMAPK1 interacts with HSP70 and HSP90 but also demonstrated that MAPK1 abundance modulates their expression. The interaction is sensitive to treatment with AMTZD, a competitive ERK inhibitor. MAPK1 also displayed kinase activity with HSP90 or HSP70 as substrates. By phosphorylating HSPs in the foldosome complex, MAPK1 may regulate the stability and activity of the foldosome which in turn plays a pivotal role in the parasitic life cycle of L. donovani. Our study therefore implicates LdMAPK1 in the post-translational modification and possibly the regulation of heat shock proteins. Conversely, HSP90 and HSP70 are identified as the first substrates of LdMAPK1.

SUBMITTER: Kaur P

PROVIDER: S-EPMC5579238 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex.

Kaur Pavneet P Garg Mansi M Hombach-Barrigah Antje A Clos Joachim J Goyal Neena N

Scientific reports 20170831 1

MAP kinases (MAPK) are the most downstream kinases in signal transduction cascades and regulate critical cellular activities such as cell proliferation, differentiation, mortality, stress response, and apoptosis. The Leishmania donovani MAPK1 (LdMAPK1) is involved in parasite viability and drug resistance, but its substrates have not been identified yet. Aiming to identify the possible targets(s) of LdMAPK1, we sought to isolate interacting partners by co-immunoprecipitation, gel electrophoresis ...[more]

PMID: 28860596

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Project description:The 90-kDa heat shock protein (HSP90) of eukaryotes is a highly abundant and essential chaperone required for the maturation of regulatory and signal proteins. In the protozoan parasite Leishmania donovani, causative agent of the fatal visceral leishmaniasis, HSP90 activity is essential for cell proliferation and survival. Even more importantly, its inhibition causes life cycle progression from the insect stage to the pathogenic, mammalian stage. To unravel the molecular impact of HSP90 activity on the parasites' gene expression, we performed a ribosome profiling analysis of L. donovani, comparing genome-wide protein synthesis patterns in the presence and absence of the HSP90-specific inhibitor radicicol and an ectopically expressed radicicol-resistant HSP90 variant. We find that ribosome-protected RNA faithfully maps open reading frames and represents 97% of the annotated protein-coding genes of L. donovani. Protein synthesis was found to correlate poorly with RNA steady-state levels, indicating a regulated translation as primary mechanism for HSP90-dependent gene expression. The results confirm inhibitory effects of HSP90 on the synthesis of Leishmania proteins that are associated with the pathogenic, intracellular stage of the parasite. Those include heat shock proteins, redox enzymes, virulence-enhancing surface proteins, proteolytic pathways, and a complete set of histones. Conversely, HSP90 promotes fatty acid synthesis enzymes. Complementing radicicol treatment with the radicicol-resistant HSP90rr variant revealed important off-target radicicol effects that control a large number of the above-listed proteins. Leishmania lacks gene-specific transcription regulation and relies on regulated translation instead. Our ribosome footprinting analysis demonstrates a controlling function of HSP90 in stage-specific protein synthesis but also significant, HSP90-independent effects of the inhibitor radicicol. IMPORTANCE Leishmania parasites cause severe illness in humans and animals. They exist in two developmental stages, insect form and mammalian form, which differ in shape and gene expression. By mapping and quantifying RNA fragments protected by protein synthesis complexes, we determined the rates of protein synthesis for >90% of all Leishmania proteins in response to the inhibition of a key regulatory protein, the 90-kDa heat shock protein. We find that Leishmania depends on a regulation of protein synthesis for controlling its gene expression and that heat shock protein 90 inhibition can trigger the developmental program from insect form to mammalian form of the pathogen.

Project description:Leishmaniasis includes multiple clinical syndromes, most notably visceral, cutaneous, and mucosal forms. Visceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease endemic to large parts of Africa and Asia, and in South-Eastern Europe (Greece, Turkey, Georgia). Visceral leishmaniasis is a parasitic zoonosis caused by species of the L. donovani complex. In the classical epidemiological model the main reservoir for VL are canines.The study included a cohort of 513 individuals of both genders (190 males and 323 females) from the ages of 1 to 70 years that were screened in ten villages across two districts in Kakheti using the Kalazar Detect™ rK39 rapid diagnostic test. The phylogenetic diversity patterns of local strains, based on the rDNA internal transcribed spacer (ITS) sequences, were assessed for samples obtained from patients with suspected L. donovani infection, from canine reservoirs and from Phlebotomus sand flies obtained from different geographical areas of Georgia and from Azerbaijan.Out of a total of 600 domestic dog blood samples 95 (15.8 %) were positive by rK39 rapid diagnostic tests. For symptomatic domestic dogs, the testing of conjunctival swabs or bone marrow aspirates revealed a higher VL incidence in Kvareli District (Kvareli; 19.4 %, n?=?329) compared with that observed for Sagarejo District (Sagarejo; 11.4 %, n?=?271). A total of 231 sand flies of both genders were collected during the 2-month period; of the 114 females, 1.75 % were PCR positive for the presence of Leishmania spp.VL infection rates remain high in both canines and humans in Georgia, with disease in several known natural foci. The genetic relationships derived from rDNA internal transcribed spacer (ITS) sequence comparisons identified genetic subgroups, revealing preliminary insights into the genetic structure of L. donovani complex members currently circulating in the South Caucasus and demonstrates the utility of ITS-based genotyping in the resource-limited country of Georgia.

Dataset Information

MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex.

Publications

MAPK1 of Leishmania donovani interacts and phosphorylates HSP70 and HSP90 subunits of foldosome complex.

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