Inhibition of HSP90 distinctively modulates the global phosphoproteome of Leishmania mexicana developmental stages.

Abstract

Heat shock protein 90 (HSP90) is an evolutionarily conserved chaperone protein that plays a central role in the folding and maturation of a large array of client proteins. In the unicellular parasite Leishmania, the etiological agent of the neglected tropical disease leishmaniasis, treatment with HSP90 inhibitors leads to differentiation from promastigote to amastigote stage, resembling the effects of established environmental triggers, low pH and heat shock. This indicates a crucial role for HSP90 in the life cycle control of Leishmania. However, the underlying molecular mechanisms remain unknown. Using a combination of treatment with the classical HSP90 inhibitor tanespimycin, phosphoproteome enrichment, and tandem mass tag (TMT) labeling-based quantitative proteomic mass spectrometry (MS), we systematically characterized the perturbing effect of HSP90 inhibition on the global phosphoproteome of Leishmania mexicana across its life cycle stages and showed that the HSP90 inhibition causes substantially distinct molecular effects in promastigote and amastigote forms.While phosphorylation of HSP90 and its co-chaperone HSP70 was decreased in amastigote, the opposite effect was observed in promastigotes. Our results showed that kinase activity and microtubule motor activity are highly represented in the negatively affected phosphoproteins of the promastigotes, whereas ribosomal proteins, protein folding, and proton channel activity are preferentially enriched in the perturbed amastigote phosphoproteome. Additionally, cross-comparison of our results with HSP90 inhibition-affected RNA-binding proteins showed that RNA helicase domains were distinctively enriched among the upregulated amastigote phosphoproteins. In addition to providing robust identification and quantification of 1,833 phosphorylated proteins across three life cycle stages of L. mexicana, this study reveals the dramatically different ways the HSP90 inhibition stress modulates the phosphoproteome of the pathogenic amastigote and provides in-depth insight into the scope of selective molecular targeting in the therapeutically relevant amastigote stage. IMPORTANCE In the unicellular parasites Leishmania spp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein leads to differentiation from promastigote to amastigote stage. Recent studies indicate potential role for protein phosphorylation in the life cycle control of Leishmania. Also, recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition in Leishmania. Phosphorylation-dephosphorylation dynamics of RBPs in higher eukaryotes serves as an important on/off switch to regulate RNA processing and decay in response to extracellular signals and cell cycle check points. In the current study, using a combination of highly sensitive TMT labeling-based quantitative proteomic MS and robust phosphoproteome enrichment, we show for the first time that HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages of Leishmania, shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. We measured changes in phosphorylation of many RBPs and signaling proteins including protein kinases upon HSP90 inhibition in the therapeutically relevant amastigote stage. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation in Leishmania, thus significantly expanding our knowledge of the posttranslational modification in Leishmania biology.