Inhibition of HSP90 distinctively modulates the global phosphoproteome ofLeishmania mexicanadevelopmental stages
Authors: Porta Exequiel O. J. , Gao Liqian , Denny Paul W. , Steel Patrick G. , Kalesh Karunakaran 
Published: July 2023

ABSTRACTHeat shock protein 90 (HSP90) is an evolutionary conserved chaperone protein that plays a central role in the folding and maturation of a large array of client proteins. In the unicellular parasiteLeishmania, the etiological agent of the neglected tropical disease leishmaniasis, treatment of the classical HSP90 inhibitor tanespimycin leads to dose- and time-dependent differentiation from promastigote to amastigote stage, eventually culminating in parasite killing. Although this suggests a crucial role of the HSP90 in the life cycle control ofLeishmania, the underlying molecular mechanism remains unknown. Using a combination of phosphoproteome enrichment and tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS), we robustly identified and quantified 1,833 phosphorylated proteins across three life cycle stages ofLeishmania mexicana(L. mexicana) parasite. Protein kinase domain was the most enriched protein domain in theL. mexicanaphosphoproteome. Additionally, this study systematically characterised the perturbing effect of HSP90 inhibition on the global phosphoproteome ofL. mexicanaacross its life cycle stages and showed that the tanespimycin treatment causes substantially distinct molecular effects in promastigote and amastigote forms. Whilst phosphorylation of HSP90 and its co-chaperon HSP70 was decreased in amastigotes, the opposite effect was observed in promastigotes. Additionally, our results showed that while 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. Our results also show that RNA helicase domain was distinctively enriched among the positively affected RNA-binding amastigote phosphoproteome. This study reveals the dramatically different ways the HSP90 inhibition stress modulates the phosphoproteome of the pathogenic amastigotes and provides in-depth insight into the scope of selective molecular targeting in the therapeutically relevant amastigote forms.IMPORTANCEIn the unicellular parasitesLeishmaniaspp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein, a master regulator of protein homeostasis, leads to differentiation from promastigote to amastigote stage, eventually culminating in parasite death. However, the underlying molecular mechanism remains unknown. Recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition inLeishmania. Phosphorylation-dephosphorylation dynamics of RNA-binding proteins (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 tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS) and robust phosphoproteome enrichment, we show for the first time that the HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages ofLeishmania, shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation inLeishmania, thus significantly expanding our knowledge of the posttranslational modification inLeishmaniabiology.