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

Abstract

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.

Keywords: HSP90; LC-MS/MS; Leishmania; RNA helicase; TMT labeling; phosphorylation; protein kinases.

Fig 1

Global changes in the phosphoproteome of L. mexicana across its log-phase promastigote (LPP), stationary-phase promastigote (SPP), and axenic amastigote (AXA) life cycle stages profiled by phosphoproteome enrichment followed by tandem mass tag labeling-based quantitative proteomic mass spectrometry. All phosphoproteome enrichment experiments were performed in three biological replicates. (A and B) Volcano plots showing differential enrichment of phosphoproteins between LPP and AXA and between SPP and AXA, respectively. A modified t-test with permutation-based false discovery rate (FDR) statistics (250 permutations, FDR = 0.05) was applied to compare the quantitative differences in the phosphoproteins between the life cycle groups. Heat shock proteins (HSPs) that showed increased phosphorylation in the AXA are highlighted in blue filled circles. The following annotations were used for the different HSP family members: HSP70rp1: E9B125 (LmxM.29.2550); HSP70: E9B099 (LmxM.28.2770); HSP: E9ARS1 (LmxM.18.1370); HSP90: E9B3L2 (LmxM.32.0316, LmxM.32.0312, LmxM.32.0314); BiP: E9AZT9 (LmxM.28.1200); and HSP70rp: E9AYA3 (LmxM.26.1240), where the entries in brackets are the gene IDs. (C) MS/MS spectrum of Thr₂₁₆ phosphorylated peptide from the L. mexicana HSP90.

Fig 2

Phosphorylation in L. mexicana protein kinome. Classification of the protein kinases in L. mexicana according to their catalytic domain types, namely, GMGC, STE, NEK, CK1, AGC, CAMK, and others. The image has no phylogenetic significance and is for illustrative purpose only. The orange dots represent protein kinases in which phosphorylation was detected in this study.

Fig 3

Molecular function gene ontology terms enriched in (A) LPP, (B) SPP, and (C) AXA phosphoproteins. CoA, coenzyme A.

Fig 4

Physicochemical properties of L. mexicana phosphorylation substrates. (A and B) Scatter plots comparing hydrophobicity and isoelectric points and molecular weights and isoelectric points, respectively, of phosphorylation substrates in log-phase promastigote (LPP) and axenic amastigote (AXA) life cycle stages. (C–E) Cumulative distributions of hydrophobicity, molecular weights, and isoelectric points, respectively, in the AXA and LPP phosphorylation substrates and the entire L. mexicana proteome. Wilcox rank-sum test P values of the comparisons of L. mexicana total proteome vs. AXA phosphorylation substrates (L. mexicana-AXA) , L. mexicana total proteome vs. LPP phosphorylation substrates (L. mexicana-LPP) and AXA vs. LPP phosphorylation substrates (AXA–LPP) are shown. CDF, cumulative distribution function.

Fig 5

Effect of HSP90 inhibition via tanespimycin treatment on L. Mexicana phosphoproteome. (A–C) Volcano plots showing differential enrichment of phosphoproteins upon tanespimycin treatment (+) Tan and no treatment (−) Tan in log-phase promastigote (LPP), stationary-phase promastigote (SPP), and axenic amastigote (AXA) life cycle stages, respectively, profiled by phosphoproteome enrichment followed by TMT labeling-based quantitative proteomic MS. All experiments were performed in three biological replicates. A modified t-test with permutation-based FDR statistics (250 permutations, FDR = 0.05) was applied to compare the quantitative differences in the phosphoproteins between the tanespimycin-treated and non-treated groups. Protein kinases are highlighted in blue filled circles. (D) Principal component analysis of the phosphoproteins (blue) and the HSP90 inhibition affected phosphoproteins (red) in the three life cycle stages based on their relative quantification profiles.

Fig 6

Correlation between RNA-binding proteins (RBPs) downregulated by HSP90 inhibition and the HSP90 inhibition-modulated phosphoproteins in L. mexicana. Venn diagrams showing comparison of the downregulated RBPs [L. mex RBPs (↓)] and upregulated [L. mex PPs (↑)] and downregulated [L. mex PPs (↓)] phosphoproteins in (A) log phase promastigote (LPP) and (B) axenic amastigote (AXA) life cycle stages. (C and D) Most enriched InterPro domains in the upregulated and downregulated L. mexicana RNA-binding phosphoproteins, respectively, in the LPP. (E and F) Most enriched InterPro domains in the upregulated and downregulated L. mexicana RNA-binding phosphoproteins, respectively, in the AXA.

Fig 7

Protein-protein interaction network of RNA-binding phosphoproteins upregulated during HSP90 inhibition in L. mexicana axenic amastigotes (AXAs) constructed using publicly available STRING database of L. major Friedlin strain. The nodes are colored according to their betweenness centrality in the network.