Quantitative Proteomics Reveals that Hsp90 Inhibition Dynamically Regulates Global Protein Synthesis in Leishmania...

Heat shock protein 90 (Hsp90) is a conserved molecular chaperone responsible for the folding and maturation of nascent proteins. Hsp90 is regarded as a master regulator of protein homeostasis in the cell, and its inhibition affects the functions of a large array of client proteins. The classical Hsp90 inhibitor tanespimycin has shown potent antileishmanial activity. Despite the increasing importance of Hsp90 inhibition in the development of antileishmanial agents, the global effects of these inhibitors on the parasite proteome remain unknown. By combining tanespimycin treatment with bioorthogonal noncanonical amino acid tagging (BONCAT) metabolic labeling and isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic mass spectrometry, for the first time, we robustly profiled the relative changes in the synthesis of hundreds of parasite proteins as functions of dose and duration of the inhibitor treatment. We showed that Hsp90 inhibition dynamically regulates nascent protein synthesis in Leishmania mexicana, with many chaperones and virulence factors showing inhibitor concentration- and treatment duration-dependent changes in relative expression. Many ribosomal proteins showed a downregulation upon severe Hsp90 inhibition, providing the first protein-level evidence that Hsp90 inhibition affects the protein synthesis capacity of the ribosome in this organism. We also provide an unbiased target validation of tanespimycin in L. mexicana using live parasite photoaffinity labeling with a novel chemical probe and quantitative proteomic mass spectrometry. We showed that the classical Hsp90 inhibitor not only engages with its presumed target, Hsp83-1, in L. mexicana promastigotes but also affects multiple proteins involved in protein synthesis and quality control in the parasite. This study defines the Leishmania parasites' response to Hsp90 inhibition at the level of nascent global protein synthesis and provides a rich resource for future studies on Leishmania spp. biology and antileishmanial drug development.IMPORTANCE Leishmania spp. are the causative agents of leishmaniasis, a poverty-related disease, which is endemic in >90 countries worldwide, affecting approximately 12 million people, with an estimated 700,000 to 1 million new cases and around 70,000 deaths annually. Inhibitors of the chaperone protein Hsp90 have shown promising antileishmanial activity. However, further development of the Hsp90 inhibitors as antileishmanials is hampered by a lack of direct information of their downstream effects on the parasite proteome. Using a combination of mass spectrometry-based quantitative proteomics and chemical and metabolic labeling, we provide the first protein-level evidence that Hsp90 inhibition affects global protein synthesis in Leishmania We also provide the precise relative quantitative changes in the expressions of hundreds of affected proteins as functions of both the concentration and duration of the inhibitor treatment. We find that Leishmania regulates its ribosomal proteins under Hsp90 inhibition while a set of virulence factors and chaperones are preferentially synthesized.