Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani

Abstract

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.

The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability

“…Both SUMO and SENP are constitutively expressed in L. donovani . RNA-seq and ribosome profiling data generated previously [ 5 ] show minor variations for SUMO protein synthesis and RNA abundance for L. donovani before and after radicicol-induced promastigote-to-amastigote differentiation ( Figure 1 D). SENP also shows a constitutive, stage-independent protein synthesis and RNA levels.…”

“…The leishmaniae differ from their human host and from most other eukaryotes by their lack of gene-specific transcription regulation [ 1 , 2 , 3 ], relying on modulated RNA stability [ 4 ], inducible translation [ 5 ] and reversible gene amplification [ 6 , 7 ] instead.…”

“…Conjugation of another modifier, small ubiquitin-like modifier (SUMO) was studied in Trypanosoma spp: SUMOylation of proteins was described for Trypanosoma cruzi [ 18 ] and T. brucei [ 19 , 20 ], where this PTM is involved in surface antigen expression and nuclear organization. A putative ortholog of SUMO is present in the L. donovani genome and expressed [ 5 , 21 ].…”

“…For SUMOylation to happen, the SUMO precursor must first undergo a proteolytic cleavage by a sentrin-specific protease (SENP), which removes the C-terminal amino acids and leaves an exposed, reactive, C-terminal di-glycine group [ 22 , 23 ]. A putative SENP ortholog is also encoded in the L. donovani genome and expressed [ 5 , 24 ]. In humans, the di-glycine is further activated by the E1 protein, transesterified to the E2 SUMO-conjugating enzyme and finally transferred to the substrate protein by the E3 SUMO ligase.…”

A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation

“…We have developed and applied an integrated chemical proteomics method that combines the BONCAT approach with bead-based affinity enrichment and iTRAQ quantitative proteomics MS for robust and sensitive profiling of starvation-responsive NSPs of Leishmania. Although the alternative, ribosome profiling [32,33] is emerging as a powerful method for global profiling of protein translation, MS-based proteomics, comparatively, provides a more direct, and therefore more reliable, readout of the cellular proteome and its changes under different perturbations [34]. Proteins are more robust during sample handling, whilst every step in the experimental protocols of ribosome profiling from cell lysis to nuclease digestion to library generation is likely to cause distortions in the data [35].…”