The C-terminal segment of Leishmania major HslU: Toward potential inhibitors of LmHslVU activity.

It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related non-proteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 µM).

The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides.

HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.

The bacterial-like HslVU protease complex subunits are involved in the control of different cell cycle events in trypanosomatids.

The trypanosomatid parasites Leishmania and Trypanosoma are responsible for the most important WHO-designated neglected tropical diseases, for which the need for cost-effective new drugs is urgent. In addition to the classical eukaryotic 20S and 26S proteasomes, these unconventional eukaryotes possess a bacterial-like protease complex, HslVU, made of proteolytic (HslV) and regulatory (HslU) subunits. In trypanosomatids, two paralogous genes are co-expressed: HslU1 and HslU2. Conflicting reports have been published with respect to subcellular localization, functional redundancy and putative roles of the different subunits of this complex in trypanosomatids. Here, we definitively established the mitochondrial localization of HslVU in L. major procyclic promastigotes and of HslV in T. brucei bloodstream trypomastigotes, the latter being the form responsible for the disease in the mammalian host. Moreover, our data demonstrate for the first time the essential nature of HslVU in the bloodstream trypomastigotes of T. brucei, in spite of mitochondrial repression at this stage. Interestingly, our work also allows distinguishing a specific role for the different members of the complex, as HslV and HslU1 appear to be involved in the control of different cell cycle events. Finally, these data validate HslVU as a promising drug target against these parasitic diseases of wide medical and economical importance.