Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates

Abstract

Colonization of host phagocytic cells by Leishmania metacyclic promastigotes involves several parasite effectors, including the zinc-dependent metalloprotease GP63. The major mode of action of this virulence factor entails the cleavage/degradation of host cell proteins. Given the potent proteolytic activity of GP63, identification of its substrates requires the adequate preparation of cell lysates to prevent artefactual degradation during cell processing. In the present study, we re-examined the cleavage/degradation of reported GP63 substrates when GP63 activity was efficiently neutralized during the preparation of cell lysates. To this end, we infected bone marrow-derived macrophages with either wild type, Δgp63, and Δgp63+GP63 L. major metacyclic promastigotes for various time points. We prepared cell lysates in the absence or presence of the zinc-metalloprotease inhibitor 1,10-phenanthroline and examined the levels and integrity of ten previously reported host cell GP63 substrates. Inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the cleavage/degradation of several previously described GP63 targets, including PTP-PEST, mTOR, p65RelA, c-Jun, VAMP3, and NLRP3. Conversely, we confirmed that SHP-1, Synaptotagmin XI, VAMP8, and Syntaxin-5 are bona fide GP63 substrates. These results point to the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates. In addition, our results indicate that the role of GP63 in Leishmania pathogenesis must be re-evaluated.

The protein map of the protozoan parasiteLeishmania (Leishmania) amazonensis,Leishmania(Viannia) braziliensisandLeishmania (Leishmania) infantumduring growth phase transition and temperature stress

“…Multiple copies of Leishmania gp63 genes are reported together at a single chromosome site [102, 103] having a large polymorphism within the same species [104], which may explain the difference between the results obtained in this study through proteomics and Western blot analysis. Inclusion of protease inhibitors in the lysis buffer did not affect the activity of gp63, corroborating gp63’s resistance to majority of protease inhibitors previously reported [97, 105], and the analysis of the proteolytic activity of the three Leishmania species under the three conditions revealed a specific protease pattern for each species and condition. Early reports of the proteolytic profile of five L. braziliensis strains isolated from patients in Colombia and Brazil with different clinical outcomes revealed 50-125 kDa proteins [106].…”

Ethanolaminephosphate cytidyltransferase is essential for survival, lipid homeostasis and stress tolerance inLeishmania major

“…While the alteration of EPCT expression had little impact on the cellular level of lipophosphoglycan (LPG), it did significantly reduce the expression of GP63, a zinc-dependent metalloprotease that plays pivotal role Leishmania infection through proteolytic cleavage of host complement proteins and subversion of macrophage signaling [47][48][49][50]. As illustrated in Fig.…”

Metalloenzyme Inhibitors against Zoonotic Infections: Focus on Leishmania and Schistosoma

“…Although the precise mechanism by which gp63 within these exosomes reaches its targets has yet to be elucidated, it is hypothesized that these vesicles may fuse with macrophages within the phagolysosomal compartment to release their contents into the cytoplasm of infected cells. Additionally, the entry of gp63 is partly mediated by a lipid raft-dependent mechanism. , …”

“…Additionally, the entry of gp63 is partly mediated by a lipid raft-dependent mechanism. 117,118 From a structural point of view gp63 shows a predominant β-sheet secondary structure (Figure 9), which can be divided in three domains: the N-terminal domain, the central domain and the C-terminal domain. The N-terminal domain consists of amino acids 100−273 with the zinc-binding domain HExxH motif containing His264 and His268 as catalytic residues.…”