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

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.