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, {Delta}gp63, and {Delta}gp63+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. AUTHORS SUMMARYIn the protozoan parasite Leishmania, the abundant zinc-dependent metalloprotease GP63 is expressed at high levels at the surface of the promastigotes forms of the parasite. Upon phagocytosis by host macrophages, this metalloprotease is released from the parasites surface and spreads across the cytosol of infected cells. There, GP63 cleaves a number of host cell proteins involved in the control of host microbicidal function and in the regulation of immune responses, thereby contributing the ability of Leishmania to impair host defence mechanisms against infection. Given the abundance and powerful proteolytic activity of GP63, it is crucial to prevent artefactual proteolysis during processing of infected cells to identify genuine GP63 substrates. In this study, we found that inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the degradation of several of previously identified GP63 substrates. These results uncover the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates.