Pierpaolo Ginefra
Proprotein Convertases (PCs) constitute a family of serine proteases that activate various
hormones, growth factors and cell adhesion molecules by mediating endoproteolytic cleavage
of their secreted inactive precursors during their transit in the secretory pathway. Their
physiological roles in most tissues, in cancer and other diseases have remained nowadays
poorly defined, partly because of technical hurdles in clearly distinguishing functionally
overlapping PC activities from each other. Related to this, a major unsolved question is where,
among the different subcellular compartments of the secretory pathway, substrate cleavage
takes place. To address these questions, we mapped intracellular PC activities of two
unrelated cell lines (HEK293T and B16F1 melanoma) by ratiometric and Förster Resonance
Energy Transfer (FRET)-based imaging of the PC-specific biosensors, called cell-linked
indicator of proteolysis (CLIP)v3 and CLIPv4 respectively, which are sorted to different
subcellular compartments by specific trafficking signals. Until now, PCs have been thought to
cleave the majority of the substrates in the trans-Golgi network (TGN). By contrast, our
analysis of compartment-specific CLIPv3 and CLIPv4 variants in cultured cells suggested that
PC activities are much higher in post-exocytic compartments. Furthermore, imaging in B16F1
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-edited clones for
endogenously expressed Furin or PC7, revealed an enrichment of Furin activity in endosomes
compared to TGN, whereas PC7 activity was confined to a distinct compartment resistant to
the pan-PC inhibitor CMK, demonstrating that endogenous Furin and PC7 are biologically
active in distinct compartments. In addition, we also demonstrated that substrate and protease
localization are rate-limiting of how efficiently a given substrate is processed.
PC inhibition represses human primary melanoma cells motility and migration in vitro and
proliferation in other skin cancer cells both in vitro and in vivo. To elucidate the roles of PC
activities in melanoma progression and differentiation, we evaluated relative contribution of
Furin and PC7 in B16F1 tumor grafts. Both proteins were required for tumor pigmentation,
whereas Furin deletion reduced tumor growth, but its loss may be rescued by PC7 deletion.
Altogether, these findings will be important to inform future therapeutic approaches and
strategies for targeting PCs to preferentially block oncogenic activities.
EPFL2018
