PC7 : une protéase sécrétoire énigmatique ayant une fonction de sheddase et un ciblage cellulaire unique

Many secretory proteins are synthesized as precursors that are cleaved at specific sites to become activated. Most of these cleavages are achieved along the secretory pathway and/or at the cell surface by the proprotein convertases (PCs). The latter are first synthesized as zymogens (pro) that are subsequently auto-catalytically cleaved in the endoplasmic reticulum (ER). However, the prodomain and PC remain non-covalently associated until they reach specific intracellular organelles where they either separate (e.g., PC7) or undergo a second cleavage before separation (e.g., Furin). PC7 is the most ancient and highly conserved PC-family member. Recent behavioral tests show that PC7 KO mice are healthy and have a normal lifespan but exhibit an anxiolytic and novelty-seeking behaviors. In contrast, PC7 knockdown is lethal in lower species such as xenopus and zebrafish. PC7 uniquely sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1. To elucidate the link between biological activities and physiological roles of PC7, we first concentrated on the cellular trafficking of PC7. Thus, we deleted the PC7 transmembrane domain (TM) and cytosolic tail (CT), resulting in a drastic reduction in the activity of PC7 on hTfR1, emphasizing the importance of the membrane localization of PC7 via its TMCT for activity. Since human and xenopus CTs are 35% identical and yet both proteins can shed hTfR1, we focused on the role of the conserved amino acids in the CT of human PC7 using deletions and site-directed mutagenesis. Immunocytochemical analysis of the subcellular localization of wild type (WT) and CT mutants, and Western blot analyses of L725 and E719,721 mutants, were used to assess the importance of these residues on the activity of PC7 on hTfR1. The data show that lack of the L725 motif leads to a major re-localization of PC7 to the cell surface, resulting in a drastic loss of activity. The loss of the negative charge of E719,721 influences the subcellular endosomal localization of PC7, both mutants results in a reduction in the activity of PC7. Finally, we demonstrate that an ExExxxL motif in the CT significantly regulates the efficacy of PC7 cleavage activity on hTfR1 in endosomes, likely by affecting the subcellular trafficking of active PC7 and/or its recycling to the TGN. Current NMR studies of a 14 aa peptide encompassing this sequence and its mutants point to a specific structural element in this motif. Analyses of possible partners to the ExExxxL motif led to the identification of AP-2as a new regulator of PC7 trafficking and hence cleavage activity. iv Our recent immunocytochemical data revealed that mature PC7 seems to separate from its inhibitory prodomain at the cell surface. However, PC7 activity is not present at the cell surface. A possibility is that mature PC7 and/or its prosegment are bound to a cell surface to regulating factor. This led us to test the possibility that the calcium-dependant Cab45 known to release soluble cargo proteins from the TGN to the cell surface. No report appeared on a possible role of Cab45 on membrane-bound cargo proteins. Cab45 could regulate the traffic and/or activity of PC7 by preventing the separation of PC7 from its prosegment. Our recent data suggests that Cab45 overexpression reduces by ~50% the activity of PC7 on hTfR1, without affecting the hTfR1 trafficking.

Keyphrases: calcium-binding protein 45 (Cab45), proprotein convertase type 7 (PC7), proprotein convertase subtilisin/kexin type 7 (PCSK7), transferrin receptor 1 (TfR1), cytosolic tail (CT), adaptor protein 2 (AP-2), cleavage, traffic