Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3

摘要

The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO FUR), which regulates furin activation at pH ~ 6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO PC1), PC1 nonetheless activates at pH ~ 5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO FUR regulates furin activation and examine why PRO FUR and PRO PC1 differ in their pH-dependent activation. Sequence analyses establish that while both PRO FUR and PRO PC1 are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO FUR is ~ 2-fold greater than that in PRO PC1, which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO FUR when compared to PRO PC1 to enhance autoproteolysis. We further demonstrate that PRO FUR and PRO PC1 are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles.