Propeptides Are Sufficient to Regulate Organelle-Specific pH-Dependent Activation of Furin and Proprotein Convertase 1/3

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 (PROFUR), which regulates furin activation at pH similar to 6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PROFPC1), PC1 nonetheless activates at pH similar to 5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PROFUR regulates furin activation and examine why PROFUR and PROPC1 differ in their-dependent activation. Sequence analyses establish that while both PROFUR and PROPC1 are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidirte content in PROFUR is similar to 2-fold greater than that in PROPC1, which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PROFUR when compared to PROPC1 to enhance autoproteolysis. We further demonstrate that PROFUR and PROPC1 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. (c) 2012 Elsevier Ltd. All rights reserved.