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

Abstract

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 which 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~6.5 within the trans Golgi network. Although this residue is conserved in the PC1 propeptide (PROPC1), PC1 nonetheless activates at pH~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 pH-dependent activation. Sequence analyses establish that while both PROFUR and PROPC1 are enriched in histidines when compared with cognate catalytic-domains and prokaryotic orthologs, histidine content in PROFUR is ~two-fold greater than 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.

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

“…A large loop, which is located between strand ␤3 and helix ␣2 of the N2 motive in mammalian PPs, is not found in plants and bacteria. This is the region where secondary cleavage occurs in mammalian PPs, by which the subtilase is released from autoinhibition (29,35,48). In SBT3, on the other hand, the first of the two internal PP cleavage sites (Asn-38/Val-39) is located within the first ␤-␣-␤ motive in a loop between strand ␤1 and helix ␣1.…”

“…In SBT3, on the other hand, the first of the two internal PP cleavage sites (Asn-38/Val-39) is located within the first ␤-␣-␤ motive in a loop between strand ␤1 and helix ␣1. Conservation of this loop and the secondary cleavage site among bacterial and plant subtilases indicates similarities with respect to the mechanism of PP degradation and protease activation (35,48).…”

“…This region corresponds to the site of PP processing in Bacillus sp. subtilases (37,48). Additional cleavage between Asp-54 and Ser-55 resulted in the generation of the smaller degradation product.…”

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

“…During secretion, the prodomain undergoes an autoprocessing maturation process before to be degraded, thus unlocking enzymatic activity. Recent data have elegantly shown that the organelle-specific, pHdependent activation of the mammalian subtilases furin and PC1/3 along their secretion route is regulated in a prodomaindependent way 6 . The finely tuned spatiotemporal regulation of subtilases prevents intracellular damage caused by prematurely activated bacterial enzymes and allows pro-protein convertases (PC) to mature their substrates in ad hoc cellular compartments in response to a specific stimulus 2,3,5,7 .…”

O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking

“…O-Glycosylation Affects the Stability of PAM-Regulated secretory pathway proteins like PAM, which enter immature secretory granules upon exiting the Golgi complex, become prohormone convertase substrates as luminal pH drops and calcium levels rise (53)(54)(55). Prohormone convertase 1-mediated cleavage at the Lys-Lys site in PAM-1 (35) increases the specific activity of PHM and allows the secretion of soluble PHM; PAM-2, which lacks exon 16 and this Lys-Lys site, remains largely intact.…”