The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin

Abstract

Background: Histidine 69 in the propeptide is a pH sensor that mediates compartment-specific furin activation. Results: Histidine 69 protonation exposes the activation loop for proteolysis only within an optimal window for pH-dependent activation. Conclusion: A small structural change functions as the trigger that regulates precise spatiotemporal activation of furin. Significance: Our work provides insights into how individual proprotein convertases encode their unique compartmentspecific activation.

Mechanism of Fine-tuning pH Sensors in Proprotein Convertases

“…Cells were kept at 37°C in a 5% CO 2 environment as described previously (32,35). For expression of ER-retained constructs, COS7 cells at 60 -80% confluence were transfected with pcDNA3.1 expression vectors containing WT PC1/3 truncated at Arg 618 with a C-terminal -KDEL sequence (32) or vectors containing the noted PRO PC1/3 variants using TransIT LT-1 transfection reagent (Invitrogen) according to the manufacturer's instructions.…”

“…Circular Dichroism Spectroscopy-Circular dichroism spectroscopy was performed on an AVIV model 215 CD spectrometer at 4°C as described previously (32,35). Briefly, after centrifugation at 100,000 ϫ g for 30 min, protein concentration was adjusted to ϳ0.3 mg/ml, and spectra were obtained using a 1-mm quartz cuvette for far-UV spectra.…”

“…50 -Activity assays were performed to determine the inhibitory capabilities of the various propeptides as described previously (32,35). Briefly, a 15 M concentration of the fluorogenic substrate (Abz-RVKRGLA-Tyr(3-NO 2 )) was incubated with serially diluted amounts of either refolded WT or mutant PRO PC1/3 in concentrations ranging from 0.15 to 3000 nM in activity assay buffer at pH 6.8, and 0.02 enzyme units (1 enzyme unit of enzyme produces 267 relative fluorescence units/min in the assay buffer) of mature PC1/3 was added to initiate the reaction.…”

“…At the near-neutral pH of the ER, the deprotonated histidine acts as a hydrophobic residue, stabilizing the packing within this pocket and keeping the pH-sensitive loop protected against cleavage. Upon entry into the trans-Golgi network, the histidine is exposed to a ϳ10-fold higher proton concentration and thus is protonated; as a result, the imidazole side chain becomes polar, disrupting the packing and driving a local conformational change that exposes the secondary cleavage site, allowing for rapid degradation and release of PRO FUR from MAT FUR (35). Propeptide domains alone encode sufficient information for regulating the organelle-specific pH-dependent activation of cognate protease domains (32).…”

“…A fundamental question has long been how the timing of activation is encoded and recognized by the protease. Our understanding of how this regulation is achieved is based on studies of profurin (32)(33)(34)(35). This furin precursor contains an 83-residue N-terminal propeptide that is requisite for folding of its cognate catalytic domain in the ER (36 -38) after completion of which the propeptide gets cleaved but subsequently remains associated as an inhibitor until reaching the trans-Golgi network.…”