Abstract
The propeptides of proprotein convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Earlier experimental work identified a conserved histidine-encoded pH sensor within the propeptide of the canonical PC, furin. To date, whether protonation of this conserved histidine is solely responsible for PC activation has remained unclear because of the observation that various PC paralogues are activated at different organellar pH values. To ascertain additional determinants of PC activation, we analyzed PC1/3, a paralogue of furin that is activated at a pH of ∼5.4. Using biophysical, biochemical, and cell-based methods, we mimicked the protonation status of various histidines within the propeptide of PC1/3 and examined how such alterations can modulate pH-dependent protease activation. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a "gatekeeper" that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion.
Highlights
Propeptides regulate the organelle-specific, pH-dependent activation of proprotein convertases
We asked whether the pH-sensing histidine is conserved in PROPC1/3 and why does MATPC1/3 not become active at the same pH as furin? We present biochemical and structural data that suggest that the conserved histidine residue plays a critical role in pH-dependent activation similar to what has been established in furin; an additional histidine residue modulates this pH sensitivity such that a more acidic environment is required for PC1/3 activation
The alignment of eight orthologues of PROFUR and PROPC1/3 indicates a high degree of sequence similarity, including stretches of completely conserved residues distributed throughout the entirety of both propeptides (Fig. 2A)
Summary
Propeptides regulate the organelle-specific, pH-dependent activation of proprotein convertases. Results: A histidine residue pair in the propeptide cooperatively defines the activation pH for proprotein convertase 1/3. The propeptides of proprotein convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a “gatekeeper” that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion
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