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

Stephanie L. Dillon; Danielle M. Williamson; Johannes Elferich; David Radler; Rajendra Joshi; Gary Thomas; Ujwal Shinde

doi:10.1016/j.jmb.2012.06.023

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

Stephanie L. Dillon, Danielle M. Williamson, Johannes Elferich, David Radler, Rajendra Joshi, Gary Thomas, Ujwal Shinde

Chemical Physiology and Biochemistry

Research output: Contribution to journal › Article › peer-review

23 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	47-62
Number of pages	16
Journal	Journal of molecular biology
Volume	423
Issue number	1
DOIs	https://doi.org/10.1016/j.jmb.2012.06.023
State	Published - Oct 12 2012

Keywords

folding
molecular dynamics
pH sensor
protease activation
subtilisin

ASJC Scopus subject areas

Biophysics
Structural Biology
Molecular Biology

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10.1016/j.jmb.2012.06.023

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

abstract = "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 ~ 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 ~ 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.",

keywords = "folding, molecular dynamics, pH sensor, protease activation, subtilisin",

author = "Dillon, {Stephanie L.} and Williamson, {Danielle M.} and Johannes Elferich and David Radler and Rajendra Joshi and Gary Thomas and Ujwal Shinde",

note = "Funding Information: We thank Mahta Nili and Parvathy Ramakrishnan for their help in cloning and the cell-based techniques and Nathan Brandt and Laura Figoski for protein expression. Special thanks to Jimmy Dikeakos for reading the manuscript. This work was supported by the National Science Foundation CAREER award ( MCB0746589 ) and Grant in Aid from the American Heart Foundation to U.S., a National Institutes of Health training grant to D.M.W., an American Heart Association pre-doctoral training grant ( 12PRE11470005 ) to J.E., and National Institutes of Health grants ( DK37274 and CA151564 ) to G.T. Simulations were performed using computing equipment in the laboratory of Michael Chapman funded by the OHSU Emerging Technology Fund.",

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language = "English (US)",

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T1 - Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3

AU - Dillon, Stephanie L.

AU - Williamson, Danielle M.

AU - Elferich, Johannes

AU - Radler, David

AU - Joshi, Rajendra

AU - Thomas, Gary

AU - Shinde, Ujwal

N1 - Funding Information: We thank Mahta Nili and Parvathy Ramakrishnan for their help in cloning and the cell-based techniques and Nathan Brandt and Laura Figoski for protein expression. Special thanks to Jimmy Dikeakos for reading the manuscript. This work was supported by the National Science Foundation CAREER award ( MCB0746589 ) and Grant in Aid from the American Heart Foundation to U.S., a National Institutes of Health training grant to D.M.W., an American Heart Association pre-doctoral training grant ( 12PRE11470005 ) to J.E., and National Institutes of Health grants ( DK37274 and CA151564 ) to G.T. Simulations were performed using computing equipment in the laboratory of Michael Chapman funded by the OHSU Emerging Technology Fund.

PY - 2012/10/12

Y1 - 2012/10/12

N2 - 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 ~ 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 ~ 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.

AB - 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 ~ 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 ~ 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.

KW - folding

KW - molecular dynamics

KW - pH sensor

KW - protease activation

KW - subtilisin

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JO - Journal of molecular biology

JF - Journal of molecular biology

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ER -

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

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