Endoproteolytic Cleavage of Its Propeptide Is a Prerequisite for Efficient Transport of Furin Out of the Endoplasmic Reticulum

Creemers, John W.M.; Vey, Martin; Schäfer, Wolfram; Ayoubi, Torik; Roebroek, Anton; Klenk, Hans‐Dieter; Garten, Wolfgang; Ven, Wim J.M. Van de

doi:10.1074/jbc.270.6.2695

Cited by 116 publications

(119 citation statements)

References 32 publications

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“…Monoclonal antibody (mAb) PS1-3, reacting with the peptide RRVSKNSKYNAES-TERESQDTVAEN in the hydrophilic loop domain of PS1 mAb 9E10 against the Myc tag (26), was kindly provided by Dr. J. Creemers. mAbs MON160, 161 and 162 and MON148 and 152 against NSP/reticulon and furin have been described (22)(23)(24)(25). mAbs against the immunoglobulinbinding protein (BiP) and against the calcium pump Serca 2a (IID8) were purchased from, respectively, StressGen (Victoria, Canada) and Affinity BioReagents (Neshanic Station, NJ).…”

Section: Constructs-mentioning

confidence: 99%

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Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins

Strooper¹,

Beullens²,

Contreras

et al. 1997

Journal of Biological Chemistry

271

187

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Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100 -250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37°C instead of 95°C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 3 Glu or Cys-410 3 Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.

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Section: Constructs-mentioning

confidence: 99%

“…The resulting cDNA codes for PS1 with the Myc tag (EQKLISEEDL) immediately after the initiator methionine, as confirmed by cDNA sequencing. Plasmids containing the cDNA for furin (22,23) or reticulon/NSP (24,25) were kindly provided by Dr. J. Creemers, Dr. A. Roebroek, and Dr. W. Van De Ven (Center for Human Genetics, Leuven, Belgium).…”

Section: Constructs-mentioning

confidence: 99%

Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins

Strooper¹,

Beullens²,

Contreras

et al. 1997

Journal of Biological Chemistry

271

187

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“…Our data clearly show that PC2-S383A is secreted as the intact zymogen from CHO-K1/7B2 cells. This result stands in contrast to data obtained for other convertases, which indicate that catalytic triad mutants of human furin (furD46A, furH87A, and furS261A) expressed in transfected COS-1 cells are retained in the ER and are unable to undergo autocatalytic maturation [9,10]. Similarly, when PC1-S382A was expressed in HEK293 cells, no processed and secreted enzyme was detected, suggesting ER retention similar to furin [11].…”

Section: Discussionmentioning

confidence: 62%

“…Interestingly, catalytic triad mutants of human furin (furD46A, furH87A, and furS261A) do not undergo autocatalytic maturation, but instead accumulate in the ER [9,10]; similar results were found for a catalytic site mutant of PC1, PC1-S382A [11]. In this paper we have investigated the fate of a catalytic site mutant of proPC2 and show that in contrast to other convertases, this active site mutant is apparently folded correctly, as evidenced by its ability to efficiently traverse the secretory pathway.…”

Section: Introductionmentioning

confidence: 99%

Processing and trafficking of a prohormone convertase 2 active site mutant

Lee

Kacprzak

Day

et al. 2007

Biochemical and Biophysical Research Communications

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Processing of most PC zymogens is required for successful folding and/or passage through the secretory pathway; active site mutants are retained in the ER and degraded. We here report that the active site serine mutant of PC2 (PC2-S383A) was efficiently secreted as the intact zymogen in CHO-K1 cells, suggesting that its propeptide can productively insert into the mutated binding pocket without causing misfolding. In AtT-20 cells, PC2-S383A was cleaved at the secondary cleavage site within the propeptide; this cleavage event was pH-dependent and was inhibited by a proprotein convertase inhibitor. In vitro digestion of PC2-S383A with various convertases indicates that this site is accessible to in trans cleavage. Abundant immunoreactive S383A PC2 was found in secretory granules, supporting the idea that this protein is efficiently trafficked through the secretory pathway.

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“…PCs are themselves synthesized as precursors with 83-106-amino-acid pro-regions that are cleaved in an autocatalytic manner [8] soon after biosynthesis of the enzyme in the ER (endoplasmic reticulum) [9,10].…”

Section: Introductionmentioning

confidence: 99%

Identification of furin pro-region determinants involved in folding and activation

Bissonnette

Charest

Longpré

et al. 2004

Biochemical Journal

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The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the transGolgi network. To identify residues that are important for proregion function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furin's pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furin's proregion.

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Endoproteolytic Cleavage of Its Propeptide Is a Prerequisite for Efficient Transport of Furin Out of the Endoplasmic Reticulum

Cited by 116 publications

References 32 publications

Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins

Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins

Processing and trafficking of a prohormone convertase 2 active site mutant

Identification of furin pro-region determinants involved in folding and activation

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