In Vitro Characterization of the Novel Proprotein Convertase PC7

Munzer, Jon Scott; Basak, Ajoy; Zhong, Mei; Mamarbachi, Aida M.; Hamelin, Josée; Savaria, Diane; Lazure, Claude; Benjannet, Suzanne; Chrétien, Michel; Seidah, Nabil G.

doi:10.1074/jbc.272.32.19672

Cited by 85 publications

(98 citation statements)

References 52 publications

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“…The 12-mer peptides were incubated in vitro overnight with 2 units of purified PCs [2 pmol amino methyl coumarin (AMC) released per minute from the peptide Pyr-RTKR-MCA) (43)], in 2 mM CaCl 2 and 25 mM Tris-Mes, pH 7.5, in a volume of 100 l. The products were separated by RP-HPLC on a Varian C 18 column (5 m, 100 Å, 4.5 ϫ 250 mm), and MS confirmed that cleavage took place exclusively after Arg 296 . HEK293 cells were grown in DMEM containing 10% heat-inactivated FCS (Canadian Life Technologies) at 37°C in 5% CO2.…”

Section: Pcr Genotyping Of Embryos and Micementioning

confidence: 99%

In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate

Essalmani

Zaid

Marcinkiewicz

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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114

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The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/ C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1 Asn in the RSRR2N cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase.Furin-like ͉ Meox2-cre ͉ Pcsk5

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Section: Pcr Genotyping Of Embryos and Micementioning

confidence: 99%

In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate

Essalmani

Zaid

Marcinkiewicz

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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114

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“…N-terminal radiosequencing (26,30) was carried out on SDS-PAGE-purified immunoprecipitates. The C-terminally flagged 72-kDa [pro-BACE F ] FG , labeled with [ 3 H]Leu and produced in the presence of ␣ 1 -PDX, had a Leu at positions 3, 7, 9, and 13 (not shown).…”

Section: Biosynthesis and Processing Of Bace-mentioning

confidence: 99%

Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding

Benjannet

Elagöz

Wickham

et al. 2001

Journal of Biological Chemistry

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278

134

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Processing of the ␤-amyloid precursor protein (␤APP) by ␤-and ␥-secretases generates the amyloidogenic peptide A␤, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the ␤-secretase ␤-amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant ␤APP sw ␤-secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylationdependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the ␣-secretase product APPs␣. Although there is little increase in the generation of A␤ by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced A␤ levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of ␤APP.Alzheimer's disease is a progressive degenerative disorder of the brain characterized by mental deterioration, memory loss, confusion, and disorientation. Among the cellular mechanisms contributing to this pathology are two types of fibrous protein deposition in the brain, intracellular neurofibrillary tangles consisting of polymerized tau protein, and abundant extracellular fibrils largely composed of ␤-amyloid 1 (for reviews see Refs. 1-3). ␤-Amyloid, also known as A␤, arises from proteolytic processing of the ␤-amyloid precursor protein (␤APP) at the ␤-and ␥-secretase cleavage sites. The cellular toxicity and amyloid-forming capacity of the two major forms of A␤ (A␤ 40 and especially A␤ 42 ) have been well documented (1-3).An alternative, anti-amyloidogenic cleavage carried out by ␣-secretase(s) is located within the A␤ peptide sequence of ␤APP, thus precluding the formation of intact insoluble A␤. This cleavage by ␣-secretase within the (His-His-GlnLys2Leu-Val) sequence of ␤APP is the major physiological route of APP maturation. The products of this reaction are a soluble 100 -120-kDa N-terminal fragment (␤APPs␣) and a C-terminal membrane-bound ϳ9-kDa segment (C83). In several recent reports, metalloproteinases such as ADAM9, -10, and -17 were shown to be involved in the ␣-secretase cleavage

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“…1, right panel), showing that the unglycosylated pro-LPC detected in these transfections is the result of translation initiation at the second ATG codon. This methionine is located 35 amino acids carboxyl-terminal of the first methionine, two amino acids aminoterminal of the major signal peptide cleavage site, and five amino acids amino-terminal of the minor signal peptide cleavage site (11). Translation initiation at this ATG codon will result in the biosynthesis of a protein without a functional signal peptide.…”

Section: Methodsmentioning

confidence: 99%

“…It is widely expressed, both during embryogenesis and adult life (8,9), and has a substrate specificity similar but not identical to that of furin (10,11). LPC is the least conserved family member, and phylogenetic analysis indicates that LPC is more related to kexin in yeast than to any other mammalian PC (12,13).…”

mentioning

confidence: 99%

Binding of BiP to the Processing Enzyme Lymphoma Proprotein Convertase Prevents Aggregation, but Slows Down Maturation

Creemers¹,

Loo²,

Plets³

et al. 2000

Journal of Biological Chemistry

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Lymphoma proprotein convertase (LPC) is a subtilisin-like serine protease of the mammalian proprotein convertase family. It is synthesized as an inactive precursor protein, and propeptide cleavage occurs via intramolecular cleavage in the endoplasmic reticulum. In contrast to other convertases like furin and proprotein convertase-1, propeptide cleavage occurs slowly. Also, both a glycosylated and an unglycosylated precursor are detected. Here we demonstrate that the unglycosylated precursor form of LPC is localized in the cytosol due to the absence of a signal peptide. Using a reducible cross-linker, we found that glycosylated pro-LPC is associated with the molecular chaperone BiP. In addition, we show that pro-LPC is prone to aggregation and forms large complexes linked via interchain disulfide bonds. BiP is associated mainly with non-aggregated pro-LPC and pro-LPC dimers and trimers, suggesting that BiP prevents aggregation. Overexpression of wild-type BiP or a dominant-negative BiP ATPase mutant resulted in reduced processing of pro-LPC. Taken together, these results suggest that binding of BiP to pro-LPC prevents aggregation, but results in slower maturation.

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In Vitro Characterization of the Novel Proprotein Convertase PC7

Cited by 85 publications

References 52 publications

In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate

In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate

Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding

Binding of BiP to the Processing Enzyme Lymphoma Proprotein Convertase Prevents Aggregation, but Slows Down Maturation

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