Role of PCSK5 Expression in Mouse Ovarian Follicle Development: Identification of the Inhibin α- and β-Subunits as Candidate Substrates

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and βB-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability

Baculovirus-infected cells do not produce the amyloid peptide of Alzheimer's disease from its precursor.

The amyloid peptide (Abeta) of Alzheimer's disease (AD) is produced by proteolytic cleavage of a larger precursor, the amyloid peptide precursor or APP. The discovery of pathogenic mutations in the APP gene provides strong evidence for the hypothesis that APP metabolism is involved in the etiology of AD. To study the metabolism of the protein, human APP has been expressed in several mammalian cell types. Insect cells, infected by a recombinant baculovirus carrying the human APP sequence, also provide an interesting expression system because these cells do not produce endogenous APP. Baculovirus-infected cells synthesize very high amounts of extracellular soluble APP, after cleavage of the transmembrane protein, as described for mammalian cells. However, we demonstrate here that insect cells do not produce Abeta from APP. These results suggest that while the enzymatic activity needed for the production of soluble APP is conserved between insect and mammalian cells, the enzymes required for the production of Abeta from APP are only expressed in mammalian cells

Missense mutations associated with familial Alzheimer's disease in Sweden lead to the production of the amyloid peptide without internalization of its precursor.

Production of soluble amyloid peptide precursor (APP) and amyloid peptide (A beta) was measured in CHO cells transfected by the wild-type APP 695 cDNA sequence or by the same sequence carrying missense mutations associated with familial Alzheimer's disease in Sweden. Deletion of the C-terminal domain of the protein corresponding to residues 654 to 695 of APP 695 not only inhibited very significantly the internalization of APP at 37 degrees C, but also led to the secretion of an uncleaved APP in the culture medium of CHO cells. This deletion did not affect A beta production from the Swedish APP but was able to inhibit the production of the wild-type APP. These results demonstrate that, in CHO cells, the internalization of the wild-type APP is needed for A beta production, while the production of the amyloid peptide from Swedish APP is independent of the internalization process

The role of presenilin-1 in the gamma-secretase cleavage of the amyloid precursor protein of Alzheimer's disease.

Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells

Données de base sur la transgenèse

National audienceLa transgenèse permet d’introduire dans le génome d’un animal un fragment d’ADN qui sera ensuite transmis de génération en génération. Elle utilise différentes approches méthodologiques. Certaines, encore limitées à peu d’espèces, permettent des modifications très fines du génome. La transgenèse, associée au développement de la biologie moléculaire, offre des applications multiples tant dans la recherche fondamentale qu’appliquée. Elle est de fait devenue un outil indispensable pour l’analyse de la régulation de l’expression des gènes et la compréhension de leur fonction

Loss of endothelial furin leads to cardiac malformation and early postnatal death.

In mammals, seven proprotein convertases (PCs) cleave secretory proteins after basic residues, and four of them are called furin-like PCs: furin, PC5, PACE4, and PC7. In vitro, they share many substrates. However, furin is essential during development since deficient embryos die at embryonic day 11 and exhibit multiple developmental defects, particularly defects related to the function of endothelial cells. To define the role of furin in endothelial cells, an endothelial cell-specific knockout (ecKO) of the Furin gene was generated. Newborns die shortly after birth, indicating that furin is essential in these cells. Magnetic resonance imaging revealed that ecKO embryos exhibit ventricular septal defects (VSD) and/or valve malformations. In addition, primary cultures of wild-type and ecKO lung endothelial cells revealed that ecKO cells are unable to grow. Growth was efficiently rescued by extracellular soluble furin. Analysis of the processing of precursors of endothelin-1 (ET-1), adrenomedullin (Adm), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein 4 (BMP4) confirmed that ET-1, Adm, and TGF-β1 are in vivo substrates of endothelial furin. Mature ET-1 and BMP4 forms were reduced by ~90% in ecKO purified endothelial cells from lungs