Background-The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in Ϸ7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results-We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E-deficient, and LDL receptor-deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor-deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions-Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective. (Circulation. 2012;125:894-901.)Key Words: atherosclerosis Ⅲ cardiovascular diseases Ⅲ cholesterol Ⅲ lipoproteins Ⅲ proprotein convertases A therosclerosis is a progressive degenerative pathology of large arteries that leads to ischemic heart diseases, which are expected to remain one of the leading causes of mortality until at least 2030. 1 The different stages of atherosclerotic plaque development have been reviewed extensively. [2][3][4][5] In brief, high levels of circulating atherogenic lipoproteins favor the accumulation of oxidized low-density lipoproteins (LDLs) in the subendothelial space. The latter are taken up by invading macrophages that, if they cannot efflux excess cholesterol to high-density lipoprotein, become foam cells. Foamy cells then release cytokines that recruit more macrophages into the building plaque. After formation of a necrotic core, plaques may ultimately become unstable; their fibrous cap can rupture and cause the formation of thrombus that can lead to death. Although inflammation is an important contributor to atherosclerosis, accumulation of oxidized LDLs in the subendothelial compartment is a key triggering event in atherosclerosis...

The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like endoprotease family and is synthesized as a preproenzyme. To investigate the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculovirus-infected insect cells, rendered soluble with 6 M guanidine HCl and 20 mM dithiothreitol, and purified by gel filtration and metal-binding affinity chromatography. Two NH 2 -terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, purified, and chemically characterized. Progress curve kinetic analysis with enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin demonstrated that both fragments were potent slow tight-binding inhibitors of either enzyme with K i in the low nanomolar range. Additional cleavages at Trp residues yielded fragment 9 -71 , which no longer represents a potent inhibitor. Upon incubation at pH 5.5 in the presence of excess 71-kDa murine PC1/3, NH 2 -terminal fragment 1-98 is cleaved at two sites, as revealed through Western blotting using NH 2 -terminal-directed PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/ 3 1-98 peptide, albeit at a much lesser extent with a micromolar K i and in a strictly competitive manner. These results suggest that the proregion of PC1/3 is an important feature in regulating its activity.

The cleavage of parathyroid hormone (PTH) from its precursor proparathyroid hormone (pro-PTH) is accomplished efficiently by the proprotein convertase furin (Hendy, G. N., Bennett, H. P. J., Gibbs, B. F., Lazure, C., Day, R., and Seidah, N. G. (1995) J. Biol. Chem. 270, 9517-9525). We also showed that a synthetic peptide comprising the ؊6 to ؉7 sequence of human pro-PTH is appropriately cleaved by purified furin in vitro. The human pro-PTH processing site Lys-Ser-Val-Lys-LysArg differs from the consensus furin site Arg-Xaa-(Lys/ Arg)-Arg that is represented by Arg-Arg-Leu-Lys-Arg in the cleavage site of pro-PTH-related peptide (proPTHrP). An earlier study demonstrated that an internally quenched fluorogenic substrate bearing an O-aminobenzoyl fluorescent donor at the NH 2 terminus and an acceptor 3-nitrotyrosine near the COOH terminus was appropriately cleaved by the convertases furin and PC1 (Jean, F., Basak, A., DiMaio, J., Seidah, N. G., and Lazure, C. (1995) Biochem. J. 307, 689 -695). Here, we have synthesized a series of internally quenched fluorogenic substrates based upon the pro-PTH and pro-PTHrP sequences to determine which residues are important for furin cleavage. Purified recombinant furin and PC1 cleaved the human pro-PTH internally quenched substrate at the appropriate site in an identical manner to that observed with the nonfluorescent peptide. Several substitutions in the P 6 -P 3 sequence were well tolerated; however, replacement of the Lys at the P 6 position with Gly and replacement of the P 3 Lys by an acidic residue led to markedly compromised cleavage by furin. Furin activity was very sensitive to substitution in P positions. Replacement of Ser at P 1 with Gly and Val at P 2 with Ala generated substrates that were less well cleaved. Substitution at the P 1 position of Val for Ser in conjunction with Ala for Val at P 2 , as well as a single substitution of Lys for Val at P 2 , generated specific inhibitors of furin cleavage. The findings of this study open the way to the rational design of inhibitors of furin with therapeutic potential.Polypeptide hormones, such as parathyroid hormone (PTH) 1 which is the major regulator of extracellular calcium homeostasis (1) and many other biologically active proteins and peptides, including PTH-related peptide (PTHrP), are initially synthesized as larger inactive precursor proteins that undergo processing to release the active moiety. The related PTH gene family member, PTHrP, was originally recognized as the major pathogenic agent in the hypercalcemia of malignancy syndrome (2). Under normal physiological conditions it subserves a variety of autocrine or paracrine roles such as modulator of the growth of various cell types including cartilage, bone, skin, and breast, or regulator of vascular smooth muscle tone (3). Among the proprotein precursors, pro-PTH is unusual in that the excised pro-segment consists of only a hexapeptide (4) located NH 2 -terminal to the 84-amino acid bioactive hormone. ProPTHrP is very similar to pro-PTH at the NH 2 terminus (5...

These results indicate that non-(1-84) PTH fragments are composed of a family of fragments which may be generated by specific or progressive cleavage at the N region. The longest fragment starts at position 4 and the shortest at position 15. A peptide starting at position 7 appears as the major component of non-(1-84) PTH fragments. The generation process is similar to the one described for smaller C-PTH fragments a number of years ago, suggesting a similar production mechanism and source for all C-PTH fragments.

Background: Proprotein convertases (PCs) activate overexpressed endothelial lipase (EL) inhibitor angiopoietin-like-3 (ANGPTL3) and inactivate EL. Results: In PC knock-out-mice, analysis of primary hepatocytes and circulating ANGPTL3 and EL fragments revealed that furin is their primary convertase. Conclusion: However, the lack of hepatocyte furin had no major impact on HDL-cholesterol or EL phospholipase activity. Significance: Inhibition/silencing of furin in hepatocytes would not affect lipid profiles.

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Abstract␤-Microseminoprotein, alternatively called prostatic secretory protein of 94 amino acids, is a hydrophilic, unglycosylated, small protein rich in conserved half-cystine residues. Originally found in human seminal plasma and prostatic fluids, its presence was later shown in numerous secretions and its homologs were described in many vertebrate species. These studies showed that this protein had rapidly evolved, but they failed to unambiguously identify its biological role. Here, we show that a protein isolated from ostrich pituitary gland is closely related to a similar one isolated from chicken serum and that the two are structurally related to the mammalian ␤-microseminoprotein. The complete 90-amino acid sequence of the ostrich molecule was established through a combination of automated Edman degradation and matrixassisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric procedures, including postsource decay (PSD) and ladder sequencing analyses. This study documents for the first time that ␤-microseminoprotein is present in aves. It is also the first report of a C-terminal amidated form for a member of this protein family and the first in which the disulfide linkages are established. Database searches using the herein-described amino acid sequence allowed identification of related proteins in numerous species such as cow, African clawed frog, zebrafish, and Japanese flounder. These small proteins show a strikingly high rate of amino acid substitutions, especially across phyla boundaries. Noticeably, no ␤-microseminoprotein-related gene could be found in the recently completed fruit fly genome, indicating that if such a gene exists in arthropods, it must have extensively diverged from the vertebrate ones.

The study objective was to measure breastfeeding rates and patterns in the Montérégie region of Québec. A survey of 632 mothers of 6-month-old infants was performed, of which 80% initiated breastfeeding, and 68% exclusively breastfed at birth. Breastfeeding rates progressively decreased with time: 63%, 56%, 51%, 44%, 39%, and 32% of mothers breastfed at 1, 2, 3, 4, 5, and 6 months, respectively. Among mothers breastfeeding at a given period, 62%, 57%, 48%, 35%, and 10% of women exclusively breastfed since birth for 1, 2, 3, 4, and 5 months, respectively. Exclusive breastfeeding for 6 months among the 200 women still breastfeeding was practically nonexistent. Introduction of nonhuman milk or solids was primarily responsible for the shift in patterns from exclusive to complementary feeding without passing through predominant breastfeeding. These findings confirm the need to prioritize effective hospital-based and community-based interventions to increase breastfeeding duration and exclusivity in the region.