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PubMed2003

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs.

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.

Amino Acid Sequence↗
PubMed2005

Secretion of soluble leptin receptors by exocrine and endocrine cells of the gastric mucosa.

Leptin is a hormone secreted by the gastric mucosa into the lumen of the stomach. It is present in its intact form in the intestine where it regulates nutrient absorption and intestinal mucosa integrity. We have identified the binding protein that protects leptin from the harsh conditions of the gastric juice. Immunoprecipitations and Western blot analyses demonstrated that leptin is present in the gastric mucosa and the gastric juice, bound to a protein corresponding to the extracellular domain of the leptin receptor. In the absence of this soluble receptor, leptin is rapidly degraded. Immunocytochemistry on rat gastric mucosa identified the cells and intracellular compartments involved in secretion of this complex. Leptin receptor extracellular domain and leptin are present along the rough endoplasmic reticulum-Golgi-granules secretory pathways and form a complex in the secretory granules of Chief and specific endocrine cells. The long-form membrane leptin receptor OB-Rb, the protease activator furin, and proprotein convertase 7 were found in Chief cell granules but not in those of endocrine cells. The shedding of the receptor occurs in the immature granules. It is concluded that in the immature secretory granules of Chief cells, furin activates proprotein convertase 7 that, in turn, cleaves the extracellular portion of membrane-bound leptin receptors. Leptin bound to its soluble receptor forms a complex that is resistant to the gastric juice. Endocrine cells, on the other hand, generate a soluble leptin receptor by mechanisms different from those of the exocrine cells.

Animals↗
PubMed1999

The exon-intron organization of the prohormone convertase PC2 gene from the insect Lucilia cuprina.

Prohormone or proprotein convertases are members of the subtilisin family of serine proteases. They are involved in the activation of precursor molecules by endoproteolytic cleavage at basic amino acid residues. Among the different members of this prohormone convertase family, the prohormone convertase 2 (PC2) is almost exclusively expressed in endocrine and neuroendocrine tissues and plays an important role in the endoproteolytic processing of prohormones. Here we describe the exon-intron organization of the PC2 gene from the insect Lucilia cuprina by characterization of PCR-amplified genomic DNA fragments. The insect PC2 gene contains 12 exons with an estimated size of over 14.5 kb. The exon sizes range from 38 bp to > 448 bp. All identified intron-exon boundaries are consistent with the GT-AG-rule. A comparison of the genomic structures of the thus far known prohormone convertase genes with that of the insect PC2 gene revealed a conservation of the positions of most introns interrupting the exons coding for the amino-terminal and catalytic domains. This conservation is consistent with the suggestion of a common evolutionary origin for the prohormone convertase gene family.

Animals↗
PubMed2002

Inhibitors of the subtilase-like pro-protein convertases (SPCs).

Following protein biosynthesis, some of the most important cellular mechanisms that generate biological diversity are the enzymatically driven post-translational modifications that ultimately lead to the formation of bioactive molecules. Within the secretory pathway, a multitude of precursor proteins are thus modified resulting in hormones, neuropeptides, growth factors, receptors and even enzymes. These modifications include cleavage at specific sites through endo- or exo-peptidase action, amidation, glycosylation and sulfation. In recent years, an important family of these processing enzymes was discovered and characterized. The so-called proprotein convertases are the products of seven distinct genes and function as endopeptidases that cleave protein precursors C-terminal to basic residue sites. They are structurally related to the bacterial subtilisin family of enzymes and are thus referred to as the subtilisin-like proprotein convertases (SPCs). Many studies have examined the inhibition of this family of enzymes, through the search of endogenous inhibitors or through the development of peptidyl, non-peptidyl or protein inhibitors. Some potent inhibitors have been discovered or engineered. While it is certain that potent inhibitors could serve as important tools to further elucidate the specific functions of each SPC, it has also been suggested that such inhibitors may be developed into lead compounds that could have important therapeutic applications. This review examines the progress made in regards to endogenous and engineered inhibitors and evidence for possible uses as molecular tools or in therapeutic applications. It is noted that although important inhibitory potencies have often been reported, there is generally insufficient evidence to demonstrate high levels of specificity. It is thus suggested that an important short-term challenge before the field will be a better understanding of the catalytic specificity of each SPC.

Drug Design↗
PubMed1999

Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1-42 maturase.

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2's subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.

Amino Acid Sequence↗
PubMed1997

Prorenin activation and prohormone convertases in the mouse As4.1 cell line.

The precise identification of prorenin-processing enzymes has been hampered by the very low abundance of juxtaglomerular cells in the kidney. Recently, an immortalized renin-producing renal tumor cell line (As4.1) has been proposed as a model to carry out such studies. Despite the fact that they contain secretory granules, we found no evidence (on the basis of enzymatic assays of renin activity in the supernatant of the cells and of immunoprecipitations experiments) that the As4.1 cells can secrete active renin through the regulated pathway. As4.1 cells produce only renin-1, as they derive from a strain of mice expressing only one renin gene. However, stable transfection of these cells with a renin-2 expression plasmid increased the capacity of this cell line to secrete active renin in the regulated pathway. Northern blot and reverse transcriptase-polymerase chain reaction amplification (RT-PCR) assays revealed that furin, PACE4 and PC5 were the only members of the proprotein convertase (PC) family to be present in these cells. As PC5 is the only such enzyme with the demonstrated ability to process mouse prorenin 2, it may constitute a candidate enzyme for the processing of prorenin-2 in mouse juxtaglomerular cells. However, it is not likely to be involved in the processing of mouse prorenin 1.

Animals↗
PubMed2005

Dual regulation of the LDL receptor--some clarity and new questions.

Upregulation of the expression of the LDL receptor (LDLR) gene does not always lead to an increase in the LDLR protein. While the mRNA expression of the LDLR and the newly studied proprotein convertase PCSK9 are coordinately upregulated in absence of sterols, the latter proteinase apparently enhances the degradation of the LDLR protein.

Animals↗
PubMed1998

alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent.

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor alpha1-antitrypsin Portland (alpha1-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged alpha1-PDX (alpha1-PDX/hf) revealed the selectivity of the alpha1-PDX/hf reactive site loop for furin (Ki, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that alpha1-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin's active site, alpha1-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg355-Ile-Pro-Arg358- downward arrow or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of alpha1-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the alpha1-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.

ADP Ribose Transferases↗
PubMed2001

Processing of immunosuppressive pro-TGF-beta 1,2 by human glioblastoma cells involves cytoplasmic and secreted furin-like proteases.

TGF-beta is a putative mediator of immunosuppression associated with malignant glioma and other types of cancer. Subtilisin-like proprotein convertases such as furin are thought to mediate TGF-beta processing. Here we report that human malignant glioma cell lines express furin mRNA and protein, exhibit furin-like protease (FLP) activity, and release active furin into the cell culture supernatant. FLP activity is not modulated by exogenous TGF-beta or neutralizing TGF-beta Abs. Exposure of LN-18 and T98G glioma cell lines to the furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, inhibits processing of the TGF-beta1 and TGF-beta2 precursor molecules and, consequently, the release of mature bioactive TGF-beta molecules. Ectopic expression of PDX, a synthetic antitrypsin analog with antifurin activity, in the glioma cells inhibits FLP activity, TGF-beta processing, and TGF-beta release. Thus, subtilisin-like proprotein convertases may represent a novel target for the immunotherapy of malignant glioma and other cancers or pathological conditions characterized by enhanced TGF-beta bioactivity.

Cell Line, Transformed↗
PubMed1999

Carboxypeptidase D is a potential candidate to carry out redundant processing functions of carboxypeptidase E based on comparative distribution studies in the rat central nervous system.

Post-translational processing is essential for the biological activation of many proteins and peptides. After precursor cleavage at specific single residues or pairs of basic residues by the proprotein convertases, the C-terminal basic residues are removed. Carboxypeptidase E was thought to be the only enzyme responsible. Recent studies with carboxypeptidase E-deficient mice, Cpe(fat)/Cpe(fat), indicated the existence of carboxypeptidase E-like carboxypeptidases, such as carboxypeptidase D. In order to define potential redundant functions in vivo, we compared the distributions of both carboxypeptidases in the rat central nervous system and selected endocrine tissues. Carboxypeptidase D messenger RNA was abundantly expressed in glial cells in the gray and white matter, while neurons in several brain regions, such as the piriform cortex, basolateral amygdala and hippocampus, also expressed high levels of carboxypeptidase D messenger RNA. Co-localization of carboxypeptidases E and D messenger RNAs was observed in many brain regions, the spinal cord and endocrine tissues. Immunohistochemistry showed the intracellular distribution of carboxypeptidase D with a perinuclear pattern. The extensive distribution of carboxypeptidase D in both glial and neuronal cells indicates the important role of carboxypeptidase D in peptide processing, possibly working together with furin, a ubiquitously expressed proprotein convertase. The co-localization of carboxypeptidases D and E suggests that carboxypeptidase D may, at least partially, compensate for carboxypeptidase E processing functions in Cpe(fat)/Cpe(fat) mice.

Adrenal Glands↗
PubMed2005

Mutations of the PC2 substrate binding pocket alter enzyme specificity.

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.

Amino Acid Sequence↗
PubMed2002

Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects.

The subtilisin-like proprotein convertases PC1/3 (SPC3) and PC2 (SPC2) are believed to be the major endoproteolytic processing enzymes of the regulated secretory pathway. They are expressed together or separately in neuroendocrine cells throughout the brain and dispersed endocrine system in both vertebrates and invertebrates. Disruption of the gene-encoding mouse PC1/3 has now been accomplished and results in a syndrome of severe postnatal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2. Mice lacking PC1/3 are normal at birth, but fail to grow normally and are about 60% of normal size at 10 weeks. They lack mature GHRH, have low pituitary growth hormone (GH) and hepatic insulin-like growth factor-1 mRNA levels and resemble phenotypically the "little" mouse (Gaylinn, B. D., Dealmeida, V. I., Lyons, C. E., Jr., Wu, K. C., Mayo, K. E. & Thorner, M. O. (1999) Endocrinology 140, 5066-5074) that has a mutant GHRH receptor. Despite a severe defect in pituitary proopiomelanocortin processing to mature adrenocorticotropic hormone, blood corticosterone levels are essentially normal. There is marked hyperproinsulinemia but without impairment of glucose tolerance. In contrast, PC2-null mice lack mature glucagon and are chronically hypoglycemic (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H. & Steiner, D. (1997) Proc. Natl. Acad. Sci. USA 94, 6646-6651). The PC1/3-null mice differ from a human subject reported with compound heterozygosity for defects in this gene, who was of normal stature but markedly obese from early life. The PC1/3-null mice are not obese. The basis for these phenotypic differences is an interesting topic for further study. These findings prove the importance of PC1/3 as a key neuroendocrine convertase.

Adrenocorticotropic Hormone↗
PubMed2003

Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.

Autosomal dominant hypercholesterolemia (ADH; OMIM144400), a risk factor for coronary heart disease, is characterized by an increase in low-density lipoprotein cholesterol levels that is associated with mutations in the genes LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B). We mapped a third locus associated with ADH, HCHOLA3 at 1p32, and now report two mutations in the gene PCSK9 (encoding proprotein convertase subtilisin/kexin type 9) that cause ADH. PCSK9 encodes NARC-1 (neural apoptosis regulated convertase), a newly identified human subtilase that is highly expressed in the liver and contributes to cholesterol homeostasis.

Amino Acid Substitution↗
PubMed2007

Molecular biology of PCSK9: its role in LDL metabolism.

Proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a newly discovered serine protease that destroys low density lipoprotein (LDL) receptors in liver and thereby controls the level of LDL in plasma. Mutations that increase PCSK9 activity cause hypercholesterolemia and coronary heart disease (CHD); mutations that inactivate PCSK9 have the opposite effect, lowering LDL levels and reducing CHD. Although the mechanism of PCSK9 action is not yet clear, the protease provides a new therapeutic target to lower plasma levels of LDL and prevent CHD.

Cholesterol, LDL↗
PubMed1994

Levels of the conversion endoproteases PC1 (PC3) and PC2 distinguish between insulin-producing pancreatic islet beta cells and non-beta cells.

PC1 (PC3) and PC2, members of the mammalian family of proprotein convertases homologous to the yeast Kex2 gene product, are both expressed in pancreatic islets of Langerhans. Recent studies have suggested that PC1 and PC2 are responsible for the conversion of proinsulin to insulin and connecting peptide (C-peptide) in the islet beta cells. However, the insulin-secreting beta cells are not the only cells present in these complex micro-organs, prompting us to evaluate the expression of PC1 and PC2 in islet beta and non-beta cells. Rat islet cells were sorted by autofluorescence-activated flow cytometry to separate beta cells from non-beta cells, and conversion endoprotease levels were analysed by Western blotting. The immunolabel ratio of PC1/PC2 in beta cells was 2.6. Non-beta cells displayed much lower levels of PC1 than beta cells, but twice as much PC2 (PC1/PC2 = 0.05). Post-translational modification of the convertases themselves was found to differ between the cell types. In particular, a 75 kDa precursor form of PC2 (pro-PC2) was found to accumulate in beta cells, whereas only the fully processed 67 kDa form was detected in the non-beta cells. Finally, the quantification of PC1 and PC2 and their precursor forms in transformed cells (insulin-producing beta-TC and glucagon-producing alpha-TC) showed that transformation appeared to be accompanied by unusually high levels of the precursors.

Animals↗
PubMed2004

No genetic linkage or molecular evidence for involvement of the PCSK9, ARH or CYP7A1 genes in the Familial Hypercholesterolemia phenotype in a sample of Danish families without pathogenic mutations in the LDL receptor and apoB genes.

A locus on chromosome 1p34.1-p32 has been linked to autosomal dominant Familial Hypercholesterolemia (FH) and is termed the third FH locus. We tested whether this third FH locus is linked to the FH phenotype in 20 Danish families, with 158 members, without pathogenic mutations in the genes, encoding the low-density lipoprotein (LDL) receptor or apolipoprotein B (apoB). We could exclude the third FH locus as a cause of FH by genetic linkage analysis in the families taken together. Since haplotype analysis of each family nevertheless suggested that the FH phenotype co-segregated in a manner consistent with linkage to the third FH locus in three small pedigrees, we performed sequencing analysis without being able to demonstrate mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, the main candidate gene in the third FH locus. By the same combination of genetic linkage and molecular analysis we could also exclude mutations in the gene for the LDL receptor adaptor protein and in the gene for cholesterol-7-alpha-hydroxylase as causes of FH in our sample. Although not indicating linkage to any known loci, our data still indicate that another dominant gene may be involved in causing a FH phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed2001

IDA-1, a Caenorhabditis elegans homolog of the diabetic autoantigens IA-2 and phogrin, is expressed in peptidergic neurons in the worm.

The closely related mammalian proteins IA-2 and phogrin are protein tyrosine phosphatase-like receptor proteins spanning the membrane of dense core vesicles of neuroendocrine tissues. They are of interest as molecular components of the secretory machinery and as major targets of autoimmunity in type I diabetes mellitus. The Caenorhabditis elegans genome has a single copy of an IA-2/phogrin homolog ida-1 III (islet cell diabetic autoantigen), which encodes the ida-1 (B0244.2) gene product as a series of 12 exons over a 10-kb region of chromosome III. The full-length sequence of the ida-1 cDNA encoded a 767-amino acid type 1 transmembrane protein of 87 kDa. The PTP catalytic site consensus sequence of IDA-1, like IA-2 and phogrin, diverged and would not be active. Expression of green fluorescent protein (GFP) under the ida-1 gene promoter showed activity in a subset of around 30 neurons with sensory functions and the uv1 cells of the vulva in hermaphrodites. Males showed additional expression in male-specific neurons. In situ experiments in rat brain showing the distribution of IA-2 and phogrin suggested a complimentary and overlapping pattern compared with the proprotein convertases PC1 and PC2. In C. elegans, IDA-1-expressing cells comprised a subset of those expressing the PC2 homolog KPC-2 (C51E3. 7), consistent with IDA-1 being a component of neuropeptide-containing dense core vesicles. The results support the hypothesis that C. elegans IDA-1 is the functional homolog of IA-2 and phogrin in mammals. Analysis of the function of IDA-1 should contribute to our understanding of the function of these proteins in signal transduction, vesicle locomotion, and exocytosis.

Amino Acid Sequence↗
PubMed2006

The C679X mutation in PCSK9 is present and lowers blood cholesterol in a Southern African population.

OBJECTIVE: Missense mutations in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) can cause familial hypercholesterolemia. However, two nonsense variants of PCSK9, Y142X and C679X, found in approximately 2% of black American subjects, are associated with a 28% reduction in mean low density lipoprotein (LDL)-cholesterol. We sought to determine the frequency and effect of these nonsense variants in an African population. METHODS AND RESULTS: PCSK9 genotypes were determined in 653 black African women attending two antenatal clinics in Zimbabwe. C679X occurred in 3.7% of subjects and was associated with a 27% reduction in LDL-cholesterol (1.6+/-0.3 mmol/L versus 2.2+/-0.7 mmol/L in non-carriers). We did not observe the Y142X variant. CONCLUSIONS: Our results show that the PCSK9 C679X variant has a marked cholesterol-lowering effect.

Adult↗