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Results for “Proprotein Convertase 1”

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PubMed1988

Calcium-dependent KEX2-like protease found in hepatic secretory vesicles converts proalbumin to albumin.

The yeast KEX2 protease is the only enzyme that has a proven role in the activation of polypeptide hormones through cleavage at pairs of basic residues. The enzyme that fulfils this role in higher eukaryotes has yet to be unequivocally identified. In this investigation, a KEX2-like calcium-dependent protease has been identified in rat hepatic microsomes. The enzyme is membrane-bound, has a pH optimum of 5-6 and converts proalbumin to albumin. More importantly, like the KEX2 protease, it meets two other exacting criteria defined by specific mutations in humans. Namely, it does not process proalbumin Christchurch (-1 Arg----Gln) which lacks one of the requisite basic residues and, whilst not itself a serine protease, it is inhibited by the reactive center variant, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) but not by normal alpha 1-antitrypsin.

Autoradiography↗

PubMed2002

Development of selectivity of alpha1-antitrypsin variant by mutagenesis in its reactive site loop against proprotein convertase. A crucial role of the P4 arginine in PACE4 inhibition.

PACE4, furin and PC6 are Ca2+-dependent serine endoproteases that belong to the subtilisin-like proprotein convertase (SPC) family. Recent reports have supported the involvement of these enzymes in processing of growth/differentiation factors, viral replication, activation of bacterial toxins and tumorigenesis, indicating that these enzymes are a fascinating target for therapeutic agents. In this work, we evaluated the sensitivity and selectivity of three rat alpha1-antitrypsin variants which contained RVPR352, AVRR352 and RVRR352, respectively, within their reactive site loop using both inhibition of enzyme activity toward a fluorogenic substrate in vitro and formation of a SDS-stable protease/inhibitor complex ex vivo. The RVPR variant showed relatively broad selectivity, whereas the AVRR and RVRR variants were more selective than the RVPR variant. The AVRR variant inhibited furin and PC6 but not PACE4. This selectivity was further confirmed by complex formation and inhibition of pro-complement C3 processing. On the other hand, although the RVRR variant inhibited both PACE4 and furin effectively, it needed a 600-fold higher concentration than the RVPR variant to inhibit PC6 in vitro. These inhibitors will be useful tools in helping us to understand the roles of PACE4, furin and PC6.

Animals↗

PubMed2000

Characterization of the kexin-like maturase of Aspergillus niger.

Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of Aspergillus niger (kexB). We constructed strains that either overexpressed KexB or lacked a functional kexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the kexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of A. niger.

Aspergillus niger↗

PubMed1998

BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development.

Bone morphogenetic protein-4 (BMP-4) is a multifunctional developmental regulator. BMP-4 is synthesized as an inactive precursor that is proteolytically activated by cleavage following the amino acid motif -Arg-Ser-Lys-Arg-. Very little is known about processing and secretion of BMPs. The proprotein convertases (PCs) are a family of seven structurally related serine endoproteases, at least one of which, furin, cleaves after the amino acid motif -Arg-X-Arg/Lys-Arg-. To examine potential roles of PCs during embryonic development we have misexpressed a potent protein inhibitor of furin, alpha1-antitrypsin Portland (alpha1-PDX) in early Xenopus embryos. Ectopic expression of alpha1-PDX phenocopies the effect of blocking endogenous BMP activity, leading to dorsalization of mesoderm and direct neural induction. alpha1-PDX-mediated neural induction can be reversed by co-expression of downstream components of the BMP-4 signaling pathway. Thus, alpha1-PDX can block BMP activity upstream of receptor binding, suggesting that it inhibits an endogenous BMP-4 convertase(s). Consistent with this hypothesis, alpha1-PDX prevents cleavage of BMP-4 in an oocyte translation assay. Using an in vitro digestion assay, we demonstrate that four members of the PC family have the ability to cleave BMP-4, but of these, only furin and PC6B are sensitive to alpha1-PDX. These studies provide the first in vivo evidence that furin and/or PC6 proteolytically activate BMP-4 during vertebrate embryogenesis.

Animals↗

PubMed2004

Genetic variants in PCSK9 affect the cholesterol level in Japanese.

Mutations in the proprotein convertase subtilisin/kexin 9 ( PCSK9) gene have been reported in affected members of two families with autosomal dominant hypercholesterolemia. To investigate the effects of common variants in PCSK9 on the cholesterol level, we conducted an association study using a large cohort representing the general population in Japan (n=1,793). Direct sequencing in all of the exonic regions identified 21 polymorphisms. After consideration of linkage disequilibrium among these polymorphisms, we selected and genotyped nine polymorphisms by the TaqMan method. The intron 1/C(-161)T and exon 9/I474 V polymorphisms were associated with levels of total cholesterol (TC) [C(-161)T, P=0.0285; I474 V, P=0.0069] and low-density lipoprotein cholesterol (LDL-C) [C(-161)T, P=0.0257; I474 V, P=0.0007]. The distributions of these polymorphisms in subjects with miocardial infarction (MI) (n=649) were not different from those in the control population. These results provide the first evidence that common variants intron 1/C(-161)T and exon 9/I474 V in PCSK9 significantly affect TC and LDL-C levels in the general population in Japan.

Base Sequence↗

PubMed2002

Protein engineering and properties of human metalloproteinase and thrombospondin 1.

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.

ADAM Proteins↗

PubMed1997

Proteolytic activation of the precursor of membrane type 1 matrix metalloproteinase by human plasmin. A possible cell surface activator.

Membrane type 1 matrix metalloproteinase (MT1-MMP) was suggested to play a critical role in the regulation of tissue invasion by normal and neoplastic cells by directly mediating the activation of pro-gelatinase A. Recently, the proteolytic activation of a pro-MT1-MMP by an intracellular proprotein convertase, furin, was reported. In this study, we found that plasmin efficiently activates the pro-MT1-MMP by cleaving immediately downstream of Arg108 and Arg111 in the multi-basic motif between its pro- and catalytic domains that participates in the activation of pro-gelatinase A. Our present data suggest that pro-MT1-MMP transported to the plasma membrane is activated by plasmin extracellularly and thus it may play an important role in the matrix degradation process.

Amino Acid Sequence↗

PubMed2004

A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family.

ADAMTS proteases are complex secreted enzymes containing a prometalloprotease domain of the reprolysin type attached to an ancillary domain with a highly conserved structure that includes at least one thrombospondin type 1 repeat. Known functions of ADAMTS proteases include processing of procollagens and von Willebrand factor as well as catabolism of aggrecan, versican and brevican. They have been demonstrated to have important roles in connective tissue organization, coagulation, inflammation, arthritis, angiogenesis and cell migration. ADAMTS can be grouped into distinct clades within which there is conservation of modular organization, protein sequence, gene structure and possibly, of substrate preference. ADAMTS proteases are synthesized as zymogens, with constitutive proprotein convertase removal of the propeptide occurring prior to secretion. Their enzymatic specificity is heavily influenced by their ancillary domain, which plays a critical role in directing these enzymes to their substrates, the cell surface and the extracellular matrix.

Amino Acid Motifs↗

PubMed1992

Proteolytic cleavages of proalbumin and complement Pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease.

We have recently purified and characterized a truncated soluble form of furin from which the predicted transmembrane domain and cytoplasmic tail were deleted (Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) J. Biol. Chem. 267, 16094-16099). Our results showed that furin resembles the yeast Kex2 protease with respect to both its enzymic properties and substrate specificity. Here we demonstrate that the soluble form of furin is capable of converting the precursors of albumin and the third component of complement (proalbumin and pro-C3, respectively) in vitro to mature proteins. Thus furin mimics the Ca(2+)-dependent proalbumin and pro-C3 convertases found in the Golgi membranes (Brennan, S. O., and Peach, R. J. (1988) FEBS Lett. 229, 167-170; Oda, K. (1992) J. Biol. Chem. 267, 17465-17471). Furthermore we show that the variant alpha 1-antitrypsin Pittsburgh, which is a specific inhibitor of the Golgi proalbumin convertase, inhibits not only the Golgi pro-C3 convertase, but also the soluble furin. These results suggest a role for furin in the cleavage of proproteins transported via the constitutive pathway.

Animals↗

PubMed2003

Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage.

The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H(592)VISKR(597) downward arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and downward arrow indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1-PDX (alpha1-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-alpha4 convertase. Mutational analysis showed that replacement of P1 Arg(597) with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH(4)Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg downward arrow, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.

Amino Acids↗

PubMed1998

Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.

We have examined the role of Furin in postimplantation-stage mouse embryos by analyzing both the expression pattern of fur mRNA and the developmental consequences of a loss-of-function mutation at the fur locus. At early stages (day 7.5), fur mRNA is abundant in extraembryonic endoderm and mesoderm, anterior visceral endoderm, and in precardiac mesoderm. 1 day later fur is expressed throughout the heart tube and in the lateral plate mesoderm, notochordal plate and definitive gut endoderm. Embryos lacking Furin die between days 10.5 and 11.5, presumably due to hemodynamic insufficiency associated with severe ventral closure defects and the failure of the heart tube to fuse and undergo looping morphogenesis. Morphogenesis of the yolk sac vasculature is also abnormal, although blood islands and endothelial precursors form. Analysis of cardiac and endodermal marker genes shows that while both myocardial precursors and definitive endoderm cells are specified, their numbers and migratory properties are compromised. Notably, mutant embryos fail to undergo axial rotation, even though Nodal and eHand, two molecular markers of left-right asymmetry, are appropriately expressed. Overall, the present data identify Furin as an important activator of signals responsible for ventral closure and embryonic turning.

Animals↗

PubMed2000

Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE).

Tumour necrosis factor alpha convertase (TACE) is a metalloprotease/disintegrin involved in the ectodomain shedding of several proteins, a process thought to be important in inflammation, rheumatoid arthritis and murine development. The characterization of the intracellular maturation and subcellular localization of endogenous TACE is decribed in the present study. Similarly to other proteolytically active metalloprotease/disintegrins, two forms of TACE are found in cells; a full-length precursor and a mature form lacking the prodomain. Prodomain removal occurs in a late Golgi compartment, consistent with the proposed role of a furin type proprotein convertase in this process. An additional form of TACE, lacking the pro and cytoplasmic domains, is detected when cell lysates are prepared in the presence of EDTA instead of a hydroxamate-based metalloprotease inhibitor or 1,10-phenanthroline. This form appears to be generated by mature TACE cleaving its own cytoplasmic tail and may explain why little mature TACE has been detected in previous studies. In cell-surface labelling experiments, mature TACE was detected on the cell surface but immunofluorescence data indicate that TACE is predominantly localized to a perinuclear compartment similar to that described for tumour necrosis factor (TNF)alpha. This raises the possibility that TACE-mediated ectodomain shedding may occur in an intracellular compartment in addition to the cell surface.

ADAM Proteins↗

PubMed2001

The Inhibitory Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using Site-directed Mutagenesis and Molecular Modeling.

Mammalian furin and yeast kexin are members of the proprotein convertase family involved in the proteolytic processing of many important precursor proteins. Here the gene coding for the subtilisin inhibitor eglin C was totally synthesized and expressed in E.coli. Substitution of residues at each position P(1), P(2) and P(4) of eglin C with a basic residue using protein engineering could make eglin C a very strong inhibitor for furin (K(i) around 10(-9) mol/L),and even more strong for kexin (K( i ) around 10(-11) mol/ L). Results indicated that (1) A basic residue Lys or Arg at P(1) site is prerequisite for the inhibitor. (2) The second mutation with basic residue at P(4) site drastically increase the inhibitory activity by two orders of magnitude. (3) A basic residue at P(2) site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition. (4) A hydrophobic residue is preferential at P(3) site. Based on the known crystal structures of subtilisin and eglin C, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results.

Journal Article↗

PubMed1999

Exposure of human islets to cytokines can result in disproportionately elevated proinsulin release.

Infiltration of immunocytes into pancreatic islets precedes loss of beta cells in type 1 diabetes. It is conceivable that local release of cytokines affects the function of beta cells before their apoptosis. This study examines whether the elevated proinsulin levels that have been described in prediabetes can result from exposure of beta cells to cytokines. Human beta-cell preparations were cultured for 48 or 72 hours with or without IL-1beta, TNF-alpha, or IFN-gamma, alone or in combination. None of these conditions were cytotoxic, nor did they reduce insulin biosynthetic activity. Single cytokines did not alter medium or cellular content in insulin or proinsulin. Cytokine combinations, in particular IL-1beta plus IFN-gamma, disproportionately elevated medium proinsulin levels. This effect expresses an altered functional state of the beta cells characterized by preserved proinsulin synthesis, a slower hormone conversion, and an increased ratio of cellular proinsulin over insulin content. The delay in proinsulin conversion can be attributed to lower expression of PC1 and PC2 convertases. It is concluded that disproportionately elevated proinsulin levels in pre-type 1 diabetic patients might result from exposure of their beta cells to cytokines released from infiltrating immunocytes. This hormonal alteration expresses an altered functional state of the beta cells that can occur independently of beta-cell death.

Adolescent↗

PubMed2000

ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.

ATF6 is a membrane-bound transcription factor that activates genes in the endoplasmic reticulum (ER) stress response. When unfolded proteins accumulate in the ER, ATF6 is cleaved to release its cytoplasmic domain, which enters the nucleus. Here, we show that ATF6 is processed by Site-1 protease (S1P) and Site-2 protease (S2P), the enzymes that process SREBPs in response to cholesterol deprivation. ATF6 processing was blocked completely in cells lacking S2P and partially in cells lacking S1P. ATF6 processing required the RxxL and asparagine/proline motifs, known requirements for S1P and S2P processing, respectively. Cells lacking S2P failed to induce GRP78, an ATF6 target, in response to ER stress. ATF6 processing did not require SCAP, which is essential for SREBP processing. We conclude that S1P and S2P are required for the ER stress response as well as for lipid synthesis.

Activating Transcription Factor 6↗

PubMed2004

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1.

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH2-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST[1-10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys13 followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST[1-10] production. Since the prosomatostatin sequence R8-Q9-F10-L11 \ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST[1-10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST[1-10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST[1-10] required the RXRXXL motif. However, this NH2-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.

Amino Acid Motifs↗

PubMed2000

Coordinate regulation of neuroendocrine convertase PC2 and peptide 7B2 in P19 neurons.

Convertases are proteases responsible for the bioactivation of many proteins and peptides having a potential role in ontogenesis. As a model to study regulation of convertases in embryo, we use the P19 embryonal carcinoma cell line, which can differentiate into various cell types. The expression of convertase PC2 and its specific binding peptide 7B2 are co-induced during neuronal differentiation of P19 cells. We investigated the possibility that expression of both proteins may be coregulated by T3 and dexamethasone, activators of nuclear receptors, isobutylmethylxanthine, and dibutyryl cAMP, activators of protein kinase A, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. Western blotting results show that expression of PC2 and 7B2 can be upregulated by modulators of the protein kinases, and upregulation needs not be strictly stoichiometric.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Distribution of proSAAS-derived peptides in rat neuroendocrine tissues.

Using a technique to identify substrates of the peptide processing enzyme carboxypeptidase E (CPE), several novel peptides were detected in the brain and pituitary of Cpe(fat)/Cpe(fat) mice and found to be derived from a single precursor, termed proSAAS. In order to gain further information regarding the potential physiological roles of these peptides, we have examined the distribution of two proSAAS-derived peptides, ARPVKEPRSLSAASAPLAETSTPLRL (SAAS) and LENSSPQAPARRLLPP (LEN), in rat neuroendocrine tissues using immunohistochemistry. Both peptides are detected throughout the brain, with the highest concentrations of SAAS peptide in the hypothalamus. In the hippocampus, both peptides are co-localized with prohormone convertase 1 in the dentate gyrus and CA1-3 region. In cerebellum, SAAS peptide is co-localized with prohormone convertase 1 in Purkinje and granular cells, whereas LEN is much more abundant in the Purkinje cells relative to the granular cells. Similarly, SAAS and prohormone convertase 1 are co-localized in the dorsal horn of the spinal cord, while LEN is mainly restricted to fibers of the white matter. In the pituitary, SAAS, LEN, and prohormone convertase 1 are detected in all three lobes. In the pancreas, SAAS, LEN, and prohormone convertase 1 are only detected in the islets, although the peptides are enriched in the peripheral cells (alpha and/or delta) while prohormone convertase 1 is only expressed in the inner cells (beta). Both SAAS and LEN are present in the adrenal medulla along with prohormone convertase 1. Taken together, these data are consistent with the proposed role for proSAAS as an endogenous inhibitor of prohormone convertase 1 in many, but not all cell types. However, the broader localization of the peptides allows for the possibility that they perform additional functions.

Adrenal Glands↗