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PubMed2003

Changes in the levels of neuropeptides and their metabolizing enzymes in the brain regions of nucleus basalis magnocellularis-lesioned rats.

The regulation mechanism of the interrelation between neuropeptides and their metabolizing enzymes in in vivo tissues is still not clear. In the present report, we attempted to measure the levels of neuropeptides and their enzymes in the frontal cortex, hippocampus, and striatum of the rat that had been bilaterally lesioned by the infusion of ibotenic acid or amyloid beta-peptide 25 - 35 (Abeta25 - 35) into the nucleus basalis magnocellularis. In the drug-treated rats, at two weeks after the infusion, the decrease of somatostatin-like immunoreactivity (SS-LI) and the increase of cholecystokinin-8S-LI were found in some brain regions relative to vehicle-treated rats. The immunoreactivities of endopeptidase 24.15 and puromycin-sensitive aminopeptidase and the leucine aminopeptidase- and aminopeptidase B-like enzyme activities did not change in the three brain regions, suggesting that the levels of those peptide-degrading enzymes do not correlate with the changes of the neuropeptide levels. The decrease of subtilisin-like proprotein convertase (SPC)-like enzyme activity was found in the hippocampus of the Abeta25 - 35-treated rats. The SS mRNA level decreased in the hippocampus in parallel with decreases in the SS-LI level and SPC-like enzyme activity. The present data indicate that some of the neuropeptide-processing enzymes may contribute to the control of neuropeptide levels.

Aminopeptidases↗
PubMed1998

Identification and characterization of spodoptera frugiperda furin: a thermostable subtilisin-like endopeptidase.

Spodoptera frugiperda (Sf9) cells are widely employed for high-level expression of heterologous recombinant genes from baculovirus vectors. Using a plasmid library encoding cDNA of Sf9 cells we have identified here the Spodoptera frugiperda analog of the proprotein convertase furin which plays an important role in posttranslational protein processing. Spodoptera frugiperda furin (Sfurin) is closest related to Drosophila melanogasterfurin with which it shares an extended cysteine-rich domain, whereas mammalian furin shows high homology only in the catalytic domain. Mammalian furin and Sfurin were further compared by expression from baculovirus vectors. Substrate specificity and inhibitor profiles are identical for Sfurin and mammalian furin, whereas calcium-dependence, pH-optimum, and thermostability differ. Cleavage of recombinant influenza virus hemagglutinin was significantly enhanced in Sf9 cells after overexpression of Sfurin.

Amino Acid Sequence↗
PubMed1997

Genetics of NIDDM in France: studies with 19 candidate genes in affected sib pairs.

As part of an ongoing search for susceptibility loci for NIDDM, we tested 19 genes whose products are implicated in insulin secretion or action for linkage with NIDDM. Loci included the G-protein-coupled inwardly rectifying potassium channels expressed in beta-cells (KCNJ3 and KCNJ7), glucagon (GCG), glucokinase regulatory protein (GCKR), glucagon-like peptide I receptor (GLP1R), LIM/homeodomain islet-1 (ISL1), caudal-type homeodomain 3 (CDX3), proprotein convertase 2 (PCSK2), cholecystokinin B receptor (CCKBR), hexokinase 1 (HK1), hexokinase 2 (HK2), mitochondrial FAD-glycerophosphate dehydrogenase (GPD2), liver and muscle forms of pyruvate kinase (PKL, PKM), fatty acid-binding protein 2 (FABP2), hepatic phosphofructokinase (PFKL), protein serine/threonine phosphatase 1 beta (PPP1CB), and low-density lipoprotein receptor (LDLR). Additionally, we tested the histidine-rich calcium locus (HRC) on chromosome 19q. All regions were tested for linkage with microsatellite markers in 751 individuals from 172 families with at least two patients with overt NIDDM (according to World Health Organization criteria) in the sibship, using nonparametric methods. These 172 families comprise 352 possible affected sib pairs with overt NIDDM or 621 possible affected sib pairs defined as having a fasting plasma glucose value of >6.1 mmol/l or a glucose value of >7.8 mmol/l 2 h after oral glucose load. No evidence for linkage was found with any of the 19 candidate genes and NIDDM in our population by nonparametric methods, suggesting that those genes are not major contributors to the pathogenesis of NIDDM. However, some evidence for suggestive linkage was found between a more severe form of NIDDM, defined as overt NIDDM diagnosed before 45 years of age, and the CCKBR locus (11p15.4; P = 0.004). Analyses of six additional markers spanning 27 cM on chromosome 11p confirmed the suggestive linkage in this region. Whether an NIDDM susceptibility gene lies on chromosome 11p in our population must be determined by further analyses.

Age Factors↗
PubMed1999

Functional genomics in Caenorhabditis elegans: An approach involving comparisons of sequences from related nematodes.

Comparative genomic analysis was used to investigate the gene structure of the bli-4 locus from two related Caenorhabditis species, C. elegans and C. briggsae. In C. elegans, bli-4 is a complex gene encoding a member of the kex2/subtilisin-like family of proprotein convertases. Genomic sequence comparisons coupled with RT-PCR analysis identified five additional coding exons that had not been identified previously using standard recombinant DNA techniques. The C. briggsae gene was able to rescue both viable blistered and developmentally arrested mutants of C. elegans bli-4, demonstrating functional conservation. In addition, deletion analysis of conserved sequences outside of coding regions, combined with phenotypic rescue experiments, identified regulatory elements that alter the expression of the bli-4 gene. These results demonstrate the utility of genomic sequence comparisons of homologous genes in related species as an effective tool with which to dissect the functional information of complex genes.

Alternative Splicing↗
PubMed2000

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28.

The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 ('a disintegrin and metalloprotease 28'), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal.

ADAM Proteins↗
PubMed2000

The cytosolic C-terminus of the glucose transporter GLUT4 contains an acidic cluster endosomal targeting motif distal to the dileucine signal.

The insulin-responsive glucose transporter GLUT4 is targeted to a post-endocytic compartment in adipocytes, from where it moves to the cell surface in response to insulin. Previous studies have identified two cytosolic targeting motifs that regulate the intracellular sequestration of this protein: FQQI(5-8) in the N-terminus and LL(489,490) (one-letter amino acid notation) in the C-terminus. In the present study we show that a GLUT4 chimaera in which the C-terminal 12 amino acids in GLUT4 have been replaced with the same region from human GLUT3 is constitutively targeted to the plasma membrane when expressed in 3T3-L1 adipocytes. To further dissect this domain it was divided into three regions, each of which was mutated en bloc to alanine residues. Analysis of these constructs revealed that the targeting information is contained within the residues TELEYLGP(498-505). Using the transferrin-horseradish peroxidase endosomal ablation technique in 3T3-L1 adipocytes, we show that mutants in which this C-terminal domain has been disrupted are more sensitive to chemical ablation than wild-type GLUT4. These data indicate that GLUT4 contains a targeting signal in its C-terminus, distal to the dileucine motif, that regulates its sorting into a post-endosomal compartment. Similar membrane-distal, acidic-cluster-based motifs are found in the cytosolic tails of the insulin-responsive aminopeptidase IRAP (insulin-regulated aminopeptidase) and the proprotein convertase PC6B, indicating that this type of motif may play an important role in the endosomal sequestration of a number of different proteins.

3T3 Cells↗
PubMed2001

Antisense inhibition of a tomato meiotic proteinase suggests functional redundancy of proteinases during microsporogenesis.

Anther development in angiosperms culminates in the programmed cell death of specific tissues to facilitate the release of pollen. Despite a wealth of morphological descriptions of this process, there have been few reports on the regulation of dehiscence or the coordination of events between tissues. We have cloned an anther-specific tomato gene encoding a serine proteinase that is expressed during meiosis and late microsporogenesis. The conceptualized tomato meiotic proteinase (TMP) is a member of a family of genes that exhibit characteristics of mammalian proprotein convertases. To examine the role of TMP in microsporogenesis, we generated transgenic plants harboring an antisense construct of the gene. Some of these plants produced little or no detectable TMP, yet no phenotypic abnormalities were observed. Zymogram analyses revealed that multiple proteinases are present in mature anthers and that proteinase activity increases as development proceeds. Taken together, these data indicate that the role of TMP during microsporogenesis, if any, may be compensated for by other proteinases.

Amino Acid Sequence↗
PubMed2002

Analysis of ten candidate genes in autism by association and linkage.

We studied the possible involvement of ten candidate genes in autism: proenkephalin, prodynorphin, and proprotein convertase subtilisin/kexin type 2 (opioid metabolism); tyrosine hydroxylase, dopamine receptors D2 and D5, monoamine oxidases A and B (monoaminergic system); brain-derived neurotrophic factor, and neural cell adhesion molecule (involved in neurodevelopment). Thirty-eight families with two affected siblings and one family with two affected half-siblings, recruited by the Paris Autism Research International Sibpair Study (PARIS), were tested using the transmission disequilibrium test and two-point affected sib-pair linkage analysis. We found no evidence for association or linkage with intragenic or linked markers. Our family sample has good power for detecting a linkage disequilibrium of 0.80. Thus, these genes are unlikely to play a major role in the families studied, but further studies in a much larger sample would be needed to highlight weaker genetic effects.

Alleles↗
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↗
PubMed2002

Furin expression in squamous cell carcinomas of the oral cavity and other sites evaluated by tissue microarray technology.

Furin is a proprotein convertase that activates many cancer development-related substrates such as growth factors, growth factor-receptors, adhesion molecules, and matrix degrading enzymes. Furin expression was studied in sections from tissue microarrays (TMA) and conventional paraffin blocks in a collection of squamous cell carcinomas (SCC) from three different sites. A total of 118 SCCs from the oral cavity, lung and esophagus as well as 34 precursor lesions (intraepithelial neoplasia) from the oral and bronchial mucosae were studied by immunohistochemistry. Furin expression was notably higher in most precursor lesions than in normal epithelia. Tumors from either the TMAs or the conventional blocks showed significant differences when compared to the mostly negative normal epithelia. High levels of furin expression were observed in approximately 50% SCCs of three different sites as well as in precursor lesions of the oral and bronchial mucosae. In addition another 30% showed low furin expression that was localized in all tumor cells including those in a basaloid position. Normal epithelia sometimes showed low level expression but the normal basal cells were always negative. These results show that furin is up-regulated in SCCs from three different organs and validates its use as a tumor marker in both invasive and pre-invasive neoplasia.

Biomarkers, Tumor↗
PubMed2004

Rational redesign of inhibitors of furin/kexin processing proteases by electrostatic mutations.

AIM: To model the three-dimensional structure and investigate the interaction mechanism of the proprotein convertase furin/kexin and their inhibitors (eglin c mutants). METHODS: The three-dimensional complex structures of furin/kexin with its inhibitors, eglin c mutants, were generated by modeller program using the newly published X-ray crystallographical structures of mouse furin and yeast kexin as templates. The electrostatic interaction energy of each complex was calculated and the results were compared with the experimentally determined inhibition constants to find the correlation between them. RESULTS: High quality models of furin/kexin-eglin c mutants were obtained and used for calculation of the electrostatic interaction energies between the proteases and their inhibitors. The calculated electrostatic energies of interaction showed a linear correlation to the experimental inhibition constants. CONCLUSION: The modeled structures give good explanations of the specificity of eglin c mutants to furin/kexin. The electrostatic interactions play important roles in inhibitory activity of eglin c mutants to furin/kexin. The results presented here provided quantitative structural and functional information concerning the role of the charge-charge interactions in the binding of furin/kexin and their inhibitors.

Animals↗
PubMed1999

Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides.

Proproteins and prohormones are the fundamental units from which bioactive proteins and peptides as well as neuropeptides are derived by limited proteolysis within the secretory pathway. Precursors are usually cleaved at the general motif (K/R)--(X)n--(K/R)down arrow, where n=0, 2, 4 or 6 and X is any amino acid and usually is not a Cys. Seven mammalian precursor convertases (PCs) have been identified: PC1, PC2, furin, PC4, PC5, PACE4 and PC7. Each of these enzymes, either alone or in combination with others, is responsible for the tissue-specific processing of multiple polypeptide precursors both in the brain and in periphery. This combinatorial mechanism generates a large diversity of bioactive molecules in an exquisitively regulated manner. The production of null mice allowed the assessment of the critical role of convertases in vivo. Thus, male PC4 (-/-) mice are infertile, furin (-/-) and PC1(-/-) mice are embryonic lethal, and PC2 (-/-) mice are mildly diabetic and runted. Interestingly, animals deficient in 7B2, a PC2-specific binding protein, exhibit a Cushing-like syndrome and die soon after birth. Recently, the first member of a new class of subtilisin--kexin-like convertases, called SKI-1, was identified. Its structure is closer to pyrolysin than to mammalian PCs and it exhibits a specificity for cleavage at the motif (R/K)--X--X--(L,T) down arrow as deduced from its ability to process sterol regulatory element binding proteins and pro-brain derived neurotrophic factor. Thus, while PCs are responsible for the processing of neuropeptides, adhesion molecules, receptors, growth factors, cell surface glycoprotein and enzymes, SKI-1 cleaves proproteins that are critical for the control of cholesterol and fatty acid metabolism and for neuronal protection and growth.

Amino Acid Sequence↗
PubMed2000

Translational regulation of proinsulin biosynthesis and proinsulin conversion in the pancreatic beta-cell.

Insulin secretion from the pancreatic beta -cell can be initiated in minutes, vary as much as 50-100-fold, and be sustained for several hours without need for changes in insulin gene transcription. Remarkably, the cellular content of the hormone and its molecular composition do not vary appreciably in the face of changes of insulin granule exocytosis. Minimal morphological changes are apparent, further indicating that the movement of lipids and membrane proteins between the granule storage pool, the plasma membrane, and Golgi are likewise tightly controlled. Such homeostasis is achieved by an interplay of signaling pathways originating from the metabolism of glucose with downstream targets at the level of translation of dense-core granule proteins, granule biogenesis, and membrane trafficking. Our scant knowledge in this area is confined mostly to a descriptive account of the fate of the major secreted components, principally insulin and the enzymes PC1, PC2, and CPH involved in the proteolytic conversion of proinsulin to insulin. A common theme seems to be the role of intracellular energy homeostasis in integrating the stimulus-secretion and stimulus-biosynthetic responses of this cell.

Amino Acid Sequence↗
PubMedDate not supplied

cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases.

Based on the concept of sequence conservation around the active sites of serine proteinases, polymerase chain reaction applied to mRNA amplification allowed us to obtain a 260-bp probe which was used to screen a mouse pituitary cDNA library. The primers used derived from the cDNA sequence of active sites Ser* and Asn* of human furin. Two cDNA sequences were obtained from a number of positive clones. These code for two similar but distinct structures (mPC1 and mPC2), each being homologous to yeast Kex2 and human furin. In situ hybridization (mPC1) and Northern blots (mPC1 = 3.0 kb and mPC2 = 2.8 and 4.8 kb) demonstrated tissue and cellular specificity of expression, only within endocrine and neuroendocrine cells. These data suggest that mPC1 and mPC2 represent prime candidates for tissue-specific pro-hormone converting proteinases.

Amino Acid Sequence↗
PubMed1995

Maintained PC1 and PC2 expression in the AtT-20 variant cell line 6T3 lacking regulated secretion and POMC: restored POMC expression and regulated secretion after cAMP treatment.

Two variant cell lines were recently established from parent AtT-20 cells. Whereas HYA.15.10.T.2 have a reduced level of secretory granules, HYA.15.6.T.3 are completely devoid of both the regulated pathway of secretion and of dense-core secretory granules. AtT-20 cells normally express the processing enzymes PC1, PC2, furin, carboxypeptidase E, and peptidylglycine alpha-amidating monooxygenase, as well as proopiomelanocortin, chromogranin B, and 7B2. We measured the expression of these mRNAs in both variant cell lines. Although some differences in mRNA level were noted, HYA.15.10.T.2 and HYA.15.6.T.3 cell lines maintained their expression of the processing enzymes and of 7B2. Furthermore, PC1 and PC2 were shown to be functionally active in the HYA.15.6.T.3 cells. In contrast, proopiomelanocortin and chromogranin B mRNA levels were no longer detectable in HYA.15.6.T.3 cells. Interestingly, stimulation of the HYA.15.6.T.3 cells with cAMP restored proopiomelanocortin mRNA, beta-endorphin immunoreactivity, and dense-core granules. Furthermore, at the ultrastructural level, beta-lipotropin immunoreactivity was detected in granules of cAMP-induced HYA.15.6.T.3 cells. Finally, depolarization of cAMP-induced HYA.15.6.T.3 cells with 56 mM potassium chloride resulted in a marked increase in the release of beta-endorphin immunoreactivity. These observations demonstrate that cAMP restores the regulated pathway of secretion in HYA.15.6.T.3 cells, which under untreated conditions do not demonstrate regulated release. These variant cell lines are unique models to understand better the relationship of the regulated pathway and the expression of the processing enzymes.

Animals↗
PubMed1991

Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA.

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).

Amino Acid Sequence↗
PubMed1999

The differential processing of proenkephalin A in mouse and human breast tumour cell lines.

We have carried out an investigation into the processing of the enkephalin-like immunoreactivity reported in breast tissue using two human breast tumour cell lines and a mouse tumour cell line. A 46 kDa form of proenkephalin (PE) has been observed in the cell lysates of two human breast tumour cell lines (MCF-7, ZR-75-1) and the mouse androgen-responsive Shionogi breast carcinoma cell line (SC115). PE processing in the cell lysates of these cells was assessed by a specific met-enkephalin RIA. The basal levels of processed PE in the MCF-7, ZR-75-1 and SC115 cell lysates were 30, 30 and 76% respectively. The processing enzymes PC1 and PC2, which have been implicated in the differential processing of PE, were detected by immunoblot analysis in these cells. PC1 was found within the cell extracts of all three cell lines. PC2 was only observed in the SC115 cell line, which may account for the higher percentage of processed PE measured. The cDNA of PC2 has been transfected into ZR-75-1 cells and this was accompanied by an increase in the level of processed PE from 30 to 76%. These breast tumour cell lines may provide a useful insight into the function of enkephalin-containing peptides in breast cancer.

Animals↗