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J W Creemers

Publications and source records attributed to J W Creemers.

At least 19 recordsLinked to original sources
PubMed2001

Lefty proteins exhibit unique processing and activate the MAPK pathway.

Lefty polypeptides, novel members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the formation of embryonic lateral patterning. Members of the TGF-beta superfamily require processing for their activation, suggesting cleavage to be an essential step for lefty activation. Transfection of different cell lines with lefty resulted in expression of a 42-kDa protein, which was proteolytically processed to release two polypeptides of 34 and 28 kDa. Since members of the proprotein convertase (PC) family cleave different TGF-beta factors and are involved in the establishment of embryonic laterality, we studied their role in lefty processing. Cotransfection analysis showed that PC5A processed the lefty precursor to the 34-kDa form in vivo, whereas furin, PACE4, PC5B, and PC7 had a limited activity. None of these PCs showed activity in the processing of the lefty polypeptide to the 28-kDa lefty form. The mutation of the consensus sequences for PC cleavage in the lefty protein allowed the lefty cleavage sites to be identified. Mutations of the sequence RGKR to GGKG (amino acids 74-77) and of RHGR to GHGR (amino acids 132-135) prevented the proteolytic processing of the lefty precursor to the 34- and 28-kDa forms, respectively. To identify the biologically active form of lefty, we studied the effect of lefty treatment on pluripotent P19 cells. Lefty did not induce Smad2 or Smad5 phosphorylation, Smad2/Smad4 heterodimerization, or nuclear translocation of Smad2 or Smad4, but activated the MAPK pathway in a time- and dose-dependent fashion. Further analysis showed the 28-kDa (but not the 34-kDa) polypeptide to induce MAPK activity. Surprisingly, the 42-kDa lefty protein was also capable of inducing MAPK activity, indicating that the lefty precursor is biologically active. The data support a molecular model of processing as a mechanism for regulation of lefty signaling.

3T3 Cells↗
PubMed2000

Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization.

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t(1/2) approximately 50 min) than that of the LPC polypeptide backbone (t(1/2) approximately 3 h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.

Amino Acid Substitution↗
PubMed2000

Binding of BiP to the processing enzyme lymphoma proprotein convertase prevents aggregation, but slows down maturation.

Lymphoma proprotein convertase (LPC) is a subtilisin-like serine protease of the mammalian proprotein convertase family. It is synthesized as an inactive precursor protein, and propeptide cleavage occurs via intramolecular cleavage in the endoplasmic reticulum. In contrast to other convertases like furin and proprotein convertase-1, propeptide cleavage occurs slowly. Also, both a glycosylated and an unglycosylated precursor are detected. Here we demonstrate that the unglycosylated precursor form of LPC is localized in the cytosol due to the absence of a signal peptide. Using a reducible cross-linker, we found that glycosylated pro-LPC is associated with the molecular chaperone BiP. In addition, we show that pro-LPC is prone to aggregation and forms large complexes linked via interchain disulfide bonds. BiP is associated mainly with non-aggregated pro-LPC and pro-LPC dimers and trimers, suggesting that BiP prevents aggregation. Overexpression of wild-type BiP or a dominant-negative BiP ATPase mutant resulted in reduced processing of pro-LPC. Taken together, these results suggest that binding of BiP to pro-LPC prevents aggregation, but results in slower maturation.

Adenosine Triphosphatases↗
PubMed2000

Processing of beta-secretase by furin and other members of the proprotein convertase family.

The amyloid peptide is the main constituent of the amyloid plaques in brain of Alzheimer's disease patients. This peptide is generated from the amyloid precursor protein by two consecutive cleavages. Cleavage at the N terminus is performed by the recently discovered beta-secretase (Bace). This aspartyl protease contains a propeptide that has to be removed to obtain mature Bace. Furin and other members of the furin family of prohormone convertases are involved in this process. Surprisingly, beta-secretase activity, neither at the classical Asp(1) position nor at the Glu(11) position of amyloid precursor protein, seems to be controlled by this maturation step. Furthermore, we show that Glu(11) cleavage is a function of the expression level of Bace, that it depends on the membrane anchorage of Bace, and that Asp(1) cleavage can be followed by Glu(11) cleavage. Our data suggest that pro-Bace could be active as a beta-secretase in the early biosynthetic compartments of the cell and could be involved in the generation of the intracellular pool of the amyloid peptide. We conclude that modulation of the conversion of pro-Bace to mature Bace is not a relevant drug target to treat Alzheimer's disease.

Amyloid beta-Protein Precursor↗
PubMed1998

Furin and proprotein convertase 7 (PC7)/lymphoma PC endogenously expressed in rat liver can be resolved into distinct post-Golgi compartments.

The intracellular compartmentalization in rat liver of the membrane-associated convertases furin and proprotein convertase 7 (PC7)/lymphoma PC (LPC) was investigated by analytical subcellular fractionation. In control animals, both enzymes were found to localize in fractions depleted of endoplasmic reticulum, cis-Golgi and lysosomal markers, but to co-distribute with the Golgi marker galactosyltransferase and the trans-Golgi network (TGN) marker TGN38. After overloading Golgi-derived vesicles with very-low-density lipoproteins (VLDL) by feeding rats with ethanol, the distribution of PC7/LPC was shifted markedly towards lower densities, in contrast with those of furin and the TGN marker. This provides support for the TGN localization of endogenously expressed furin and indicates that, at steady state, a considerable proportion of PC7/LPC may be associated with vesicles derived from the TGN.

Animals↗
PubMed1998

Molecular and cellular regulation of prohormone processing.

The processing of prohormones involves cleavage at specific basic amino acids by members of the subtilisin-like serine endoprotease family, followed by trimming of the COOH terminus by carboxypeptidase E. The enzymes are regulated by the intra-organelle ionic environment, through post-translational processing and by interaction with endogenous inhibitors. Much has been learned about their catalytic function and cell biology from in vitro gene transfer experiments using chimeric molecules and by site-directed mutagenesis. Further insight into their molecular properties and physiological function has been gained recently from the study of in vivo mutants.

Amino Acid Sequence↗
PubMed1997

Biosynthesis, distinct post-translational modifications, and functional characterization of lymphoma proprotein convertase.

Proprotein convertases are responsible for the endoproteolytic processing of prohormones, neuropeptide precursors, and other proproteins within the constitutive and regulated secretory pathways. Cleavage occurs carboxyl-terminally of basic amino acid motifs, such as RX(K/R)R, RXXR, and (R/K)R. As already available for the other known mammalian members of this enzyme family, we here define structural and functional features of human lymphoma proprotein convertase (LPC). Analysis of expression of recombinant LPC in stably transfected Chinese hamster ovary cells reveals biosynthesis of a 92-kDa nonglycosylated precursor (proLPC) and a 102-kDa endoglycosidase H-sensitive glycosylated form of proLPC. Only the latter is further processed and after propeptide removal converted into a complexly N-glycosylated mature form of LPC of about 92 kDa. Co-expression experiments of truncated LPC with an active site mutant of LPC (LPCS265A) indicate that prodomain removal of LPC occurs via an autoproteolytic, intramolecular mechanism, as was demonstrated before for some of the other members of this enzyme family. Prodomain removal is shown to be required for LPC to exit the endoplasmic reticulum. As far as subcellular localization is concerned, immunocytochemical, ultrastructural, and biochemical analyses show that LPC is concentrated in the trans-Golgi network, associated with membranes, and not secreted. Carboxyl-terminal domains are critically involved in this cellular retention, because removal of both the hydrophobic region and the cytoplasmic tail of LPC results in secretion. Of interest are the observations that LPC is not phosphorylated like furin but is palmitoylated in its cytoplasmic tail. Finally, substrate specificity of LPC is similar to that of furin but not identical. Whereas for furin a basic substrate residue at position P-2 is dispensable, it is essential for LPC. For optimal LPC substrate processing activity, an arginine at position P-6 is preferred over an arginine at P-4.

Animals↗
PubMed1997

Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene.

Human obesity has an inherited component, but in contrast to rodent obesity, precise genetic defects have yet to be defined. A mutation of carboxypeptidase E (CPE), an enzyme active in the processing and sorting of prohormones, causes obesity in the fat/fat mouse. We have previously described a women with extreme childhood obesity (Fig. 1), abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism and elevated plasma proinsulin and pro-opiomelanocortin (POMC) concentrations but a very low insulin level, suggestive of a defective prohormone processing by the endopeptidase, prohormone convertase 1 (PC1; ref. 4). We now report this proband to be a compound heterozygote for mutations in PC1. Gly-->Arg483 prevents processing of proPC1 and leads to its retention in the endoplasmic reticulum (ER). A-->C+4 of the intro-5 donor splice site causes skipping of exon 5 leading to loss of 26 residues, a frameshift and creation of a premature stop codon within the catalytic domain. PC1 acts proximally to CPE in the pathway of post-translational processing of prohormones and neuropeptides. In view of the similarity between the proband and the fat/fat mouse phenotype, we infer that molecular defects in prohormone conversion may represent a generic mechanism for obesity, common to humans and rodents.

Amino Acid Sequence↗
PubMed1996

Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2.

The mammalian subtilisin-like endoproteases furin and PC2 catalyze similar reactions but in different parts of the cell: furin in the trans-Golgi network and PC2 in dense-core granules. To map targeting domains within PC2, chimeras were constructed of the pro-, catalytic, and middle domains of furin with the carboxyl-terminal domain of PC2 (F-S-P) or of the pro- and catalytic domains of furin with the middle and carboxyl-terminal domains of PC2 (F-N-P). Their behavior in stable transfected AtT-20 cells was compared to a furin mutant truncated after the middle domain (F-S), wild-type furin, and with wild-type PC2. F-S-P, F-N-P, and F-S were catalytically active and underwent post-translational proteolysis and N-glycosylation with similar kinetics to wild-type furin. The truncated furin mutant was not stored intracellularly, whereas both chimeras, like PC2, showed intracellular retention and regulated release. Immunofluorescence and immuno-electron microscopy showed the presence of the chimeras and PC2 in dense-cored secretory granules together with proopiomelanocortin immunoreactivity. PC2 was sorted more efficiently than F-S-P, and the inclusion of the middle domain (F-N-P) further enhanced intracellular retention. It is concluded that sorting of PC2 into the regulated pathway depends on its carboxyl terminus. The middle domain may provide additional sorting determinants or a conformational framework for expression of the sorting signal.

Animals↗
PubMed1996

Human lactase-phlorizin hydrolase is not processed by furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the family of subtilisin-like proprotein processing proteases.

Human lactase-phlorizin hydrolase (LPH, EC 3.2.1.23/62) is synthesized as a single-chain precursor glycoprotein (pro-LPH) with a relative molecular mass of just over 200 kDa. Maturation to the mature enzyme (m-LPH, 160 kDa) occurs after passage of pro-LPH through the Golgi complex and involves the proteolytic removal of a 849 amino acid propeptide. The role of this propeptide as well as its removal is not fully understood and the proteolytic enzyme or enzymes involved are unknown. We studied the potential role of five different members of the family of subtilisin-like proprotein processing proteases in the maturation process of human LPH using a vaccinia virus based coexpression system in pig kidney PK(15) cells. Infected/transfected PK(15) cells expressed full-length pro-LPH but no maturation to m-LPH was observed. Coexpression of human pro-LPH with human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A in PK(15) cells did not result in maturation of the enzyme. Cleavage and secretion of von Willebrand factor precursor (pro-vWF) was used as a positive control. None of the five proprotein processing proteases tested were capable of cleaving human pro-LPH, strongly suggesting that they are not involved in the maturation of this enzyme.

Animals↗
PubMed1995

Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum.

The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.

Amino Acid Sequence↗
PubMed1995

Amyloid precursor protein is not processed by furin, PACE 4, PC1/3, PC2, PC4 and PC5/6 of the furin family of proprotein processing enzymes.

Proteolytic cleavage of the amyloid precursor protein (APP) has previously been shown to release its extracellular domain into the medium. The identification of the responsible proteinase(s), termed secretase(s), is a high priority in ongoing Alzheimer research. This is hampered by the unusual characteristics of these enzyme(s) and by the fact that they cleave only membrane associated APP. We report here, using a vaccinia virus based expression system, that pig kidney PK(15) cells express full-length, membrane bound APP695, but that secretion of APP is low. This heterologous expression system allows to assay candidate secretases in a cellular context by simple co-transfection of the APP and candidate secretase cDNA containing plasmids. Eight different members of the mouse and human furin family of proprotein processing enzymes were tested in this assay, but none of them enhanced the secretion of APP. Secretion of von Willebrand's factor was used as a positive control.

Amyloid↗
PubMed1995

The Dfur2 gene of Drosophila melanogaster: genetic organization, expression during embryogenesis, and pro-protein processing activity of its translational product Dfurin2.

The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.

Amino Acid Sequence↗
PubMed1994

Homology modelling of the catalytic domain of human furin. A model for the eukaryotic subtilisin-like proprotein convertases.

A model is presented for the three-dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin-like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca(2+)-binding sites and salt bridges. A detailed prediction of the substrate-binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S1, S2 and S4 subsites of the substrate-binding region of furin are identified which appear to be of particular importance, while residues of the S2', S3, S5 and S6 subsites may also contribute to substrate binding. Based on this model, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors.

Amino Acid Sequence↗
PubMed1994

Expression of the dibasic proprotein processing enzyme furin is directed by multiple promoters.

The prototype mammalian proprotein processing enzyme furin is shown to be encoded by three distinct FUR mRNA isoforms which differ only in their 5'-untranslated regions. By primer extension analysis, the transcription start sites of the three mRNA isoforms were defined. The genomic regions located immediately upstream of the three alternative transcriptional start sites were shown to possess promoter activity in transfection experiments using the luciferase encoding gene as reporter. In a liver cell line, the P1 promoter appeared to be the strongest; in a lung cell line, the P1A promoter. Human FUR promoter P1 but not P1A or P1B was transactivated by transcription factor C/EBP beta. Other members of this family of bZIP transcription factors, C/EBP alpha and C/EBP delta, were not able to transactivate the P1 promoter. Promoter P1A and P1B have characteristics of promoters of housekeeping genes. They lack TATA or CAAT boxes upstream of the transcription start site but are very GC-rich and contain several SP1 sites. Promoter P1, on the other hand, has a TATA box in the proximal promoter region. In electromobility shift assays and DNase I footprinting analysis, transcription factor SP1 was found to bind to the proximal region of the P1 promoter. Altogether, our results indicate that expression of the human FUR gene is directed by alternative promoters, housekeeping (GC-rich) as well as regulated (TATA-containing) promoters, suggesting that their differential use may be a mechanism to modulate levels of the furin enzyme.

Base Sequence↗
PubMed1994

Furin-mediated proprotein processing activity: involvement of negatively charged amino acid residues in the substrate binding region.

Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amino acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residues. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or in the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neutral residues. Analysis of processing by these furin mutants of wild-type and cleavage mutants of pro-von Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for specific cleavage activity.

Amino Acid Sequence↗
PubMed1993

Proprotein processing activity and cleavage site selectivity of the Kex2-like endoprotease PACE4.

Proprotein processing activity of the Kex2-like mammalian endoprotease PACE4 and its cleavage selectivity for sites with basic amino acid residues were determined. Using a recombinant vaccinia virus-based expression system, PACE4 was expressed in pig kidney PK(15) cells and, like two other Kex2-like endoproteases furin and PC6A, shown to correctly process the precursor of von Willebrand factor (pro-vWF). Furthermore, characteristics of the cleavage site selectivity of PACE4 were compared to those of furin and PC6A using the vWF cleavage site mutants vWFR-1G, vWFK-2A, and vWFR-4A as substrates. Cleavage site selectivity of PACE4 and PC6A appeared to be similar but they differed from that of furin.

Amino Acid Sequence↗