[Activation of proprotein and kexin-family processing protease].
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Amino Acid Sequence↗
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Endoproteases other than furin have a role in hepatic proprotein processing.
The enzyme or enzymes responsible for dibasic-directed proprotein processing in the liver have not yet been unequivocally identified, although there are a number of potential candidates. We have compared a Kex2-like proalbumin convertase activity present in rat liver ER/Golgi membranes with recombinant furin, a candidate hepatic convertase. Using a series of mutant recombinant proalbumins as substrates the biochemically identified convertase and furin had very similar specificities with both preferring a substrate with an ArgXaaArgArg processing motif. Kinetic studies with normal and -4R proalbumin suggested however that the proalbumin convertase was not identical to furin. This was confirmed in immunoabsorption studies which demonstrated that furin only accounts for approximately half of the convertase activity. Therefore at least two proprotein convertases with overlapping specificities are involved in hepatic proprotein processing.
Amino Acid Sequence↗
Endoplasmic reticulum Ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell.
The role of intracellular Ca2+ in the proteolytic processing and intracellular transport of secretory granule proproteins was investigated by pulse-chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ with EGTA and by blocking the transport of Ca2+ into cells with the Ca2+-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conversion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, indicating that the process requires transport of Ca2+ into the endoplasmic reticulum. This was supported by the finding that proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to post-endoplasmic-reticulum compartments. Similarly, the inhibition of proinsulin processing was reversed by re-introduction of medium Ca2+ around the time of radiolabelling, but not after 15 min of chase incubation. Ca2+ depletion also decreased proteolytic maturation of the prohormone convertases PC1, PC2 and carboxypeptidase H. Secretion experiments suggested that the rate and extent of proinsulin transport into secretory granules were inhibited marginally by Ca2+ depletion, whereas those of the convertases were markedly impeded. Inhibition of proinsulin conversion by Ca2+ depletion was thus not simply related to the Ca2+-dependencies of mature PC1 and PC2, but also to a requirement for endoplasmic reticulum Ca2+ in proteolytic maturation of the convertases and in their transfer to secretory granules. The results also suggest that the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.
Animals↗
Altered proglucagon processing in an alpha-cell line derived from prohormone convertase 2 null mouse islets.
The endoproteolytic processing of proproteins in the secretory pathway depends on the expression of selected members of a family of subtilisin-like endoproteases known as the prohormone convertases (PCs). The main PC family members expressed in mammalian neuroendocrine cells are PC2 and PC1/3. The differential processing of proglucagon in pancreatic alpha-cells and intestinal L cells leads to production of distinct hormonal products with opposing physiological effects from the same precursor. Here we describe the establishment and characterization of a novel alpha-cell line (alphaTC-DeltaPC2) derived from PC2 homozygous null animals. The alphaTC-DeltaPC2 cells are shown to be similar to the well characterized alphaTC1-6 cell line in both morphology and overall gene expression. However, the absence of PC2 activity in alphaTC-DeltaPC2 leads to a complete block in the production of mature glucagon. Surprisingly, alphaTC-DeltaPC2 cells are able to efficiently cleave the interdomain site in proglucagon (KR 70-71). Further analysis reveals that alphaTC-DeltaPC2 cells, unlike alphaTC1-6 cells, express low levels of PC1/3 that lead to the generation of glicentin as well as low amounts of oxyntomodulin, GLP-1, truncated GLP-1, and N-terminally extended GLP-2. We conclude that alphaTC-DeltaPC2 cells provide additional evidence for PC2 as the major convertase in alpha-cells leading to mature glucagon production and provide a robust model for further analysis of the mechanisms of proprotein processing by the prohormone convertases.
Animals↗
The developmental expression in the rat CNS and peripheral tissues of proteases PC5 and PACE4 mRNAs: comparison with other proprotein processing enzymes.
Many peptides modulating cellular growth and differentiation in development are first synthesized as precursors that require proteolytic processing by the "prohormone convertase" (PC) family of endoproteases. Using in situ hybridization, we have here determined that two recently identified PC members, PC5 and PACE4, are expressed prenatally in spatial and temporal patterns that are each unique and distinct from those of previously characterized PCs. PC5 mRNA is first detected at e9 in highly restricted regions of the neural tube, in caudal myotomes, and at the materno-embryonic junction of the uterus. At e10, restricted PC5 mRNA expression is detected in the optic and otic vesicles, the roof of midbrain, and trunk myotomes. By midgestation (e13-e16), PC5 mRNA expression in the developing nervous system has expanded to multiple regions including hippocampus, thalamus, hypothalamus, brain stem, and spinal cord. By midgestational stages, PACE4 mRNA is expressed in multiple regions of the developing nervous system, generally distinct from PC5, and including a uniquely high level of expression in the ventricular zone of the hippocampus. In several peripheral organ systems, including lung and gut, we observed remarkably complementary patterns of PC5 and PACE4 expression. In addition, PACE4 transcripts are expressed in the heart and liver, whereas PC5 is expressed in the adrenal and kidney primordia. These results suggest that both PC5 and PACE4 may be involved in neuropeptide precursor processing in the developing nervous system and peripheral tissues with the general nonoverlapping expression patterns suggesting that PC5 and PACE4 may process distinct sets of proprotein substrates.
Animals↗
Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.
A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Enhanced TGFbeta1 maturation in high five cells coinfected with recombinant baculovirus encoding the convertase furin/pace: improved technology for the production of recombinant proproteins in insect cells.
One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFbeta1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFbeta1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFbeta1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFbeta1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFbeta1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFbeta1 by coexpressing prohTGFbeta1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFbeta1 increased in a dose-dependent fashion reaching up to 7. 8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFbeta1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.
Animals↗
Differential localization of prohormone convertases PC1 and PC2 in two distinct types of secretory granules in rat pituitary gonadotrophs.
Prohormone convertases PC1 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. There is a report that prohormone convertase exists in the rat anterior pituitary gonadotrophs, where it had previously been considered that proprotein processing does not take place. In addition to luteinizing hormone and follicle-stimulating hormone, rat pituitary gonadotrophs contain chromogranin A (CgA) and secretogranin II (SgII), two members of the family of granin proteins, which have proteolytic sites in their molecules. In the present study we examined whether there is a close correlation between subcellular localization of prohormone convertases and granin proteins. Ultrathin sections of rat anterior pituitary were immunolabeled with anti-PC1 or -PC2 antisera and then stained with immunogold. Immunogold particles for PC1 were exclusively found in large, lucent secretory granules, whereas those for PC2 were seen in both large, lucent and small, dense granules. The double-immunolabeling also demonstrated colocalization of PC2 and SgII in small, dense granules and of PC1, PC2, and CgA in large, lucent granules. These immunocytochemical results suggest that PC2 may be involved in the proteolytic processing of SgII and that both PC1 and PC2 may be necessary to process CgA.
Animals↗
Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids.
The prohormone convertases (PCs) are processing enzymes that activate proproteins via cleavage at specific single or pairs of basic residues. The hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) are primary sites of biosynthesis of several neuroendocrine hormone precursors, including provasopressin (pro-AVP), pro-oxytocin (pro-OT), and procorticotrophin-releasing hormone (pro-CRH), which require post-translational processing to yield active products. Using in situ hybridization, we observed PC1 and PC5 mRNAs in PVN and SON magnocellular neurons, while PC2 mRNA was observed in both magnocellular and parvocellular PVN neurons as well as magnocellular SON neurons. Similar to furin, PC7 mRNA was expressed throughout the PVN and SON, whereas PACE4 mRNA levels were undetectable. Both immunohistochemical and Western blot studies were performed to demonstrate the presence of PC proteins and forms in the PVN and SON. Using double-labeling in situ hybridization, we examined the cellular colocalization of each PC mRNA with pro-AVP, pro-OT, and pro-CRH mRNAs in PVN and SON. PC1 mRNA was colocalized with both AVP and OT mRNA in PVN and SON magnocellular neurons. All AVP, OT, and CRH neurons expressed PC2. In contrast, PC5 mRNA was colocalized only with OT mRNA. We examined the effects of adrenalectomy (ADX) on PVN PC mRNA levels. PC1 mRNA levels were increased selectively within CRH/AVP parvocellular neurons but were unchanged in PVN magnocellular AVP or OT neurons. These results established the anatomical organization of each convertase and proneuropeptide substrates in the PVN and SON and suggested potential roles for each enzyme under resting and stimulated conditions.
Adrenalectomy↗
Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases.
Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH3 cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.
Animals↗
Nucleotide sequence and analysis of the mouse SPC3 promoter region.
Insulin is converted from the higher molecular weight proprotein, proinsulin by highly specific proteolytic cleavage at two dibasic amino acid sites. SPC3 and SPC2, two recently identified prohormone convertase that are specifically expressed in beta cells and other neuroendocrine cells, appear to be responsible for those cleavages. We have sequenced the 5'-upstream region of the SPC3 gene and examined its promotor/enhancer activity and most of several deletion mutants in several cell lines. This region contains no CAAT box but has several non-functional TATA-like sequences and several putative transcriptional regulatory elements, including AP-1, Sp1 and cAMP response elements. These features are not unlike those of the human SPC2 upstream region. In beta TC3 insulinoma cells, the sequence between the EcoRI (620 bp) and NsiI (702 bp) sites seems to be important for gene expression, while the sequence between the NsiI and DraI (775 bp) sites may contain strong enhancer element(s).
Amino Acid Sequence↗
Maturation of HIV envelope glycoprotein precursors by cellular endoproteases.
The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.
Amino Acid Sequence↗
Developmental expression of proprotein-processing endoprotease furin in rat pancreatic islets.
The expression of furin in pancreatic beta-cells induces faster cell growth and a decrease in differentiated beta-cell-specific characteristics. During the development of rat pancreatic islets, the prohormone convertases, PC2 and PC3, appear during late gestation and are expressed long after birth. We investigated the developmental change in another yeast Kex2 family endoprotease, furin, in rat islets in relation to islet cell growth. Furin had appeared by gestational day 18 and was distributed in islets as well as exocrine tissues. The expression of furin in islets increased toward the neonatal stage. Bromodeoxyuridine (BrdU) incorporation was also elevated in the perinatal period. On postnatal days 10 and 20, staining characteristics were attenuated. On day 25, immediately after weaning, furin staining began to localize in the beta-cell region, and staining in the alpha-cell region became fainter. On day 270, the staining in the alpha-cell region disappeared, and staining in the beta-cell region remained positive, but was weaker. We conclude that furin expression was greatest during the perinatal period, when BrdU incorporation into islets was maximal. Furin expression continued, however, even after the BrdU incorporation decreased. Thus, furin appears to control the proliferation as well as differentiation of islet cells.
Aging↗
Differential effects of GnRH and androgens on Cres mRNA and protein in male mouse anterior pituitary gonadotropes.
The Cres gene defines a new subgroup in the family 2 cystatins of cysteine protease inhibitors. However, unlike typical cystatins, CRES does not inhibit cysteine proteases but rather inhibits the serine protease prohormone convertase 2, an enzyme with roles in proprotein processing in the neuroendocrine system. Cres is expressed in the gonadotropes and colocalizes with LHbeta, suggesting a role in the regulation of gonadotrope secretion. Our present studies were carried out to examine the regulation of Cres mRNA and protein expression by GnRH and steroid hormones, thus providing clues regarding its role in gonadotropes. Castration profoundly reduced Cres mRNA, while replacement with estradiol (E(2)), testosterone (T), or dihydrotestosterone (DHT) further decreased Cres, suggesting negative regulation by GnRH or steroid hormones. The administration of Antide, a GnRH antagonist, resulted in a 3-fold increase in Cres mRNA, supporting a negative regulation by GnRH. Because all hormonal manipulations in vivo resulted in alterations in steroid hormones, organ culture was used to assess the effects of GnRH independent of steroids. Mouse pituitaries cultured in the absence of GnRH or steroids showed high Cres mRNA levels, while DHT or E(2) resulted in decreases of 25% and 68%, respectively. However, an 85% decrease in Cres mRNA occurred following the administration of GnRH, demonstrating that GnRH, and to a lesser degree E(2), negatively regulate Cres mRNA in gonadotropes. Examination of CRES protein by immunohistochemistry showed that levels were profoundly reduced following castration, while DHT and in part T, but not E(2), restored CRES levels. Castrated mice treated with Antide showed little effect. However, castrated mice treated with Antide + DHT showed a dramatic recovery of CRES, suggesting that androgens act directly at the level of the gonadotrope to regulate CRES protein. Together, our studies suggest that Cres mRNA and protein are low at peak gonadotrope secretory activity, possibly as a means to allow proprotein processing events to occur that are integral to gonadotrope function.
Androgens↗
Proprotein-processing endoprotease furin controls growth of pancreatic beta-cells.
We have previously reported that in the well-differentiated beta-cell line MIN6 cells, the beta-cell-specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, betaTC3, betaHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINm5F and furin cDNA-introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors alpha1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific alpha1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.
Animals↗
Human NRD convertase: a highly conserved metalloendopeptidase expressed at specific sites during development and in adult tissues.
We report the cloning of the human homologue of the rat metalloprotease N-arginine dibasic convertase (NRD convertase). This endopeptidase is responsible for the processing, at the Arg-Lys dibasic site on the N-terminal side of the arginine residue, of propeptides and proproteins. Comparisons of the human and rat full-length cDNAs show similarity and identity of 94 and 91%, respectively. In humans NRD convertase is predominantly expressed in heart, skeletal muscle, and testis. We have also studied the expression of this gene in mouse at various developmental stages and found that the neural tissue is the almost exclusive site of expression in early development (between E 10.5 and E 16.5). To gain information about the possibility that defects in this gene are linked to inherited neuromuscular disorders, we determined the chromosomal location of the human NRD convertase gene by FISH analysis, showing that the gene resides at 1p32.2.
Adult↗
Identification of proteases with shared functions to the proprotein processing protease Krp1 in the fission yeast Schizosaccharomyces pombe.
Many secretory proteins are synthesized as inactive proproteins that undergo proteolytic activation as they travel through the eukaryotic secretory pathway. The best characterized family of processing enzymes are the prohormone convertases or kexins, and these are responsible for the processing of a wide variety of prohormones and other precursors. Recent work has identified other proteases that appear to be involved in proprotein processing, but characterization of these enzymes is at an early stage. Krp1 is the only kexin identified in the fission yeast Schizosaccharomyces pombe, in which it is essential for cell viability. We have used a genetic screen to identify four proteases with specificities that overlap Krp1. Two are serine proteases, one is a zinc metalloprotease (glycoprotease) and one is an aspartyl protease that belongs to the recently described yapsin family of processing enzymes. All four proteases support the growth of a yeast strain lacking Krp1, and each is able to process the P-factor precursor, the only substrate currently known to be processed by Krp1.
Amino Acid Sequence↗
Proprotein-processing endoprotease furin decreases regulated secretory pathway-specific proteins in the pancreatic beta cell line MIN6.
Prohormone convertases PC2 and PC3, yeast Kex2-family endoproteases specific to the regulated secretory pathway, cleave proinsulin to insulin in the secretory granules of pancreatic beta cells. The well-differentiated beta cell line MIN6 expresses PC2 and PC3 and another regulated secretory pathway-specific protein chromogranin A. Furin, another yeast Kex2 endoprotease, exists in the trans-Golgi networks of many cell types. The beta cell line RINm5F (a cell line that is less differentiated than the MIN6 cell line) does not express the regulated pathway-specific proteins, but strongly expresses furin. We suspected that furin expression may cause the decrement of regulated secretory pathway-specific proteins. To test this hypothesis, we expressed a furin cDNA with a metallothionein promoter in MIN6 cells. With Zn2+ stimulation of furin expression, the messages of PC2, PC3, and chromogranin A decreased, and the processing of proinsulin to mature insulin became less efficient. The furin-expressing MIN6 cells exhibited less insulin content and weakened insulin secretion in response to a high glucose concentration. The conditioned medium from furin-expressing MIN6 cells also exerted a decrease of PC2 and PC3 expression in unaltered MIN6 cells. Thus, proteins cleaved by furin inside the cells or by truncated furin shed into the culture medium appear to cause decreased PC2 and PC3 expression, insulin content, and glucose-responsive insulin secretion in MIN6 cells.
Amino Acid Sequence↗