Pairs Of Basic Amino Acids Research Articles

Processing of prothyrotropin-releasing hormone (Pro-TRH) by bovine intermediate lobe secretory vesicle membrane PC1 and PC2 enzymes.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Dissecting the factors involved in the pathogenesis of sodium retention in cirrhosis with ascites using TIPS: [formula omitted]. Depts of Medicine∗, Radiology† & Nuclear Cardiology‡, Toronto Hospital, Toronto, Canada

Endothelin (ET), a potent endothelium-derived vasoconstrictor peptide [1] contributes to the regulation of glomerular function both through changes in hemodynamics and through changes in glomerular configuration. Three structurally and pharmacologically distinct isoforms of the peptide have been identified with a molecular weight of 2.5 kDa, each consisting of 21 amino acids. These isopeptides share a common structure with a hydrophobic C-terminal end and two disulfide bonds, thus forming a hairpin loop at the amino terminus. ET-1 differs from ET-2 by only two amino acids, and from ET-3 by six amino acids. Each ET isopeptide is encoded by separate genes at distinct chromosomal loci [2].All ETs are synthesized via proteolytical cleavage from specific prohormones (Fig. 1). Prepro ETs are long polypeptide chains consisting of ∼203 amino acids [1] which exhibit specific differences among the isopeptides. ET isopeptide precursors are proteolytically cleaved by processing endopeptidases that recognize paired basic amino acids (Arg-Arg or Lys-Arg) and thus form pro or big ET. Pro ET-1 is then converted to mature ET-1 by cleavage of the Trp21-Val22 bond via a highly specific endothelin-1-converting enzyme, which is a Phosphoramidon-sensitive, membrane bound metalloprotease (Fig. 1) [3–5]. Pro ET-3 is less susceptible to this protease, so that the presence of other types of endothelin-converting enzyme are suspected [5].ETs bind to two G-protein coupled receptors of ∼43 to ∼73 kD molecular weight [6–8], consisting of ∼415 to ∼427 amino acids and having seven transmembrane domains [6, 7]. The ETA receptor binds ET-1 with 2- to 10-fold higher affinity than ET-2 and > 100-fold higher affinity than ET-3 [6–9], whereas the ETB receptor has similar affinities to all three peptides [7, 8].Little is known about the fate of ET. ETs are either inactivated by receptor internalization or by enzymatic cleavage of the peptide prior to receptor coupling [10–13].

The Yeast Proprotein Convertase Encoded by YAP3 Is a Glycophosphatidylinositol-anchored Protein That Localizes to the Plasma Membrane

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a approximately 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.

Neurochemical characterization of preprotachykinin B(50–79) immunoreactivity in the rat

Preprotachykinin B (PPT-B) contains two peptide sequences which are flanked by pairs of dibasic amino acids: the decapeptide neurokinin B and a 30 amino acid non-tachykinin peptide consisting of the amino acids 50–79 of PPT-B. Whereas the existence of neurokinin B is well established in brain and peripheral tissues, native PPT-B(50–79) has not been identified so far. We have previously studied the distribution of PPT-B(50–79)-immunoreactivity in the rat brain using antibodies directed against synthetic PPT-B(50–79). Now we adapted a radioimmunoassay for characterizing neuro-chemically PPT-B(50–79)-immunoreactivity in the rat. In the brain concentrations ranging from 2 to 180 fmol/mg wet tissue weight were measured using synthetic PPT-B(50–79) as standard. The highest concentrations were observed in the interpeduncular nucleus and in the hypothalamus (180 and 90 fmol/mg tissue, respectively). Intermediate concentrations (15 to 60 fmol/mg tissue) were present in cortical areas, in the hippocampus, the spinal cord and in the olfactory bulb. Modest levels were detected in the cerebellum. Considerably lower concentrations of PPT-B(50–79)-immunoreactivity were observed in peripheral tissues. They were highest in the adrenal medulla and in the urinary bladder (3.0 and 1.2 fmol/mg tissue, respectively). This distribution, as observed by radioimmunoassay, correlated to that previously revealed by immunocytochemistry. Tissue concentrations of total PPT-B(50–79) immunoreactivity, however, were slightly higher than those of neurokinin B. Gel filtration chromatography on Sephadex G50 and reversed phase HPLC revealed at least three PPT-B(50–79) immunoreactive peaks. About 90% of the PPT-B(50–79)-immunoreactivity was contained within 2 peaks of apparently higher molecular weight than PPT-B(50–79). A minor portion of PPT-B(50–79)-immunoreactivity comigrated with the synthetic peptide, suggesting that only minor amounts of PPT-B(50–79) are formed in vivo. The processing enzyme(s) cleaving protachykinin B at the pair of basic amino acids (Lys 80-Arg 81) located between PPT-B(50–79) and neurokinin B may not be acting at the Arg 48-Arg 49 site (followed by -Leu 50) at the amino terminal end of PPT-B(50–79).

Processing of proendothelin-1 by human furin convertase

Endothelin-1 (ET-1) is the most potent vasoactive peptide known to date. The peptide is initially synthesized as an inactive precursor (proET-1) which undergoes proteolysis at specific pairs of basic amino acids to yield bigET-1. Production of ET-1 then proceeds by cleavage of bigET-1 by the endothelin converting enzyme (ECE). Here, we demonstrate that the in vitro cleavage of proET-1 by furin, a mammalian convertase involved in precursor processing, produced bigET-1. Upon further processing, bigET-1 was converted to biologically active ET-1. Furthermore, we demonstrate that the furin inhibitor, decanoyl-Arg-ValLys-Arg chloromethylketone, abolished production of ET-1 in endothelial cells.

Processing of human factor I in COS-1 cells co-transfected with factor I and paired basic amino acid cleaving enzyme (PACE) cDNA

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed from of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.

Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin

Secretoneurin is a recently characterized neuropeptidepresent in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.

Processing Specificity and Biosynthesis of the Drosophila melanogaster Convertases dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2

Pro-protein and pro-hormone convertases are subtilisin/kexin-like enzymes implicated in the activation of numerous precursors by cleavage at sites mostly composed of pairs of basic amino acids. Six members of this family of enzymes have been identified in mammals and named furin (also called PACE), PC1 (also called PC3), PC2, PACE4, PC4, and PC5 (also called PC6). Multiple transcripts are produced for all the mammalian convertases, but only in the cases of PC4, PACE4, and PC5 does differential splicing result in the modification of the C-terminal sequence of these enzymes. A similar molecular diversity is also observed for the convertases of Hydra vulgaris, Caenorhabditis elegans, and Drosophila melanogaster. In the third species, two genes homologous to human furin called Dfur1 and Dfur2 have been identified. The Dfur1 gene undergoes differential splicing to generate three type I membrane-bound proteins called dfurin1, dfurin1-CRR, and dfurin1-X, which differ only in their C-terminal sequence. By using recombinant vaccinia viruses that express each of the dfurin proteins, we investigated the potential effect of the C-terminal domain on their catalytic specificities. For this purpose, these enzymes were coexpressed with the precursors pro-7B2, pro-opiomelanocortin, and pro-dynorphin in a number of cell lines, and the processed products obtained were characterized. Our studies demonstrate that these proteases display cleavage specificities similar to that of mammalian furin but not to that of PC2. In contrast, we noted significant differences in the biosynthetic fates of these convertases. All dfurins undergo rapid removal of their transmembrane domain within the endoplasmic reticulum, resulting in the release of several truncated soluble forms. However, in the media of cells containing secretory granules, such as GH4C1 and AtT-20, dfurin1-CRR and dfurin2 predominate over dfurin1, whereas dfurin1-X is never detected. While pro-segment removal occurs predominantly in the trans-Golgi network for all the dfurins, in the presence of brefeldin A, only dfurin1-CRR and dfurin2 can undergo partial zymogen cleavage. The conclusions drawn from the results of this study may well be applicable to the mammalian convertases PC4, PACE4, and PC5, which also display C-terminal sequence heterogeneity.

Role of cathepsin B as prorenin processing enzyme in human kidney.

Renin is synthesized from an inactive precursor, prorenin, through cleavage at a pair of basic amino acids catalyzed by prorenin processing enzyme (PPE). A lysosomal protease, cathepsin B, has been suggested to be a strong candidate for PPE. However, there still remains a possibility that other protease(s) can also catalyze prorenin processing. We studied the subcellular distribution of PPE in human renal cortex using pure recombinant prorenin as a PPE assay substrate. PPE and renin activities, and cathepsin B activity and protein were colocalized in the lysosomal fraction. The PPE activity was completely inhibited by a cathepsin B specific inhibitor, CA074. Taken together with the immunohistochemical data showing that cathepsin B and PPE are colocalized in dense secretory granules of juxtaglomerular cells of kidney, we conclude that cathepsin B is the authentic PPE in human kidney.

Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

Purification and characterization of a novel membrane-bound arginine-specific serine proteinase from porcine intestinal mucosa

A novel membrane-bound serine proteinase has been purified from the microsomal membranes of porcine intestinal mucosa. It was solubilized from the microsomal membrane fraction with 1% sodium deoxycholate, then purified by a series of column chromatographic steps on DE52, butyl-Toyopearl, Bio-Gel P-150, Mono Q, and benzamidine-Sepharose in the presence of 0.02% Lubrol PX. Its molecular mass was estimated to be 50 kDa both by SDS-polyacrylamide gel electrophoresis under non-reducing conditions and by gel filtration, and to be 32 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions, suggesting that the enzyme may exist as a homodimer in which two subunits are linked by disulfide bond(s). It had a pH optimum at around 9 and did not require Ca2+ for activity. It cleaved several peptide 4-methylcoumaryl-7-amide substrates almost exclusively after arginine residues, the best substrate among those tested being t-butyloxycarbonyl-Gln-Ala-Arg-4-methylcoumaryl-7-amide. Various neuropeptides were also cleaved by this enzyme after arginine, mainly between paired basic amino acid residues, Arg-Arg or Arg-Lys. Activity toward protein substrates was scarcely detected. Further, its partial amino acid sequences were highly homologous, but not identical, with those of trypsin-type serine proteinases. These results indicate that the present enzyme is a novel arginine-specific trypsin-like endopeptidase, possibly involved as a processing proteinase in the production of certain gastrointestinal neuropeptides or peptide hormones from their precursors, or their specific degradation.

Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed "pigeon PMV-1 viruses".

The amino acid sequence at the F2/F1 cleavage site was determined for 15 strains of the so-called pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) which showed close antigenic identity, determined by their reactions with a panel of 28 monoclonal antibodies, but considerable variation in their pathogenicity for chickens. Thirteen of the isolates possessed the motif 112G-R-Q-K-R-F117. This motif was seen for one virus which had initially low pathogenicity and remained unaltered when virulence of the virus for chickens was increased by bird to bird passage. The two other viruses had the sequence 112R-R-Q-K-R-F117 at the cleavage site which is more typical of virulent viruses, however, pathogenicity index tests indicated that these isolates were of moderate and low pathogenicity. The nucleotide sequence coding for the HN/HN0 extension region was determined for two of the PPMV-1 isolates. In both cases a stop codon was present indicating that the product for these viruses would be HN571. We conclude that the wide variation in pathogenicity of the variant PPMV-1 for chickens is not related to variation in the amino acid motif at the F2/F1 cleavage site nor due to production of HN0 which may also influence pathogenicity. The high virulence of some of the viruses examined confirms that a double pair of basic amino acids in the region of the F2/F1 cleavage site is not necessary for the full expression of virulence.

Proopiomelanocortin-Derived Peptides

Cloning of the cDNA of POMC mRNA led to the characterization of the ACTH precursor and established the molecular links between ACTH, beta LPH and gamma LPH, and beta end. Further previously unidentified POMC-derived peptides were also unravelled, including the N-terminal fragment and the JP. POMC is a precursor polypeptide that contains the amino acid sequences of numerous small polypeptide products. It contains eight pairs of basic amino acids and one sequence of four basic amino acids, which are the sites of cleavage for the recently identified prohormone convertases (PC1 and PC2), which have different specificities. PC1 but not PC2 is found in corticotroph cells of the anterior pituitary and cleaves POMC to generate NT, JP, ACTH, beta LPH and small amounts of gamma LPH and beta end. In the melanotroph cells of the intermediate pituitary, both PC1 and PC2 are present. The proteolysis of POMC is thus more extensive, resulting in a series of smaller peptides including gamma MSHs, CLIP, alpha MSH, beta MSH, beta end, and beta end. Equimolar amounts (because they are all products of the same precursor) of the POMC peptides are released into the blood by exocytosis from corticotroph cells of the anterior pituitary. No physiological role has been definitively identified for any of these products other than ACTH. POMC peptides have been assayed in human blood by specific radioimmunoassay (RIA). In most circumstances, the concentrations of all these peptides are perfectly correlated. Therefore, for practical reasons, LPH RIA is widely used as an indicator of overall corticotroph function. In rare cases, the correlation between the POMC peptides is disrupted by a general disorder selectively modifying the metabolism of the different members of the family as in chronic renal failure, or in tumors where POMC processing is abnormal. Examples include pituitary macroadenomas, some silent corticotroph adenomas, and most frequently non-pituitary tumors responsible for ectopic ACTH syndrome. These pathologies can lead to the formation and secretion into blood of CLIP and beta MSH, or alternatively POMC may be underprocessed and secreted intact. An elegant approach, specific IRMA, has been developed to directly recognize unprocessed POMC in blood.

Frog diazepam-binding inhibitor: peptide sequence, cDNA cloning, and expression in the brain.

Three peptides derived from diazepam-binding inhibitor (DBI) were isolated in pure form from the brain of the frog Rana ridibunda. The primary structures of these peptides showed that they correspond to mammalian DBI-(1-39), DBI-(58-87), and DBI-(70-87). A set of degenerate primers, whose design was based on the amino acid sequence data, was used to screen a frog brain cDNA library. The cloned cDNA encodes an 87-amino acid polypeptide, which exhibits 68% similarity with porcine and bovine DBI. Frog DBI contains two paired basic amino acids (Lys-Lys) at positions 14-15 and 62-63 and a single cysteine within the biologically active region of the molecule. Northern blot analysis showed that DBI mRNA is expressed at a high level in the brain but is virtually absent in peripheral tissues. The distribution of DBI mRNA and DBI-like immunoreactivity in the frog brain was studied by in situ hybridization and immunocytochemistry. Both approaches revealed that the DBI gene is expressed in ependymal cells and circumventricular organs lining the ventricular cavity. Since amphibia diverged from mammals at least 250 million years ago, the data show that evolutionary pressure has acted to conserve the structure of DBI in the vertebrate phylum. The distribution of both DBI mRNA and DBI-like immunoreactivity indicates that DBI is selectively expressed in glial cells.

Renal actions of endothelin: Linking cellular signaling pathways to kidney disease

Endothelin (ET), a potent endothelium-derived vasoconstrictor peptide [1] contributes to the regulation of glomerular function both through changes in hemodynamics and through changes in glomerular configuration. Three structurally and pharmacologically distinct isoforms of the peptide have been identified with a molecular weight of 2.5 kDa, each consisting of 21 amino acids. These isopeptides share a common structure with a hydrophobic C-terminal end and two disulfide bonds, thus forming a hairpin loop at the amino terminus. ET-1 differs from ET-2 by only two amino acids, and from ET-3 by six amino acids. Each ET isopeptide is encoded by separate genes at distinct chromosomal loci [2]. All ETs are synthesized via proteolytical cleavage from specific prohormones (Fig. 1). Prepro ETs are long polypeptide chains consisting of ∼203 amino acids [1] which exhibit specific differences among the isopeptides. ET isopeptide precursors are proteolytically cleaved by processing endopeptidases that recognize paired basic amino acids (Arg-Arg or Lys-Arg) and thus form pro or big ET. Pro ET-1 is then converted to mature ET-1 by cleavage of the Trp 21 -Val 22 bond via a highly specific endothelin-1-converting enzyme, which is a Phosphoramidon-sensitive, membrane bound metalloprotease (Fig. 1) [3–5]. Pro ET-3 is less susceptible to this protease, so that the presence of other types of endothelin-converting enzyme are suspected [5]. ETs bind to two G-protein coupled receptors of ∼43 to ∼73 kD molecular weight [6–8], consisting of ∼415 to ∼427 amino acids and having seven transmembrane domains [6, 7]. The ET A receptor binds ET-1 with 2- to 10-fold higher affinity than ET-2 and > 100-fold higher affinity than ET-3 [6–9], whereas the ET B receptor has similar affinities to all three peptides [7, 8]. Little is known about the fate of ET. ETs are either inactivated by receptor internalization or by enzymatic cleavage of the peptide prior to receptor coupling [10–13].

Cloning, nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110,016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5.5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.

Kex2-like endoproteases involved in processing of precursors for bioactive peptides and proteins.

Endoproteolytic processing of precursor proteins typically at specific pairs of basic amino acids is a key step in the production of bioactive peptides and proteins. The endrproteases involved in this process belong to a new family of Ca2+ -dependent serine proteases related to the yeast Kex2 protease. These include furin, PC2, PC3, PC4, PACE4 and PC6, all of which have been characterized structurally and functionally.

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 neural 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.

The Matrix Protein of Newcastle Disease Virus Localizes to the Nucleus via a Bipartite Nuclear Localization Signal

The Newcastle disease virus matrix (M) protein expressed from a cDNA clone is observed in the nucleus of transfected cells, displaying a localization pattern identical to that observed in virus-infected cells. To identify the nuclear localization signal (NLS) in the Mprotein, M gene mutants encoding deletion and amino acid substitution proteins were constructed and expressed transiently in COS-1 cells. Protein products were examined for intracellular localization using indirect immunofluorescence. Two basic amino acid clusters in the M protein were found to be required for nuclear localization since deletion of these basic clusters or substitution with random amino acids resulted in cytoplasmic localization. Substitution of pairs of basic amino acids with non-basic residues revealed that components from both basic regions are required for nuclear localization. This interdependence between two basic clusters suggests that the NLS in the M protein belongs to the newly described class of "bipartite" NLSs. Unlike most NLSs, Mprotein sequences containing the critical basic amino acid clusters fused to two different cytoplasmic reporter proteins failed to transport these proteins to the nucleus.

Parastatin (porcine chromogranin A347-419), a novel chromogranin A-derived peptide, inhibits parathyroid cell secretion.

Chromogranin A (CgA), previously referred to as secretory protein-I, is a 50-kilodalton protein present in secretory granules of many endocrine and neuroendocrine cells. In the parathyroid it is present and cosecreted with PTH in response to hypocalcemia. CgA appears to be a precursor of bioactive peptides including pancreastatin, beta-granin, vasostatin, and chromostatin. The presence of several highly conserved pairs of basic amino acids, putative cleavage sites, in the CgA molecule suggests that other yet unidentified bioactive peptides might exist within the molecule. We tested this speculation by subjecting porcine parathyroid CgA to digestion by endoproteinase Lys-C. Resulting CgA-derived peptides were isolated by reverse-phase C18 HPLC and tested for their ability to affect low-Ca2+ stimulated secretion by porcine parathyroid cells. We characterized one peptide, which we named parastatin, that inhibited secretion of both PTH and CgA in a dose-dependent fashion over the range of 0.2-0.6 microM. Parastatin migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mol wt of 11,000. Edman degradation yielded the sequence L-S-F-R-A-P-A-Y-G-F-R-G-P-G-L corresponding to residues 347-361 of porcine CgA. Amino acid analysis of endoproteinase Lys-C and endoproteinase Asp-N-generated fragments indicated that parastatin corresponds to residues 347-419 of CgA. A synthetic NH2-terminal fragment of rat parastatin corresponding to residues 1-19 was an inhibitory as intact porcine parastatin on parathyroid gland secretion. These results extend the concept that CgA is a precursor of biologically active peptides.