Carboxyl-terminal Peptide Research Articles

Purification, Characterization, and Localization of Yeast Cox17p, a Mitochondrial Copper Shuttle

Cox17p was previously shown to be essential for the expression of cytochrome oxidase in Saccharomyces cerevisiae. In the present study COX17 has been placed under the control of the GAL10 promoter in an autonomously replicating plasmid. A yeast transformant harboring the high copy construct was used to purify Cox17p to homogeneity. Purified Cox17p contains 0.2-0.3 mol of copper per mol of protein. The molar copper content is increased to 1.8 after incubation of Cox17p in the presence of a 6-fold molar excess of cuprous chloride under reduced conditions. An antibody against Cox17p was obtained by immunization of rabbits with a carboxyl-terminal peptide coupled to bovine serum albumin. The antiserum detects Cox17p in both the mitochondrial and soluble protein fractions of wild type yeast and of the transformant overexpressing Cox17p. Exposure of intact mitochondria to hypotonic conditions causes most of Cox17p to be released as a soluble protein indicating that the mitochondrial fraction of Cox17p is localized in the intermembrane space. These results are consistent with the previously proposed function of Cox17p, namely in providing cytoplasmic copper for mitochondrial utilization.

Proteolysis of the carboxyl-terminal GPI signal independent of GPI modification as a mechanism for selective protein secretion.

Variable amounts of soluble forms of a variety of glycosyl-phosphatidylinositol (GPI)-anchored proteins occur extracellularly, but the molecular mechanisms governing their release are not entirely clear. When the GPI-anchored folate receptor (FR) type beta was expressed transiently in human 293 fibroblasts, there was a roughly equal distribution of [3H]folic acid binding protein between the cell surface and the medium after 24 h over a wide range of expression levels of FR-beta. The difference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 293 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-existing levels of cell surface FR were unaltered suggesting the absence of a precursor-product relationship between the membrane-associated FR-beta and the soluble protein in the medium. [35S]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl-terminal peptides between FR-beta and FR-alpha revealed that the nature of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of soluble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of apparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constructs of FR-beta, in which the carboxyl-terminal signal for GPI modification was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-beta were identified as nonglycosylated (29.3 kDa) or glycosylated polypeptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [3H]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is efficiently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secretion. Such carboxyl-terminal sequences could represent a simple adaptation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tissue-specific release.

Identification of a Ganglioside Recognition Domain of Tetanus Toxin Using a Novel Ganglioside Photoaffinity Ligand

Tetanus toxin entry into vertebrate motorneurons may involve binding of neuronal surface gangliosides containing the "1b" substructure (a NeuAcalpha2,8NeuAc group on an internal galactose residue). The domains of tetanus toxin involved in ganglioside binding are known to reside within the carboxyl-terminal half of the toxin's heavy chain ("C fragment"). We developed a novel photoaffinity reagent based upon the structure of the 1b ganglioside GD1b (125I-azido-GD1b) to define the ganglioside-binding domains of tetanus toxin. Using this ligand, we observed radiolabeling of tetanus toxin C fragment which could be specifically inhibited by a ganglioside of the 1b series (GT1b), but not by a non-1b series ganglioside (GM3). When tetanus toxin C fragment was proteolyzed with clostripain, whether before or after reaction with 125I-azido-GD1b, a radiolabeled band was observed by SDS-polyacrylamide gel electrophoresis autoradiography, which was selectively inhibited by GT1b. Protein sequencing of proteolyzed tetanus toxin C fragment co-migrating with that band revealed the carboxyl-terminal 34 amino acid residues of tetanus toxin. Matrix-assisted laser desorption/ionization mass spectrometry of a photoaffinity labeled synthetic polypeptide representing the 34-amino acid domain revealed modification at a single residue (His1293). We propose that this domain of tetanus toxin is sufficient for ganglioside binding.

S-Acylation and plasma membrane targeting of the farnesylated carboxyl-terminal peptide of N-ras in mammalian fibroblasts.

We have used a series of fluorescent lipid-modified peptides, based on the farnesylated C-terminal sequence of mature N-ras [-GCMGLPC(farnesyl)-OCH3], to investigate the membrane-anchoring properties of this region of the protein and its reversible modification by S-acylation in cultured mammalian fibroblasts. The farnesylated peptide associates with lipid bilayers (large unilamellar phospholipid vesicles) with high affinity but in a rapidly reversible manner. Additional S-palmitoylation of the peptide suppresses its ability to desorb from, and hence to diffuse between, lipid bilayers on physiologically significant time scales. NBD-labeled derivatives of the farnesylated N-ras C-terminal heptapeptide, when incubated with CV-1 cells in culture, are taken up by the cells and reversibly S-acylated in a manner similar to that observed previously for the parent protein. The S-acylation process is highly specific for modification of a cysteine rather than a serine residue but tolerates replacement of the peptide-linked farnesyl moiety by other hydrophobic groups. Fluorescence microscopy reveals that in CV-1 cells the S-acylated form of the peptide is localized preferentially to the plasma membrane, as has been observed for N-ras itself. This plasma membrane localization is unaffected by either reduced temperature (15 degrees C) or exposure to brefeldin A, treatments which inhibit various trafficking steps within the secretory pathway. These results suggest that in mammalian cells the plasma membrane localization of mature N-ras is maintained by a 'kinetic trapping' mechanism based on S-acylation of the protein at the level of the plasma membrane itself.

A βγ Dimer Derived from G13 Transduces the Angiotensin AT1 Receptor Signal to Stimulation of Ca2+ Channels in Rat Portal Vein Myocytes

A G protein composed of alpha13, beta1, and gamma3 subunits selectively couples the angiotensin AT1A receptors to increase cytoplasmic Ca2+ concentration ([Ca2+]i) in rat portal vein myocytes (Macrez-Leprêtre, N., Kalkbrenner, F., Morel, J. L., Schultz, G., and Mironneau, J. (1997) J. Biol. Chem. 272, 10095-10102). We show here that Gbetagamma transduces the signal leading to stimulation of L-type Ca2+ channels. Intracellular dialysis through the patch pipette of a carboxyl-terminal anti-betacom antibody and a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase 1 inhibited the stimulation of Ca2+ channels and the increase in [Ca2+]i evoked by angiotensin II. The Gbetagamma binding peptide did not prevent the dissociation of the heterotrimeric G protein into its subunits, as it did not block activation of phospholipase C-beta by Galphaq in response to stimulation of alpha1-adrenoreceptors. Transient overexpression of the beta-adrenergic receptor kinase 1 fragment and of Galpha subunits also inhibited the angiotensin II-induced increase in [Ca2+]i. Both anti-alpha13 antibody and carboxyl-terminal alpha13 peptide abrogated the angiotensin II-induced stimulation of Ca2+ channels. We conclude that activation of angiotensin AT1 receptors requires all three alpha, beta, and gamma subunits of G13 for receptor-G protein interaction, whereas the transduction of the signal to L-type Ca2+ channels is mediated by Gbetagamma.

Characterization and phylogenetic significance of rhinoceros luteinizing hormone beta (LH β) subunit messenger RNA structure, complementary DNA sequence and gene copy number

The luteinizing hormone (LH) β subunit gene is expressed in the pituitary glands of all mammals, whereas the closely related chorionic gonadotropin (CG) β subunit genes have been identified only in primates and equids, and are expressed in placenta. In the case of horses, there is a single-copy equine (e) luteinizing hormone/chorionic gonadotropin hormone β subunit gene ( eLH/CGβ) that (1) is expressed in both pituitary gland and placenta, (2) encodes a characteristic carboxyl terminal peptide (CTP) extension, and (3) transcribes an atypically elongated 5′-untranslated region ( UTR) in both pituitary and placenta. However, it is not known whether similar expression patterns and gene locus characteristics may be exhibited by other members of the order Perissodactyla (equid, rhinoceros and tapir species). To begin to investigate these possibilities, we undertook analysis of the rhinoceros (rn or rhino) LH/(CG?)β gene locus and the rnLHβ cDNA. Total RNA isolated from the pituitary gland of a female white rhino was used as template for amplifying rnLHβ cDNA by reverse transcription-polymerase chain reaction. Following cloning of the amplified cDNA, nucleotide (nt) and deduced amino acid sequences were determined. The first in-frame stop codon occurred at codon position +122, suggesting that the rnLH β subunit does not contain a CTP. To assess gene copy number, Southern blot analysis of Indian rhino genomic DNA was performed. The resulting simple hybridization pattern indicated that, as in the horse and donkey, there is a single-copy gene at the rnLH/(CG?)β gene locus. Primer extension mapping of the pituitary transcriptional start site of the rnLHβ subunit gene revealed an 8 nt 5′- UTR which is similar to that reported for the majority of mammalian LH β transcripts. Northern analysis was consistent with the transcriptional start site findings. We postulate from these data that rhinos diverged from equids prior to the occurrence of the mutations causing CTP expression and adoption of a non-consensus 5′- UTR/proximal promoter region. However, these findings do not rule out the possibility of expression of a placental CG β subunit lacking a CTP in rhinos.

Human Thyroid-stimulating Hormone (hTSH) Subunit Gene Fusion Produces hTSH with Increased Stability and Serum Half-life and Compensates for Mutagenesis-induced Defects in Subunit Association

The human thyroid-stimulating hormone (hTSH) subunits alpha and beta are transcribed from different genes and associate noncovalently to form the bioactive hTSH heterodimer. Dimerization is rate-limiting for hTSH secretion, and dissociation leads to hormone inactivation. Previous studies on human chorionic gonadotropin (hCG) and human follicle-stimulating hormone had shown that it was possible by subunit gene fusion to produce a bioactive, single chain hormone. However, neither the stability nor the clearance from the circulation of such fused glycoprotein hormones has been studied. We show here that genetic fusion of the hTSH alpha- and beta-subunits using the carboxyl-terminal peptide of the hCG beta-subunit as a linker created unimolecular hTSH whose receptor binding and bioactivity were comparable to native hTSH. Interestingly, the fused hTSH had higher thermostability and a longer plasma half-life than either native or dimeric hTSH containing the hCG beta-subunit-carboxyl-terminal peptide, suggesting that dimer dissociation may contribute to glycoprotein hormone inactivation in vivo. In addition, we show for the first time that synthesis of hTSH as a single polypeptide chain could overcome certain mutagenesis-induced defects in hTSH secretion, therefore enabling functional studies of such mutants. Thus, in addition to prolongation of plasma half-life, genetic fusion of hTSH subunits should be particularly relevant for the engineering of novel analogs where desirable features are offset by decreased dimer formation or stability. Such methods provide a general approach to expand the spectrum of novel recombinant glycoprotein hormones available for in vitro and in vivo study.

Backfoot is a novel homeobox gene expressed in the mesenchyme of developing hind limb.

Homeobox genes play important roles in pattern formation during development. Here, we report the cloning and temporal and spatial expression patterns of a novel homeobox gene Backfoot (BFT for the human gene, and Bft for the mouse gene), whose expression reveals an early molecular distinction between forelimb and hind limb. BFT was identified as a sequence-specific DNA-binding protein. In addition to the homeodomain, it shares a carboxyl-terminal peptide motif with other paired-like homeodomain proteins. Northern hybridization analysis of RNAs from human tissues revealed that human BFT is highly expressed in adult skeletal muscle and bladder. During midgestation embryogenesis, mouse Bft is expressed in the developing hind limb buds, mandibular arches, and Rathke's pouch. The expression of Bft begins prior to the appearance of hind limb buds in mesenchyme but is never observed in forelimbs. At later stages of limb development, the expression is progressively restricted to perichondrial regions, most likely in tendons and ligaments. The timing and pattern of expression suggest that Bft plays multiple roles in hind limb patterning, branchial arch development, and pituitary development. Bft is likely identical to a mouse gene, Ptx1, that was recently isolated by Lamonerie et al. ([1996] Genes Dev. 10:1284-1295) and that has been suggested to play a role in pituitary development.

Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent.

Assembly of terminal complement complexes (TCC) C5b-7, C5b-8, and C5b-9 on target cells during acute and chronic inflammation induces hydrolysis of plasma membrane phospholipids and heterotrimeric G protein activation. TCC also stimulate a variety of cellular activities, which include cytokine synthesis, proto-oncogene activation, and mitotic signaling. Now we report that sublytic TCC induced Ras, Raf-1, and extracellular signal-regulated kinase (ERK) 1 activation in JY25 B cell line. When cells were exposed to C5b-9, GTP-bound Ras in anti-C5b-9 immunoprecipitates was increased 3.2-fold at 2 min, while GTP-bound Ras in anti-Ras immunoprecipitates was increased 2-fold at 10 min. Both C5b-9 and C5b-7, but not C5b6, increased Raf-1 kinase activity maximum 3.3-fold at 2 min and 2.8-fold at 5 min, respectively. ERK1 activity was 2-fold increased by C5b-9 at 2 min and by C5b-7 at 10 min, over the C5b6 level. The role of mitogen-activated protein kinase (MAPK) pathway on TCC-inducible mitotic signaling was evaluated by assessing DNA synthesis and activator protein 1 (AP-1) DNA-binding activity. The MAPK/ERK-specific inhibitor PD 098,059 abolished the C5b-9-induced DNA synthesis. Involvement of G protein in the activation of MAPK pathway by TCC was indicated by inhibition of Raf-1 and ERK1 kinase activity, as well as the DNA synthesis by pretreatment of cells with pertussis toxin. Overexpression of beta-adrenergic receptor kinase 1 carboxyl-terminal peptide in JY25 cells also inhibited Raf-1 and ERK1 activity, indicating a direct involvement of G betagamma subunits in the signal transduction generated through activation of MAPK pathway by TCC assembly in the plasma membrane.

Characterization of Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B as in Vitro Substrates for Farnesyl Protein Transferase and Geranylgeranyl Protein Transferase Type I

Ras proteins are small GTP-binding proteins which are critical for cell signaling and proliferation. Four Ras isoforms exist: Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B. The carboxyl termini of all four isoforms are post-translationally modified by farnesyl protein transferase (FPT). Prenylation is required for oncogenic Ras to transform cells. Recently, it was reported that Ki-Ras4B is also an in vitro substrate for the related enzyme geranylgeranyl protein transferase-1 (GGPT-1) (James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 6221-6226). In the current studies, we compared the four isoforms of Ras as substrates for FPT and GGPT-1. The affinity of FPT for Ki-Ras4B (Km = 30 nM) is 10-20-fold higher than that for the other Ras isoforms. Consistent with this, when the different Ras isoforms are tested at equimolar concentrations, it requires 10-20-fold higher levels of CAAX-competitive compounds to inhibit Ki-Ras4B farnesylation. Additionally, we found that, as reported for Ki-Ras4B, N-Ras and Ki-Ras4A are also in vitro substrates for GGPT-1. Of the Ras isoforms, N-Ras is the highest affinity substrate for GGPT-1 and is similar in affinity to a standard GGPT-1 substrate terminating in leucine. However, the catalytic efficiencies of these geranylgeranylation reactions are between 15- and 140-fold lower than the corresponding farnesylation reactions, largely reflecting differences in affinity. Carboxyl-terminal peptides account for many of the properties of the Ras proteins. One interesting exception is that, unlike the full-length N-Ras protein, a carboxyl-terminal N-Ras peptide is not a GGPT-1 substrate, raising the possibility that upstream sequences in this protein may play a role in its recognition by GGPT-1. Studies with various carboxyl-terminal peptides from Ki-Ras4B suggest that both the carboxyl-terminal methionine and the upstream polylysine region are important determinants for geranylgeranylation. Furthermore, it was found that full-length Ki-Ras4B, but not other Ras isoforms, can be geranylgeranylated in vitro by FPT. These findings suggest that the different distribution of Ras isoforms and the ability of cells to alternatively process these proteins may explain in part the resistance of some cell lines to FPT inhibitors.

Bone morphogenetic protein-2 stimulates differentiation of cultured spinal ligament cells from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is characterized by heterotopic bone formation occurring in spinal ligament, causing severe compression myelopathy. In order to investigate the mechanism of OPLL development, we isolated spinal ligament cells from OPLL patients as well as non-OPLL patients, and established 10 OPLL cell lines and 7 non-OPLL cell lines, respectively. We analyzed the effects of bone morphogenetic protein-2 (BMP-2) on these cells with respect to alkaline phosphatase (AP) activity, DNA synthesis, and collagen production. BMP-2 caused a significant increase of AP activity in 4 OPLL cell lines, whereas the activity did not change in any non-OPLL cells. Among OPLL cells, BMP-2 stimulated DNA synthesis in four cell lines and procollagen type I carboxyl-terminal peptide (PICP) synthesis in five cell lines. Some non-OPLL cells also responded to BMP-2, as there was an increase of DNA synthesis in three cell lines and PICP synthesis in one cell line. These data collectively indicate that BMP-2 preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. OPLL cells, therefore, exhibit a different response to BMP-2 than non-OPLL cells, suggesting that the expression of BMP receptor(s) and/or the signal transduction initiated by BMP-2 in the spinal ligament cells of OPLL patients somewhat deviate from those in normal spinal ligament cells. Such abnormal characteristics of OPLL cells as described here provide some clues to the clarification of the pathogenesis of OPLL.

Makatoxin I, a Novel Toxin Isolated from the Venom of the Scorpion Buthus martensi Karsch, Exhibits Nitrergic Actions

Buthus martensi Karsch venom exhibits nitrergic action in rat anococcygeus muscle (ACM). We have purified a novel toxin, makatoxin I (MkTx I), which exhibits nitrergic action, to homogeneity from this venom by a combination of gel-filtration, cation-exchange chromatography, and reverse-phase chromatography. Its purity was assessed by capillary electrophoresis and mass spectrometry. Its molecular weight was found to be 7031.71 +/- 2.88 as calculated from electrospray mass spectrographic data. The complete amino acid sequence was elucidated by sequencing of reduced and S-pyridylethylated toxin and a carboxyl-terminal peptide, P55-64, generated by the cleavage of toxin with endoproteinase Lys-C. The complete sequence of MkTx I is GRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPDKVPIRISGSCR. This toxin is composed of 64 amino acid residues and contains 8 half-cystine residues. Structurally, MkTx I has high similarity to Bot I and Bot II when compared with toxins from other scorpion species. The effects of MkTx I on nitrergic responses were investigated using the rat isolated ACM mounted in Krebs solution (37 degrees C, 5% CO2 in O2). MkTx I (2 microg/ml) markedly relaxed the carbachol-precontracted ACM; the relaxation was inhibited by the stereoselective inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (50 microM). Thus, MkTx I is the first alpha-toxin that can mediate nitrergic responses in the rat isolated ACM.

The COOH-terminal Peptide Binding Domain Is Essential for Self-association of the Molecular Chaperone HSC70

We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 10(5) M-1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties.

Post-translational Processing and Renal Expression of Mouse Indian Hedgehog

The full-length mouse Indian hedgehog (Ihh) cDNA was cloned from an embryonic 17.5-day kidney library and was used to study the post-translational processing of the peptide and temporal and spatial expression of the transcript. Sequence analysis predicted two putative translation initiation sites. Ihh translation was initiated at both initiation sites when expressed in an in vitro transcription/translation system. Expression of an Ihh mutant demonstrated that the internal translation initiation site was sufficient to produce the mature forms of Ihh. Ihh post-translational processing proceeded in a fashion similar to Sonic and Drosophila hedgehog; the unprocessed form underwent signal peptide cleavage as well as internal proteolytic processing to form a 19-kDa amino-terminal peptide and a 26-kDa carboxyl-terminal peptide. This processing required His313 present in a conserved serine protease motif. Ihh transcript was detected by in situ RNA hybridization as early as 10 days postcoitum (dpc) in developing gut, as early as 14.5 dpc in the cartilage primordium, and in the developing urogenital sinus. In semiquantitative reverse transcription-polymerase chain reaction experiments, Indian hedgehog transcript was first detected in the mouse metanephros at 14.5 dpc; transcript abundance increased with gestational age, becoming maximal in adulthood. In adult kidney, Ihh transcript was detected only in the proximal convoluted tubule and proximal straight tubule.

Pyruvate-extended Amino Acid Derivatives as Highly Potent Inhibitors of Carboxyl-terminal Peptide Amidation

Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We now introduce the first potent inhibitors for peptidylamidoglycolate lyase. These inhibitors, which can be viewed as pyruvate-extended N-acetyl amino acids, constitute a novel class of compounds. They were designed to resemble likely transient species along the reaction pathway of PGL catalysis. A general synthetic procedure for preparation of pyruvate-extended N-acetyl amino acids or peptides is described. Since these compounds possess the 2,4-dioxo-carboxylate moiety, their solution tautomerization was investigated using both NMR and high performance liquid chromatography analyses. The results establish that freshly prepared solutions of N-Ac-Phe-pyruvate consist predominantly of the enol tautomer, which then slowly tautomerizes to the diketo form when left standing for several days in an aqueous medium; upon acidification, formation of the hydrate tautomer occurs. Kinetic experiments established that these novel compounds are highly potent, pure competitive inhibitors of PGL. Kinetic experiments with the ascorbate-dependent copper monooxygenases, PAM and dopamine-beta-monooxygenase, established that these compounds also bind competitively with respect to ascorbate; however, pyruvate-extended N-acyl-amino acid derivatives possessing hydrophobic side chains are much more potent inhibitors of PGL than of PAM. Selective targeting of N-Ac-Phe-pyruvate so as to inhibit the lyase, but not the monooxygenase, domain was demonstrated with the bifunctional amidating enzyme of Xenopus laevis. The availability of potent inhibitors of PGL should facilitate studies regarding the possible biological role of alpha-hydroxyglycine-extended peptides.

Serum Levels of Osteocalcin and Type I Procollagen in Children with Celiac Disease

Bone metabolism may be disturbed in children with celiac disease. Two markers of bone turnover were used: the level of osteocalcin (BGP) and the level of carboxylterminal peptide of type I procollagen (PICP). BGP and PICP were measured by radioimmunoassays in 18 untreated children with celiac disease (mean age: 22.9 +/- 15.6 months) and in 15 control subjects (mean age 28.5 +/- 21 months). All the patients were rechecked after 1 month and again after 3 months from beginning of a gluten-free diet (GFD). Compared with controls at diagnosis our patients had significantly lower serum levels of BGP and PICP (p = 0.003 and p = 0.018 by Student's t test, respectively). These levels increased markedly during the 1st 3 months of GFD. The alteration in calcium phosphate homeostasis caused by celiac disease directly affects the synthesis of both components of the connective matrix of bone. Measurements of BGP and PICP provide a reliable and rapidly obtainable index of normalization of the processes of bone growth which can be achieved with a GFD.

Serum Levels of Osteocalcin and Type I Procollagen in Children with Celiac Disease

Background:Bone metabolism may be disturbed in children with celiac disease.Methods:Two markers of bone turnover were used: the level of osteocalcin (BGP) and the level of carboxylterminal peptide of type I procollagen (PICP). BGP and PICP were measured by radioimmunoassays in 18 untreated children with celiac disease (mean age: 22.9 ± 15.6 months) and in 15 control subjects (mean age 28.5 ± 21 months). All the patients were rechecked after 1 month and again after 3 months from beginning of a gluten‐free diet (GFD).Results:Compared with controls at diagnosis our patients had significantly lower serum levels of BGP and PICP (p = 0.003 and p = 0.018 by Student's t test, respectively). These levels increased markedly during the 1st 3 months of GFD.Conclusions:The alteration in calcium phosphate homeostasis caused by celiac disease directly affects the synthesis of both components of the connective matrix of bone. Measurements of BGP and PICP provide a reliable and rapidly obtainable index of normalization of the processes of bone growth which can be achieved with a GFD.

The Influence of the Proliferating Cell Nuclear Antigen-interacting Domain of p21CIP1 on DNA Synthesis Catalyzed by the Human and Saccharomyces cerevisiae Polymerase δ Holoenzymes

In eukaryotes, processive DNA synthesis catalyzed by DNA polymerases delta and epsilon (pol delta and epsilon) requires the proliferating cell nuclear antigen (PCNA). It has recently been shown that in humans (h), the PCNA function, required for both DNA replication and nucleotide excision repair, can be inactivated by p21(CIP1) due to a specific interaction between hPCNA and the carboxyl terminus of p21(CIP1). In this report, we show that Saccharomyces cerevisiae (S. cerevisiae) PCNA-dependent pol delta-catalyzed DNA synthesis was inhibited less efficiently than the human system by the intact p21(CIP1) protein and was unaffected by the p21(CIP1) carboxyl-terminal peptide (codons 139-160). This species-specific response of PCNA to p21(CIP1)-mediated inhibition of DNA synthesis results from a marked difference in the ability of h and S. cerevisiae PCNA to interact with p21(CIP1). As shown by binding studies using the surface plasmon resonance technique, hPCNA binds both full-length p21(CIP1) and the p21(CIP1) peptide-(139-160) stoichiometrically with a similar affinity (KD approximately 2.5 nM) while S. cerevisiae PCNA binds p21(CIP1) with approximately 10-fold less affinity and does not interact with the p21(CIP1) peptide-(139-160).

Stimulation of Platelet Activation and Aggregation by a Carboxyl-terminal Peptide from Thrombospondin Binding to the Integrin-associated Protein Receptor

Thrombospondin, a major secretory product of the alpha-granules of activated platelets, is a large trimeric glycoprotein that plays an important role in platelet aggregation. On resting platelets, thrombospondin binds to a single receptor in a cation-independent manner, but upon platelet activation it binds at least two further, distinct receptors that are both dependent upon divalent cations. Each of these receptors on the platelet surface binds to different regions of the thrombospondin molecule, and such binding may be responsible for the multifunctional role of thrombospondin in aggregation. We show here that a peptide from the carboxyl terminus of thrombospondin, RFYVVMWK, directly and specifically induces the activation and aggregation of washed human platelets from different donors at concentrations of 5-25 microM. At lower concentrations the peptide synergizes with suboptimal concentrations of ADP to induce aggregation. Peptide affinity chromatography and immunoprecipitation with a monoclonal antibody were used to identify the receptor for the carboxyl-terminal peptide as the integrin-associated protein. The integrin-associated protein remained bound to the RFYVVMWK-containing peptide column when washed with a scrambled peptide in the presence of 5 mM EDTA, indicating a divalent cation-independent association. It is suggested that integrin-associated protein is the primary receptor for thrombospondin on the surface of resting platelets and is implicated in potentiating the platelet aggregation response.

Production of Bioactive Salmon Calcitonin from the Nonendocrine Cell Lines COS-7 and CHO

Takahashi, K.-I., Y.-C. Liu, N. Hayashi, F. Goto, M. Kato, H. Kawashima and T. Takeuchi. Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO. Peptides 18(3) 439–444, 1997.—To produce bioactive salmon calcitonin from the conventional nonendocrine cell lines, COS-7 and CHO, we devised a salmon calcitonin expression vector by combining the amino-terminus of human calcitonin precursor with a salmon calcitonin sequence, inserting the efficient furin-cleavable processing sequence Arg-X-Arg-X-Lys-Arg before salmon calcitonin, and deleting the carboxyl-terminal extension peptide. This chimeric calcitonin precursor terminates at glycine to easily receive an amidation reaction. COS-7 and CHO produced a high level of bioactive calcitonin by the resorption pit formation assay. Although amidating activity is highly expressed in CHO, but only a little in COS-7 cells, both cells produced a similar level of bioactive calcitonin. Thus, the engineered salmon calcitonin expression vector enables nonendocrine cells even with low amidation activity to produce bioactive calcitonin.