Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties.

Hemofiltrate CC chemokine (HCC)-1 is a recently described human chemokine that is constitutively expressed in numerous tissues and is present at high concentrations in normal plasma. Using a cell line expressing CC chemokine receptor (CCR)5 as a bioassay, we isolated from human hemofiltrate an HCC-1 variant lacking the first eight amino acids. HCC-1[9-74] was a potent agonist of CCR1, CCR3, and CCR5 and promoted calcium flux and chemotaxis of T lymphoblasts, monocytes, and eosinophils. It also blocked entry of HIV-1 strains using CCR5 as coreceptor. Limited tryptic digestion of HCC-1 generated the active variant. Conditioned media from several tumor cell lines activated HCC-1 with a high efficiency, and this activity could be inhibited by serine protease inhibitors. Our results indicate that HCC-1 represents a nonfunctional precursor that can be rapidly converted to the active chemokine by proteolytic processing. This process represents an additional mechanism by which tumor cells might generate chemoattractant molecules and recruit inflammatory cells. It might also affect HIV-1 replication in infected individuals and play an important role in AIDS pathogenesis.

HCC-1, a novel chemokine from human plasma.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways.

Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different chromosomal translocation, has recently been described. In this study, the kinase specificities of the Bcr-Abl and Tel-Abl proteins were compared to the physiological Abl family kinases c-Abl and Arg (abl related gene). Using short peptides which correspond to the target epitopes in known substrate proteins of Abl family kinases, we found a higher catalytic promiscuity of Bcr-Abl and Tel-Abl. Similar to Bcr-Abl, Tel-Abl was found in complexes with the adapter protein CRKL. In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells. GTPase analysis with a Ras-GTP-specific precipitation assay showed constitutive elevation of GTP-loaded Ras in cells expressing the leukaemic Abl proteins. The mitogenic MAPK/Erk kinases as well as Akt/PKB, a kinase implicated to negatively regulate apoptosis, were also constitutively activated by both Bcr-Abl and Tel-Abl. The results indicate that the leukaemic Abl-fusion proteins have catalytic specificities different from the normal kinases c-Abl and Arg and that Tel-Abl is capable to activate at least some pathways which are also upregulated by Bcr-Abl.

The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins.

Adapter proteins function by mediating the rapid and specific assembly of multi-protein complexes during the signal transduction which guards proliferation, differentiation and many functions of higher eukaryotic cells. To understand their functional roles in different cells it is important to identify the selectively interacting proteins in these cells. Two novel candidates for signalling partners of Crk family adapter proteins, the hematopoietic progenitor kinase 1 (HPK1) and the kinase homologous to SPS1/STE20 (KHS), were found to bind with great selectivity to the first SH3 domains of c-Crk and CRKL. While KHS bound exclusively to Crk family proteins, HPK1 also interacted with both SH3 domains of Grb2 and weakly with Nck, but not with more than 25 other SH3 domains tested. The interaction of HPK1 with c-Crk and CRKL was studied in more detail. HPK1-binding to the first SH3 domain of CRKL is direct and occurs via proline-rich motifs in the C-terminal, non-catalytic portion of HPK1. In vitro complexes were highly stable and in vivo complexes of c-Crk and CRKL with HPK1 were detectable by co-immunoprecipitation with transiently transfected cells but also with endogenous proteins. Furthermore, c-Crk II and, to a lesser extent, CRKL were substrates for HPK1. These results make it likely that HPK1 and KHS participate in the signal transduction of Crk family adapter proteins in certain cell types.

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

1H-NMR studies of the natriuretic peptide urodilatin: sequence-specific resonance assignment.

The recently discovered 32 amino-acid natriuretic peptide urodilatin was chemically synthesized and subjected to two-dimensional proton nuclear magnetic resonance (NMR) spectroscopy studies in aqueous solution in order to determine the structural state of urodilatin. In contrast to earlier studies on very closely related peptides, such as cardiodilatin (CDD/ANP-99-126) and brain natriuretic peptide (BNP), spectra of urodilatin were extremely well resolved even in millimolar concentration in H2O so that the complete sequence specific resonance assignments could be achieved. No long range NOEs could be detected, except between residues close to the single cystine bond. This leads to the conclusion that urodilatin in aqueous solution is a random coil peptide with the exception of the region around the cystine bond.

hDIP--a potential transcriptional regulator related to murine TSC-22 and Drosophila shortsighted (shs)--is expressed in a large number of human tissues.

We have cloned a 420 bp cDNA from a human fetal brain cDNA library in lambda encoding the human homologue of a DSIP-immunoreactive leucine zipper protein (DIP) isolated from porcine brain. The derived human protein (hDIP) shares a significant sequence identity with parts of the murine TSC-22 and Drosophila shs, both proteins which are discussed as functioning as transcriptional regulators. A similar role of hDIP is partially confirmed by the results of an RT-PCR analysis, demonstrating the widespread distribution of the protein among different human tissues.

Hemofiltrate CC chemokines with unique biochemical properties: HCC-1/CCL14a and HCC-2/CCL15.

The hemofiltrate CC chemokines CCL14a (formerly HCC-1), CCL14b (formerly HCC-3), and CCL15 (formerly HCC-2) are encoded by mono- as well as bicistronic transcripts from a tandem gene arrangement on human chromosome 17q11.2. The transcription and splicing into several mono- and bicistronic transcripts of this gene complex are unique for human genes. No corresponding mechanism is known in nonprimate mammalian species such as mice and rats. The extremely high concentration of CCL14a in human plasma is exceptional for chemokines and led to the identification of this chemokine. Several molecular forms of CCL14a have been isolated and investigated. The mature propeptide CCL14a(1-74) is a low-affinity agonist of CCR1 which is converted to a high-affinity agonist of CCR1 and CCR5 on proteolytic processing by serine proteases. In contrast, CCL15 is characterized using molecular forms deduced from the mRNA/cDNA and shown to activate cells via CCR1 and CCR3, also dependent on the amino-terminal length. Hemofiltrate CC chemokines are chemoattractants for different types of leukocytes including monocytes, eosinophils, T cells, dendritic cells, and neutrophils. In this review, we emphasize the genomic organization, expression patterns, and biochemical properties of CCL14a, CCL14b, and CCL15. We report results of significance for the development of therapeutic strategies, especially concerning HIV infection and inflammatory diseases.

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat.

BACKGROUND: Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS: RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS: GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION: GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.

Structural and phylogenetic characterization of human SLURP-1, the first secreted mammalian member of the Ly-6/uPAR protein superfamily.

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.

Role of the prosequence of guanylin.

Guanylin is a guanylyl cyclase (GC)-activating peptide that is mainly secreted as the corresponding prohormone of 94 amino acid residues. In this study, we show that the originally isolated 15-residue guanylin, representing the COOH-terminal part of the prohormone, is released from the prohormone by cleavage of an Asp-Pro amide bond under conditions applied during the isolation procedures. Thus, the 15-residue guanylin is probably a non-native, chemically induced GC-activating peptide. This guanylin molecule contains two disulfide bonds that are absolutely necessary for receptor activation. We demonstrate that the folding of the reduced 15-residue guanylin results almost completely in the formation of the two inactive disulfide isomers. In contrast, the reduced form of proguanylin containing the entire prosequence folds to a product with the native cysteine connectivity. Because proguanylin lacking the 31 NH2-terminal residues of the prosequence folds only to a minor extent to guanylin with the native disulfide bonds, it is evident that this NH2-terminal region contributes significantly to the correct disulfide-coupled folding. Structural studies using CD and NMR spectroscopy show that native proguanylin contains a considerable amount of alpha-helical and, to a lesser extent, beta-sheet structural elements. In addition, a close proximity of the NH2- and the COOH-terminal regions was found by NOESY. It appears that this interaction is important for the constitution of the correct conformation and provides an explanation of the minor guanylyl cyclase activity of proguanylin by shielding the bioactive COOH-terminal domain from the receptor.

Structure determination of human and murine beta-defensins reveals structural conservation in the absence of significant sequence similarity.

Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian beta-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human beta-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that beta-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the beta-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because beta-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.

Regulated, side-directed secretion of proguanylin from isolated rat colonic mucosa.

Guanylin, an activator of the guanylyl cyclase C receptor in the apical membrane of intestinal epithelium, modulates intestinal fluid and electrolyte transport. The bioactive 15-amino acid peptide originally isolated from rat intestine represents the C-terminal part of a longer, 115-residue prepropeptide. The aim of the present study was to characterize the direction and molecular form in which guanylin is secreted from the colonic mucosa, as well as the mechanisms that trigger its secretion. Isolated rat colonic mucosa was mounted in Ussing chambers, allowing the separate determination of apical and basolateral release. After HPLC purification, two different molecular forms of guanylin were identified in the apical incubation media by combining a bioassay for guanylyl cyclase C activation, a specific guanylin enzyme-linked immunosorbent assay and mass spectrometry, as well as sequence analysis: a bioactive form coeluting with synthetic 15-residue guanylin and the 94-residue propeptide, guanylin-22-115. The basal concentration of proguanylin at the apical side of epithelia was about 15-fold higher, compared with that of the small, bioactive peptide. In the basolateral incubation media, no proguanylin and only very low amounts of bioactive guanylin were detected. Incubation with carbachol led to a significant increase of about 7-fold in the release of proguanylin to both sides of the isolated epithelia. On the apical side, a concomitant increase of the small, bioactive peptide was observed; whereas, on the basolateral side, its concentration remained unchanged. Vasoactive intestinal peptide or the NO-donor S-nitroso-N-acetylpenicillamine did not affect guanylin secretion. Our results suggest that, in the intestine, guanylin is secreted mainly to the luminal side of the epithelium. The peptide is released as a 94-residue propeptide, which is then processed to a smaller, bioactive form (luminocrine secretion). Carbachol stimulates the release of proguanylin to both sides of the intestinal mucosa, but a parallel increase in the bioactive C-terminal derivative only occurs on the apical side. In vivo, the basolateral release could be a source of circulating proguanylin, which might be processed proteolytically to the active peptide in distant target tissues (endocrine secretion).

Does a synthetic peptide containing the leucine-zipper domain of c-myb form an alpha-helical structure in solution?

We have examined a synthetic peptide containing the putative leucine zipper domain of the chicken c-myb proto-oncogene using circular dichroism (CD) spectroscopy. The peptide adopts an alpha-helical structure only at low temperatures and in the presence 2,2,2-trifluoroethanol.

Radioimmunoassay for circulating human guanylin.

A highly specific and sensitive radioimmunoassay for circulating human guanylin (guanylin-22-115) has been developed. Antibodies were raised against the amino-terminus (positions 4-16) of the peptide. Western blot analysis confirmed that the antibody selected for radioimmunoassay recognizes circulating high molecular weight (10.3 kDa) guanylin. Extraction and purification of guanylin from blood hemofiltrate and from blood plasma showed that circulating guanylin is detectable in corresponding amounts by the radioimmunoassay and by a specific bioassay. In 30 healthy subjects, the mean plasma concentration of immunoreactive (IR) guanylin was 42 +/- 3 fmol/ml. In 22 patients with chronic renal insufficiency, the concentrations of IR-guanylin were significantly enhanced (1,074 +/- 24 fmol/ml), indicating that kidneys metabolize and/or eliminate the circulating hormone.

Casocidin-I: a casein-alpha s2 derived peptide exhibits antibacterial activity.

Here we report the isolation and characterization of an antibacterial peptide from bovine milk inhibiting the growth of Escherichia coli, and Staphylococcus carnosus. The primary structure of the peptide was revealed as a 39-amino-acid-containing fragment of bovine alpha s2-casein (position 165-203) by means of Edman amino acid sequencing and mass spectrometry. Since human milk does not contain any casein-alpha s2, these findings could explain the different influence of human and bovine milk on the gastrointestinal flora of the suckling.

Mapping the heparin-binding domain of boar spermadhesins.

Boar spermadhesins are a group of seminal plasma, heparin-binding proteins which appear to be involved in sperm capacitation and gamete interaction. Using a proteolytic protection assay we have identified regions of AQN-1, AQN-3, PSP-I and AWN which remain attached to a heparin-Sepharose column following in-column digestion of bound spermadhesins with chymotrypsin and elastase. In addition, the complete amino acid sequence of spermadhesin AWN was synthesized as overlapping peptides, and their ability to bind to a heparin-Sepharose column and to inhibit the interaction of soluble heparin with purified ELISA plate-coated AWN was tested. Both approaches gave similar results and as a whole showed that different regions of AWN may converge in its tertiary structure to form a composite heparin-binding site. The conformational heparin-binding surface resides on the GFCC'C'' face of the proposed structural model for AWN and is in an opposite location to the carbohydrate-binding region of the spermadhesin.

A disulfide conjugate between anti-tetanus antibodies and HIV (37-72)Tat neutralizes tetanus toxin inside chromaffin cells.

Conjugates between anti-tetanus F(ab')2 fragments and the (37-72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108-15 neurohybridoma cells and Rev-2-T-6 lymphoma cells. The uptake could not be inhibited by competition with (37-72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.

Translocation of acylated pardaxin into cells.

Acylated pardaxin is translocated through the cytoplasmic membrane and is accumulated in the nucleoli of NG108-15 and chromaffin cells. The uptake is time- and dose-dependent and temperature-sensitive. However, the binding of acylated 125I-pardaxin cannot be reduced by competition with pardaxin acylated with Rudinger's reagent. In this respect, acylated pardaxin resembles the Tat protein 37-71 fragment. Metabolic inhibitors do not significantly reduce the uptake of acylated 125I-pardaxin. Acylated pardaxin might be useful as a vector to translocate other molecules.

The circulating bioactive form of human guanylin is a high molecular weight peptide (10.3 kDa).

Guanylin is a peptide isolated from rat intestine that stimulates intestinal guanylate cyclase. We describe here the purification of circulating guanylin from human hemofiltrate. By N-terminal protein sequence analysis 47 amino acids were determined. This sequence corresponds to the positions 22 to 68 of the prohormone deduced from the cDNA sequence of human proguanylin. Mass spectral analysis of the circulating peptide showed the molecular weight to be 10,336 Da, which corresponds to the mass calculated from position 22 to the C-terminus of the peptide predicted from the cDNA sequence. Circulating guanylin markedly increased the cyclic GMP content of T84 cells. Our data show that the hormonal form of guanylin is circulating as a 10.3-kDa peptide in human blood.