Sequencing refractory regions in bird genomes are hotspots for accelerated protein evolution.

BACKGROUND: Approximately 1000 protein encoding genes common for vertebrates are still unannotated in avian genomes. Are these genes evolutionary lost or are they not yet found for technical reasons? Using genome landscapes as a tool to visualize large-scale regional effects of genome evolution, we reexamined this question. RESULTS: On basis of gene annotation in non-avian vertebrate genomes, we established a list of 15,135 common vertebrate genes. Of these, 1026 were not found in any of eight examined bird genomes. Visualizing regional genome effects by our sliding window approach showed that the majority of these "missing" genes can be clustered to 14 regions of the human reference genome. In these clusters, an additional 1517 genes (often gene fragments) were underrepresented in bird genomes. The clusters of "missing" genes coincided with regions of very high GC content, particularly in avian genomes, making them "hidden" because of incomplete sequencing. Moreover, proteins encoded by genes in these sequencing refractory regions showed signs of accelerated protein evolution. As a proof of principle for this idea we experimentally characterized the mRNA and protein products of four "hidden" bird genes that are crucial for energy homeostasis in skeletal muscle: ALDOA, ENO3, PYGM and SLC2A4. CONCLUSIONS: A least part of the "missing" genes in bird genomes can be attributed to an artifact caused by the difficulty to sequence regions with extreme GC% ("hidden" genes). Biologically, these "hidden" genes are of interest as they encode proteins that evolve more rapidly than the genome wide average. Finally we show that four of these "hidden" genes encode key proteins for energy metabolism in flight muscle.

An siRNA screen identifies RSK1 as a key modulator of lung cancer metastasis.

We performed a kinome-wide siRNA screen and identified 70 kinases altering cell migration in A549 lung cancer cells. In particular, ribosomal S6 kinase 1 (RSK1) silencing increased, whereas RSK2 and RSK4 downregulation inhibited cell motility. In a secondary collagen-based three-dimensional invasion screen, 38 of our hits cross-validated, including RSK1 and RSK4. In two further lung cancer cell lines, RSK1 but not RSK4 silencing showed identical modulation of cell motility. We therefore selected RSK1 for further investigation. Bioinformatic analysis followed by co-immunoprecipitation-based validation revealed that the actin regulators VASP and Mena interact with RSK1. Moreover, RSK1 phosphorylated VASP on T278, a site regulating its binding to actin. In addition, silencing of RSK1 enhanced the metastatic potential of these cells in vivo using a zebrafish model. Finally, we investigated the relevance of this finding in human lung cancer samples. In isogenically matched tissue, RSK1 was reduced in metastatic versus primary lung cancer lesions. Moreover, patients with RSK1-negative lung tumours showed increased number of metastases. Our results suggest that the findings of our high-throughput in vitro screen can reliably identify relevant clinical targets and as a proof of principle, RSK1 may provide a biomarker for metastasis in lung cancer patients.

Peptidomics of the pars intercerebralis-corpus cardiacum complex of the migratory locust, Locusta migratoria.

The pars intercerebralis-corpora cardiaca system (PI-CC) of insects is the endocrinological equivalent of the hypothalamus-pituitary system of vertebrates. Peptide profiles of the pars intercerebralis and the corpora cardiaca were characterized using simple sampling protocols in combination with MALDI-TOF and electrospray ionization double quadrupole time of flight (ESI-Qq-TOF) mass spectrometric technologies. The results were compared with earlier results of conventional sequencing methods and immunocytochemical methods. In addition to many known peptides, several m/z signals corresponding to putative novel peptides were observed in the corpora cardiaca and/or pars intercerebralis. Furthermore, for a number of peptides evidence was provided about their localization and MALDI-TOF analysis of the released material from the corpora cardiaca yielded information on the hormonal status of particular brain peptides.

Pyrokinin neuropeptides in a crustacean. Isolation and identification in the white shrimp Penaeus vannamei.

Identification of substances able to elicit physiological or behavioural processes that are related to reproduction would greatly contribute to the domestication of commercially important crustaceans that do not reproduce easily in captivity. Crustaceans are thought to release urine signals used for chemical communication involved in courtship behaviour. In contrast to insects, very little is known about the endocrinological processes underlying this phenomenon. Therefore, an extract of 3500 central nervous systems of female white shrimp Penaeus vannamei was screened for myotropic activity in order to purify pyrokinin-like peptides that belong to the pyrokinin/PBAN neuropeptide family. Members of this family regulate reproductive processes in insects, including pheromone biosynthesis. Purification of these pyrokinins was achieved by a combination of reversed-phase and normal-phase chromatography. Subsequent characterization by mass spectrometry, Edman degradation and peptide synthesis resulted in the elucidation of two novel peptides. Pev-PK 1 has the primary sequence DFAFSPRL-NH(2) and a second peptide (Pev-PK 2) is characterized as the nonapeptide ADFAFNPRL-NH(2). Pev-PK 1 contains the typical FXPRL-NH(2) (X = G, S, T or V) C-terminal sequence that characterizes members of the versatile pyrokinin/PBAN family. Pev-PK 2 displays an Asn residue at the variable X position of the core pyrokinin sequence. These crustacean pyrokinins are the first to be found in a noninsect. The synthetic peptides display myotropic activity on the Leucophaea maderae as well as on the Astacus leptodactylus hindgut.

Identification of

The neuropeptides inducing dark color in albino nymphs of the migratory locust Locusta migratoria were isolated from the larval brain of the silkworm, Bombyx mori and from the adult corpora cardiaca (CC) of the cricket Gryllus bimaculatus, respectively, and their amino acid sequences identified. The two peptides isolated from the two different species are identical to [Arg(7)] corazonin, a neuropeptide known to be present in a cockroach and others. This peptide induces a dark color in albino nymphs of L. migratoria at fmol levels, and a high dose of >/=100 pmol caused albino locusts to turn completely black, but it influenced neither body color nor metamorphosis in B. mori and G. bimaculatus. Therefore, the physiological functions of [Arg(7)] corazonin in the silkworm and the cricket remain unknown. The present study demonstrated the usefulness of the albino strain of L. mirgatoria as a specific bioassay system for this peptide.

Identification of a new tachykinin from the midgut of the desert locust, Schistocerca gregaria, by ESI-Qq-oa-TOF mass spectrometry.

This paper reports the purification of a tachykinin isoform from the midgut of the desert locust, Schistocerca gregaria. One hundred locust midguts were extracted in an acidified methanolic solvent, after which three HPLC column systems were used to obtain a pure peptide. A tachykinin immunoassay was used to monitor all collected fractions. After each purification step the purity of the sample was monitored by MALDI-TOF mass spectrometry. The pure peptide was sequenced by ESI-Qq-oa-TOF mass spectrometry. Edman degradation-based automated microsequencing and chemical synthesis confirmed the sequences. The midgut peptide, GNTKKAVPGFYGTRamide (Scg-midgut-TK), belongs to the tachykinin family with identified members in all vertebrate phyla and some invertebrate phyla: arthropods, annelids and molluscs. Scg-midgut-TK is the first tachykinin purified from midguts of the desert locust Schistocerca gregaria. In comparison to locust brain tachykinins, the midgut tachykinin is N-terminally extended. Similar to neuropeptide gamma, an N-terminally extended mammalian tachykinin, first isolated from rabbit intestine, the present identified locust intestinal tachykinin contains a putative dibasic cleavage site.

Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.

The carboxyl methyltransferase, which is claimed to exclusively methylate the carboxyl group of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A (Leu(309)), was purified from porcine brain. On the basis of tryptic peptides, the cDNA encoding the human homologue was cloned. The cDNA of this gene encodes for a protein of 334 amino acids with a calculated M(r) of 38 305 and a predicted pI of 5.72. Database screening reveals the presence of this protein in diverse phyla. Sequence analysis shows that the novel methyltransferase is distinct from other known protein methyltransferases, sharing only sequence motifs supposedly involved in the binding of adenosylmethionine. The recombinant protein expressed in bacteria is soluble and the biophysical, catalytic, and immunological properties are indistinguishable from the native enzyme. The methylation of PP2A by the recombinant protein is restricted to Leu(309) of PP2A(C). No direct effects on phosphatase activity changes were observed upon methylation of the dimeric or trimeric forms of PP2A.

Identification of cyk, a cyclin B2 kinase, as a novel calcium/calmodulin-dependent protein kinase II and its role during Xenopus laevis oocyte maturation.

Recently, we have partially purified and characterized a specific cell cycle-regulated cyclin B2 kinase (cyk) from prophase oocytes of Xenopus laevis after an ATP-gamma-S activation step (R. Derua, I. Stevens, E. Waelkens, A. Fernandez, N. Lamb, W. Merlevede, and J. Goris, 1997, Exp. Cell Res. 230, 310-324). In the present paper we describe its purification to homogeneity. We could identify the kinase as a special form of calcium/calmodulin-dependent protein kinase II (CaMKII), consisting of five isoforms with molecular masses ranging from 52 to 83 kDa. At least three of them could be considered as novel. Using an in vivo assay with a synthetic peptide (cyktide), an activation of the kinase was shown at about 50% maturation. Further evidence for this observation came from the injection of the calcium chelator BAPTA and the specific cyk/CaMKII inhibitor AIP. A delay of oocyte maturation of at least 1 h was observed. Besides serine 53, a second cyk phosphorylation site in cyclin B2 was identified as threonine 41. Site-directed mutagenesis of these sites indicated that phosphorylation of these sites in Xenopus cyclin B2 was not required for the hallmark functions of cyclin B2.

Vascular endothelial growth factor measured in platelet poor plasma allows optimal separation between cancer patients and volunteers: a key to study an angiogenic marker in vivo?

BACKGROUND: Serum VEGF levels are elevated in cancer patients and are used as a tumor marker in different malignancies. We have measured VEGF levels in different blood compartments in cancer patients and healthy volunteers in order to assess the most suitable way of processing blood for measuring VEGF as a marker of tumor-angiogenesis. PATIENTS AND METHODS: VEGF concentrations were analyzed by an enzyme-linked immunosorbent assay in serum (VEGFS), EDTA plasma (VEGFEDTA), citrated plasma (VEGFC), CTAD-plasma (VEGFCTAD), platelet poor plasma (VEGFPPP), platelet rich plasma after induction of platelet activation (VEGFPRP). Platelet activation was assessed by measuring PF4 concentrations in different plasma samples. RESULTS: We observed higher VEGFS (P = 0.0027), VEGFEDTA (P = 0.003) and VEGFPPP (P = 0.0007) levels in cancer patients than in volunteers; VEGFPRP concentrations showed no significant difference (P = 0.208). Analysis of the correlation between VEGFplt and VEGFS in cancer patients showed a similar correlation in a comparable VEGFS concentration range as in the volunteers. When comparing VEGFC to VEGFCTAD, we find significantly higher VEGF and PF4 levels in citrated plasma (VEGF: P = 0.00019; PF4: P = 0.00023). CONCLUSIONS: It is likely that VEGFS in cancer patients encompass platelet-delivered VEGF and VEGF from other sources, notably from (neo)-angiogenesis in tumoral tissue. The best discrimination between volunteers and cancer patients was observed in PPP. As generating plasma can induce platelet activation, with consequent VEGF release from platelets, we suggest that to assess free circulating VEGF, CTAD plasma should be used.

Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant.

In response to crowding, locusts develop characteristic black patterns that are well discernible in the gregarious phase at outbreaks. We report here a dark-color-inducing neuropeptide (dark-pigmentotropin) from the corpora cardiaca of two plague locusts, Schistocerca gregaria and Locusta migratoria. The chromatographic isolation of this neuropeptide was monitored by using a bioassay with an albino mutant of L. migratoria. The neurohormone, consisting of 11 amino acids, is identical to [His7] corazonin, previously isolated from corpora cardiaca of another acridid without known function. The present results show that even in isolated (solitary) nymphs, [His7] corazonin induces gregarious black patterns. Its primary structure shows some similarity with the vertebrate melanophore stimulating hormone.

The kinin peptide family in invertebrates.

Kinins comprise a family of peptides that were first found in the central nervous system of insects and recently also in mollusks and crustaceans. After the isolation of the first members of the kinin family, the leukokinins from Leucophaea maderae, leukokinin-related peptides were found in the cricket Acheta domesticus and the locust Locusta migratoria, all through their ability to induce Leucophaea maderae hindgut contraction. Subsequently, kinins were found in the mosquitoes Culex salinarius and Aedes aegypti and in the earworm Helicoverpa zea. The first noninsect member of this family was isolated from a mollusk, the pond snail Lymnaea stagnalis. Most recently our group has isolated the first kinins from crustaceans. Six kinins were isolated from the white shrimp Penaeus vannamei. To date, 35 members of this family have been isolated. The first relatively small family of insect kinins has grown into an expanding and rather large family with members in insects, crustaceans, and mollusks. In this paper we discuss the kinin family in terms of method of isolation, structure, in vitro and in vivo activity, distribution, receptors, and signal transduction. We will compare the crustacean and insect members of the kinin family, using the data available on crustacea.

Identification of one tachykinin- and two kinin-related peptides in the brain of the white shrimp, Penaeus vannamei.

This paper reports the purification of three myotropic neuropeptides from the white shrimp Penaeus vannamei. The central nervous systems of 3500 shrimps were extracted in an acidified solvent, after which four to five HPLC column systems were used to obtain pure peptides. A cockroach hindgut muscle contraction bioassay was used to monitor all collected fractions. The pure peptides were submitted to Edman degradation based automated microsequencing. Mass spectrometry and chemical synthesis confirmed the sequences. Ala-Pro-Ser-Gly-Phe-Leu-Gly-Met-Arg-NH2 (Pev-tachykinin, 934.1 Da) belongs to the tachykinin family with identified members in all vertebrate classes and some invertebrate classes: arthropods, annelids and molluscs. A very specific Pev-tachykinin antiserum was developed, which labels 4 neurosecretory cells in the brain. Ala-Ser-Phe-Ser-Pro-Trp-Gly-NH2 (Pev-kinin 1, 749.8 Da) and Asp-Phe-Ser-Ala-Trp-Ala-NH2 (Pev-kinin 2, 694.7 Da) are the first crustacean kinins. Pev-kinin 2 is the first kinin with a Trp-Ala-NH2 instead of a kinin-typical Trp-Gly-NH2 carboxyterminus.

Characterization and physiological importance of a novel cell cycle regulated protein kinase in Xenopus laevis oocytes that phosphorylates cyclin B2.

We have partially purified a specific cyclin B2 kinase (cyk) from prophase oocytes of Xenopus laevis after an ATP-gamma-S activation step. Phosphopeptide analysis identified Ser53 as the major in vitro phosphorylation site for cyk in cyclin B2. Using a synthetic peptide derived from cyclin B2 encompassing Ser53 (cyktide) as a substrate, cyk was shown to be activated during progesterone-induced maturation, with a peak of activity between 40 and 50% maturation. A sustained high cyk activity was observed in oscillating egg extracts. Microinjection of cyk-phosphorylated cyclin B2 into prophase oocytes accelerated progesterone-induced maturation by about 2 h, indicating that cyclin B2 is a relevant substrate for cyk and that the function of cyk is situated upstream of cdc2-cyclin B activation. Microinjection of cyk-phosphorylated cyktide or a combination of cyk and cyclin B1 into G2 fibroblasts induced significant changes in cell morphology, reminiscent of a premature prophase-like phenotype. Similarly, addition of cyk-phosphorylated cyktide in cyclin B1-dependent interphase extracts resulted in histone H1 kinase activation.

Analysis of the disulfide linkage pattern in circulin A and B, HIV-inhibitory macrocyclic peptides.

Circulin A and B are members of a family of macrocyclic peptides, originally isolated from the tropical tree Chassalia parvifolia, that have been shown to display anti-HIV activity. Complete structural elucidation of these highly constrained peptides was difficult due to their cyclic amide backbone and the presence of six disulfide-linked cysteines. In the present study, the disulfide pairing motif of circulin A and circulin B was determined. Since the circulins were resistant to enzymatic proteolysis, cysteine residue pairings were identified by analysis of the complex mixture of cleavage products that resulted from partial acid hydrolysis of the native peptides. Combined utilization of HPLC, fast atom bombardment mass spectrometry and peptide recognition software ("F-MASS" and "F-LINK" programs) were employed to identify the cleavage products. Thus, we were able to unambiguously identify the disulfide linkage pattern in circulin A and circulin B as Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6, where the numbers on the cystine residues refer to their respective order in the peptides.

Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.

Phosphorylation and negative regulation of the transcriptional activator CREM by p34cdc2.

Transcription factors that bind to cAMP-responsive elements (CREs) regulate the expression of target genes in response to activation of the adenylyl cyclase pathway. It is generally thought that activation is obtained through direct phosphorylation by the cAMP-dependent protein kinase-A. We have isolated the gene CRE modulator (CREM), which encodes multiple members of the CRE-binding protein family, by cell-specific alternative splicing. Various isoforms have been characterized, encoding both repressors (CREM alpha, -beta, and -gamma) as well as activators (CREM tau). Here we show that the function of the activator CREM tau is regulated by the p34cdc2 kinase. Multiple serine and threonine residues are phosphorylated in vivo as well as in vitro by p34cdc2. Although there is no effect of p34cdc2-mediated phosphorylation on CREM tau DNA binding, we observed a dramatic effect on the trans-regulatory function. Coexpression of a constitutively active p34cdc2 mutant shows that the trans-activation potential of CREM tau is strongly reduced by p34cdc2. This represents the first example of negative regulation of a transcription factor of this class by p34cdc2.

Specificity of the polycation-stimulated (type-2A) and ATP,Mg-dependent (type-1) protein phosphatases toward substrates phosphorylated by P34cdc2 kinase.

p34cdc2 kinase, a critical regulator of the cell cycle, has been shown to recognize the consensus sequence S/TP in proteins such as histone H1, the retinoblastoma gene product RB and the carboxyl-terminal domain of eukaryotic RNA polymerase II. Using phosphorylated synthetic peptides, representing the p34cdc2 phosphorylation sites in these proteins and histone H1 protein as substrates, we investigated the substrate specificity of the different oligomeric forms of the polycation-stimulated (PCS/type-2A) protein phosphatase and the active catalytic subunit of the ATP,Mg-dependent (AMDc/type 1) protein phosphatase. The results show that the oligomeric structure of the PCS phosphatases is an important determinant for efficient dephosphorylation. The trimeric PCSH1 and PCSM phosphatases are about 10-20-fold-better histone H1 phosphatases than the dimeric PCSH2 and PCSL phosphatases and about 100-fold better than the catalytic subunit (PCSC), suggesting a regulatory role for the 72-kDa, 65-kDa and 55-kDa subunits. The RB peptide = INGS(P)PRT(P)PRRGQNR, is preferred over phosphorylase a (8-fold) by the PCSH1 phosphatase and is about a 40-fold and 95-fold-better substrate for the PCSH1 phosphatase than for the PCSM and PCSL phosphatases, respectively. The primary structure surrounding the S/T(P)P motif, by itself a strong negative determinant for dephosphorylation, can harbour positive features which relieve the constraint imposed by the carboxyl-terminal proline. Thus, the RB peptide INGS(P)PRT(P)PRRGQNR, in which the T(P)P configuration is preferred over the S(P)P sequence, is an extremely good and specific substrate for the PCSH1 phosphatase (Km = 10 microM, Vmax = 3882 nmol.min-1.mg-1). The AMDC phosphatase is a poor phosphatase for all the phosphopeptides tested, unless Mn2+ is added. Its histone H1 phosphatase activity is much less sensitive than its phosphorylase a and phosphopeptide phosphatase activity to inhibition by the modulator or inhibitor-1. The results strongly suggest a role for the trimeric PCSH1 phosphatase in reversing the p34cdc2 phosphorylations.