Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

螺旋藻丝氨酸/苏氨酸激酶基因家族的克隆与表达分析

螺旋藻（Spirulina)，是丝状不形成异型胞的蓝藻，由于其所蕴涵的高品质营养成分而成为一属具有重要经济价值的微藻。丝氨酸/苏氨酸激酶（serine/threonine kinases，STK）系统在螺旋藻的信号转导中发挥了重要作用。螺旋藻生活环境复杂，其中温度是中国北方温带地区螺旋藻大规模养殖的主要限制性因素。本文克隆了一条螺旋藻STK基因，并初步从STK激酶基因家族的角度探索了螺旋藻对高温环境的适应。主要结果如下： 1. 构建了插入片段为1-2kb的螺旋藻基因组文库。基于该文库用简并引物PCR的方法得到了一条螺旋藻STK基因5'端长度为504bp的基因组序列。 2．以本实验室构建的螺旋藻基因组草图为基础，在基因组水平上研究了螺旋藻STK基因家族的特点。发现螺旋藻草图中STK基因家族共包含33个基因，这些基因基于蛋白结构域分析可以分为三大类群。序列保守性分析发现，螺旋藻STK激酶具有与真核STK激酶相同的保守域，此外螺旋藻STK激酶还具有特有的保守氨基酸。 3．在中国北方温带地区，螺旋藻大规模养殖的主要限制性因素是温度。由于北方地区温度较低，螺旋藻规模养殖普遍存在年养殖期较短的问题，造成设备浪费，若采用加温措施又增加养殖成本；而在夏季晴天中午，又存在高温胁迫的问题。跨膜蛋白是信号转导的第一阶段，因此我们设置了不同温度来诱导螺旋藻，对其中7个跨膜STK基因进行半定量RT-PCR分析，结果显示STK2103在不同温度下表达量不同，推测该蛋白参与了螺旋藻温度相关的信号转导过程。 本文将基础研究与实验验证相结合，为螺旋藻的信号转导研究提供了线索。

两种蓝藻真核型Ser/Thr激酶基因功能的初步验证

Ser/Thr蛋白激酶（serine/threonine kinases，STK）在真核生物的信号转导通路中具有重要作用，而且已经成为对抗肿瘤、结核等多种人类疾病的药物作用靶点。上世纪九十年代，有研究发现STK在原核生物的信号转导中也发挥重要作用。本论文以聚球藻PCC7942（Synechococcus sp. PCC7942）和钝顶螺旋藻（Spirulina platensis）为材料，对几个真核型的Ser/Thr蛋白激酶基因的功能进行了初步验证。 蓝藻兼具细菌和植物的特点，具有成熟的转化体系，为真核生物基因功能的研究提供了新的模式宿主。聚球藻PCC7942是一种单细胞的淡水蓝藻，具有天然的外源DNA转化系统，是蓝藻分子遗传学研究的模式生物。通过基因敲除及表达差异分析发现聚球藻PCC7942中的Ser/Thr蛋白激酶stk196参与高温胁迫的信号传递。钝顶螺旋藻是原核丝状蓝藻，由于其蕴涵高品质营养成分而成为一类具有重要经济价值的微藻，该研究利用半定量RT-PCR方法，分析四个具有跨膜结构域的Ser/Thr蛋白激酶在正常生长温度下和经低温、高温诱导后表达量的变化情况，发现stk2103在低温诱导后的表达量降低，高温诱导后的表达量升高，提示该基因的表达可能参与了钝顶螺旋藻对温度的适应。 蓝藻中真核型Ser/Thr蛋白激酶功能的研究为我们进一步研究真核生物的Ser/Thr蛋白激酶功能提供了借鉴，并对植物抗逆胁迫的研究提供重要的理论依据。

Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio) by using the combinative methods of suppressive subtraction hybridization, SMART cDNA synthesis and RACE-PCR. The full-length cDNA is 1870 bp. Its 5 ' untranslated region is 210 bp, followed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of ANNATG. The open reading frame encodes a protein with 329 amino acids. It has 670 bp of 3 ' untranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but not in previtellogenic oocytes (phase I oocytes). RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary, spleen and testis, but not in liver. And its mRNA could be detected in the oocytes from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart, kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtellogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription.

Involvement of p90 ribosomal S6 kinase in termination of cell cycle arrest during development of Artemia-encysted embryos

Artemia has evolved a unique developmental pattern of encysted embryos to cope with various environmental threats. Cell divisions totally cease during the preemergence developmental stage from gastrula to prenauplius. The molecular mechanism of this, however, remains unknown. Our study focuses on the involvement of p90 ribosomal S6 kinase (RSK), a family of serine/threonine kinase-mediating signal transduction downstream of mitogen-activated protein kinase cascades, in the termination of cell cycle arrest during the post-embryonic development of Artemia-encysted gastrula. With immunochemistry, morphology, and cell cycle analysis, the identified Artemia RSK was established to be specifically activated during the post-embryonic and early larval developmental stages when arrested cells of encysted embryos resumed mitoses. In vivo knockdown of RSK activity by RNA interference, kinase inhibition, and antibody neutralization consistently induced defective larvae with distinct gaps between the exoskeleton and internal tissues. In these abnormal individuals, mitoses were detected to be largely inhibited in the affected regions. These results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G(2)/M phase) arrest and promotion of mitogenesis. Our findings may, thus, provide insights into the regulation of cell division during Artemia post-embryonic development and reveal further aspects of RSK functions.

A colorimetric assay for screening microcystin class compounds in aquatic systems

Secondary metabolites produced by water-blooming cyanobacteria in eutrophic waters include some potent hepatotoxins, These compounds also have tumour-promoting properties, attributable to their inhibition and activation of protein phosphatases and kinases respectively. The inhibitory effect of these toxins on protein phosphatases have been employed in a commonly used radiometric assay, involving the use of a P-32-labeled substrate, for the detection and quantitation of these compounds. This paper investigates and describes a colorimetric method in which the activity of protein phosphatase 2A is determined by measuring the rate of colour production from the release of yellow p-nitrophenol using p-nitrophenyl phosphate as the substrate. Results of this study suggest that the colorimetric protein phosphatase inhibition assay is a simple, inexpensive tool for screening substances that may have tumour-promoting characteristics in aquatic systems. The detection limit of the colorimetric method is comparable to the radiometric assay. (C) 1998 Elsevier Science Ltd. All rights reserved.

Differential gene expression of protein kinases in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp

A modified mRNA differential display method has been applied to studying differential expression of protein kinase genes in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp. Total RNA was reverse transcribed using downstream 3' primers T(12)MA, T(12)MG and T12MC respectively. Then the reverse transcription products were amplified using upstream 5' kinase-specific primer designed according to protein kinase conserved sequence. The PCR products had different patterns and numbers of: cDNA bands on polyacrylamide:gel. Totally 21 cDNAs fragments were recovered and cloned. Two of them were confirmed to be particularly expressed in oocytes of amphimictic crucian carp, and another was specific for gynogenetic silver crucian carp.

Robustness and dissipation of mitogen-activated protein kinases signal transduction network: Underlying funneled landscape against stochastic fluctuations

We uncovered the underlying energy landscape of the mitogen-activated protein kinases signal transduction cellular network by exploring the statistical natures of the Brownian dynamical trajectories. We introduce a dimensionless quantity: The robustness ratio of energy gap versus local roughness to measure the global topography of the underlying landscape. A high robustness ratio implies funneled landscape. The landscape is quite robust against environmental fluctuations and variants of the intrinsic chemical reaction rates.

An anticoagulant serine protease from the wasp venom of Vespa magnifica

Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin. contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X. (c) 2008 Elsevier Ltd. All rights reserved.

A serine proteinase inhibitor from frog eggs with bacteriostatic activity

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K-i) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs. (C) 2007 Elsevier Inc. All rights reserved.

An inhibitor of c-Jun N-terminal kinases (CEP-11004) counteracts the anti-HIV-1 action of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating protein possessing multiple biological and pharmacological activities. One of its major actions is inhibition of human immunodeficiency virus (HIV) replication. The mechanism is still not clear. It is

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2 alpha kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2 alpha kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2 alpha kinases might play similar roles and that PoPKR is the predominant kinase. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G(4)

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH similar to 32 aa similar to E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has <50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.