Supra-domains: evolutionary units larger than single protein domains

Domains are the evolutionary units that comprise proteins, and most proteins are built from more than one domain. Domains can be shuffled by recombination to create proteins with new arrangements of domains. Using structural domain assignments, we examined the combinations of domains in the proteins of 131 completely sequenced organisms. We found two-domain and three-domain combinations that recur in different protein contexts with different partner domains. The domains within these combinations have a particular functional and spatial relationship. These units are larger than individual domains and we term them "supra-domains". Amongst the supra-domains, we identified some 1400 (1203 two-domain and 166 three-domain) combinations that are statistically significantly over-represented relative to the occurrence and versatility of the individual component domains. Over one-third of all structurally assigned multi-domain proteins contain these over-represented supra-domains. This means that investigation of the structural and functional relationships of the domains forming these popular combinations would be particularly useful for an understanding of multi-domain protein function and evolution as well as for genome annotation. These and other supra-domains were analysed for their versatility, duplication, their distribution across the three kingdoms of life and their functional classes. By examining the three-dimensional structures of several examples of supra-domains in different biological processes, we identify two basic types of spatial relationships between the component domains: the combined function of the two domains is such that either the geometry of the two domains is crucial and there is a tight constraint on the interface, or the precise orientation of the domains is less important and they are spatially separate. Frequently, the role of the supra-domain becomes clear only once the three-dimensional structure is known. Since this is the case for only a quarter of the supra-domains, we provide a list of the most important unknown supra-domains as potential targets for structural genomics projects.     

Genome-wide survey and genetic characteristics of Ophichthus evermanni based on Illumina sequencing platform

Ophichthidae fishes limit to continental shelf of all tropical and subtropical oceans and contain more than 350 species, representing the greatest specialization diversity in the order Anguiliformes. In the present study, we conducted a genome survey sequencing (GSS) analysis of Ophichthus evermanni by Illumina sequencing platform to briefly reveal its genomic characteristics and phylogenetic relationship. The first de novo assembled 1.97 Gb draft genome of O. evermanni was predicted based on K-mer analysis without obvious nucleotide bias. The heterozygosity ratio was 0.70%, and the sequence repeat ratio was calculated to be 43.30%. A total of 9016 putative coding genes were successfully predicted, in which 3587 unigenes were identified by gene ontology (GO) analysis and 4375 unigenes were classified into cluster of orthologous groups for enkaryotic complete genomes (KOG) functional categories. About 2,812,813 microsatellite motifs including mono-, di-, tri-, tetra-, penta- and hexanucleotide motifs were identified, with an occurrence frequency of 23.32%. The most abundant type was dinucleotide repeat motifs, accounting for 49.19% of the total repeat types. The mitochondrial genome, as a byproduct of GSS, was assembled to investigate the evolutionary relationships between O. evermanni and its relatives. Bayesian inference (BI) phylogenetic tree inferring from concatenated 12 protein-coding genes (PCGs) showed complicated relationships among Ophichthidae species, indicating a polyphyletic origin of the family. The results would achieve more thorough genetic information of snake eels and provide a theoretical basis and reference for further genome-wide analysis of O. evermanni.     

Genome-wide survey of tyrosine phosphatases in thirty mammalian genomes

The age of genomics has given us a wealth of information and the tools to study whole genomes. This, in turn, has facilitated genome-wide studies among organisms that were relatively less studied in the pre-genomic era or are non-model organisms. This paves the way to the discovery of interesting evolutionary patterns, which are brought to light by genome-wide surveys of protein superfamilies. Phosphorylation is a post-translational modification that is utilised across all clades of life, and acts as an important signalling switch, regulating several cellular processes. Tyrosine phosphatases, which are found predominantly in eukaryotes, act on phosphorylated tyrosine residues and sometimes on other substrates. Extending on our previous effort to look for tyrosine phosphatases in the human genome, we have looked for sequences of the cysteine-based tyrosine phosphatase superfamily in thirty mammalian genomes from all across Mammalia and validated the sequences with the presence of the signature catalytic motif. Domain architecture annotation, followed by in-depth analysis, revealed interesting taxon-specific patterns such as subtle differences between the protein families in marsupials and early mammals versus placental mammals. Finally, we discuss an interesting case of loss of the tyrosine phosphatase domain from a gene product in the course of eutherian evolution.     

Proteome-wide functional classification and identification of prokaryotic transmembrane proteins by transmembrane topology similarity comparison

We propose a new method for classifying and identifying transmembrane (TM) protein functions in proteome-scale by applying a single-linkage clustering method based on TM topology similarity, which is calculated simply from comparing the lengths of loop regions. In this study, we focused on 87 prokaryotic TM proteomes consisting of 31 proteobacteria, 22 gram-positive bacteria, 19 other bacteria, and 15 archaea. Prior to performing the clustering, we first categorized individual TM protein sequences as "known," "putative" (similar to "known" sequences), or "unknown" by using the homology search and the sequence similarity comparison against SWISS-PROT to assess the current status of the functional annotation of the TM proteomes based on sequence similarity only. More than three-quarters, that is, 75.7% of the TM protein sequences are functionally "unknown," with only 3.8% and 20.5% of them being classified as "known" and "putative," respectively. Using our clustering approach based on TM topology similarity, we succeeded in increasing the rate of TM protein sequences functionally classified and identified from 24.3% to 60.9%. Obtained clusters correspond well to functional superfamilies or families, and the functional classification and identification are successfully achieved by this approach. For example, in an obtained cluster of TM proteins with six TM segments, 109 sequences out of 119 sequences annotated as "ATP-binding cassette transporter" are properly included and 122 "unknown" sequences are also contained.     

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases

Tau is a neuronal microtubule-associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site-specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.     

丝氨酸/苏氨酸蛋白磷酸酶与DNA损伤应答

DNA损伤应答(DNA damage response,DDR)是维持基因组稳定性的核心机制,对DDR的研究不仅有助于阐明癌症发生发展的机理,同时也为癌症治疗和抗癌新药开发提供生物学基础。蛋白质翻译后修饰,尤其是蛋白激酶介导的磷酸化修饰和蛋白磷酸酶介导的去磷酸化修饰,参与调控绝大多数的生命活动过程,包括DDR。对蛋白激酶ATM/ATR/CHK2/CHK1介导的DDR的研究已经比较透彻,但是对蛋白磷酸酶在DDR中的功能研究还有待加强和深入。比较全面地综述丝氨酸/苏氨酸蛋白磷酸酶在DDR中的功能并探讨在抗癌新药开发中的前景。     

基因组范围的蛋白质功能研究方法初探

基因组大规模测序的进展使人们获得了大量新基因数据,对这些数据进行基因组范围的规模化功能分析的新方法应运而生.对其中的结构域融合分析法、系统进化特征法、簇分析法,结构分析法,插入突变法和综合分析法做一简要介绍和初步探讨.     

Survey for g-proteins in the prokaryotic genomes: prediction of functional roles based on classification

The members of the family of G-proteins are characterized by their ability to bind and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). Despite a common biochemical function of GTP hydrolysis shared among the members of the family of G-proteins, they are associated with diverse biological roles. The current work describes the identification and detailed analysis of the putative G-proteins encoded in the completely sequenced prokaryotic genomes. Inferences on the biological roles of these G-proteins have been obtained by their classification into known functional subfamilies. We have identified 497 G-proteins in 42 genomes. Seven small GTP-binding protein homologues have been identified in prokaryotes with at least two of the diagnostic sequence motifs of G-proteins conserved. The translation factors have the largest representation (234 sequences) and are found to be ubiquitous, which is consistent with their critical role in protein synthesis. The GTP_OBG subfamily comprises of 79 sequences in our dataset. A total of 177 sequences belong to the subfamily of GTPase of unknown function and 154 of these could be associated with domains of known functions such as cell cycle regulation and t-RNA modification. The large GTP-binding proteins and the alpha-subunit of heterotrimeric G-proteins are not detected in the genomes of the prokaryotes surveyed.     

水稻ECT基因家族的全基因组研究

真核生物mRNA转录后修饰可调控许多基因的遗传信息,植物m⁶A甲基化研究正成为关注的新热点。m⁶A结合蛋白 (m⁶A readers) 调节m⁶A修饰的特异性,通常具有YTH (YT521-B homology) 结构域,在拟南芥中被命名为ECT结构域 (evolutionarily conserved C-terminal region ECT domain) 。目前ECT基因已在拟南芥和水稻等植物中检测到,但该基因家族在水稻中的成员及生物学功能还缺乏研究。本研究通过水稻ECT基因家族的全基因组分析,鉴定出12个OsECT基因,具有1个保守的基序,多位于蛋白质氨基酸序列C-端。共线性分析表明,在水稻基因组内OsECT-c与OsECT-e发生了重复事件,在物种间ECT同源基因对可能是在双子叶和单子叶植物分化后形成。同源基因对OsECT的Ka/Ks < 1,表明OsECT基因家族在进化过程中可能经历了较强的纯化选择压力。表达模式分析显示,OsECT-b、OsECT-c、OsECT-e和OsECT-j在水稻生长初期各个组织均保持较高的表达水平,OsECT-g在干旱处理后表达量显著下调。因此,OsECT基因在水稻生长发育和逆境胁迫中可能发挥着重要作用。本研究为今后OsECT基因在水稻的节水抗旱机制研究和相关抗逆育种提供了重要的理论基础。     

Expression of protein phosphatases during postnatal development of rabbit heart

Protein phosphatases play a major role in the regulation of L-type calcium current (I_Ca) in heart cells. We previously showed developmental differences in the effects of inhibitors of protein phosphatases (PP's) on the modulation of I_Ca, with greater stimulatory effects on I_Ca observed in newborn than in adult ventricular cells. We hypothesized that this developmental difference might be due to greater expression and levels of PP 1 and PP 2A in newborn than in adult ventricular cells. We thus determined the mRNA expression of and subunits of PP 1 and the subunit of PP 2A in adult and newborn rabbit ventricles and levels of PP 1 and PP 2A in total homogenates, particulate membranes, and in soluble fraction prepared from isolated ventricular myocytes from adult and newborn rabbits. RT-PCR analysis demonstrated the presence of mRNA of these subunits of PP's in both newborn and adult ventricles. Northern blot analysis using ³²P labeled cDNA probes specific for PP 1, PP 1 and PP 2A showed that the expression of steady state mRNA levels for PP 1, PP 1 and PP 2A were much higher in newborn compared to adult rabbit ventricles. mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) in rabbit ventricles were measured as controls. GAPDH did not show significant developmental changes while mRNA for SERCA was higher in adult compared to newborns. Western blot analysis showed that PP 1 and PP 2A protein levels were also much higher in newborn compared to adult rabbit ventricular cells. Immunoblot analysis in particulate membranes and soluble fraction showed that PP1 was mainly membrane bound while PP 2 was present only in soluble fraction. These findings suggest that the two major protein phosphatases (PP 1 and PP 2A) in heart are expressed at much higher levels in newborn and decline to lower levels in adult ventricular myocytes. The presence of high levels of PP's and particularly PP 1 in newborn cells may be responsible for the greater dependence of newborn cells on the inhibition of PP as a mechanism of action of -agonist isoproterenol on I_Ca.     

Arabidopsis genome analysis as exemplified by analysis of chromosome 4

During the last decade the small cruciferous plant Arabidopsis thaliana has become a model organism for flowering plants. Sequencing and analysis of the Arabidopsis genome is nearing completion. Beside an overview on methods and strategies for Arabidopsis genome analysis, a summary of the results from the first analysis is presented.This includes an overview on chromosomal organisation and topological features as well as a first comparison with other genomes.     

利用蛋白质序列模式识别改善谷氨酸棒杆菌基因组注释

即使细菌基因组的基因结构较为简单,但在注释过程中也可能出现基因遗漏的现象。当潜在基因在高质量数据库中没有显著同源序列时,基于知识库的基因预测方法就会遇到困难。本文希望通过系统扫描基因组所有可能ORF的蛋白质序列模式来搜索遗漏基因。为验证该方法的可行性,作者系统分析了重要的工业发酵微生物谷氨酸棒杆菌的基因组,发现了25个候选疑似基因。它们具有显著的蛋白质序列模式,但在Swiss-Prot中元显著同源序列,并且在GenBank中仍未注释。深入分析发现,25个候选疑似基因中19个为可能基因,3个为可能假基因,3个为疑似基因序列。这些结果说明本文的分析方法可以有效地用于无显著同源序列基因的搜索。     

Incorporating functional annotation information in prioritizing disease associated SNPs from genome wide association studies

With recent advances in genotyping and sequencing technologies,many disease susceptibility loci have been identified.However,much of the genetic heritability remains unexplained and the replication rate between independent studies is still low.Meanwhile,there have been increasing efforts on functional annotations of the entire human genome,such as the Encyclopedia of DNA Elements(ENCODE)project and other similar projects.It has been shown that incorporating these functional annotations to prioritize genome wide association signals may help identify true association signals.However,to our knowledge,the extent of the improvement when functional annotation data are considered has not been studied in the literature.In this article,we propose a statistical framework to estimate the improvement in replication rate with annotation data,and apply it to Crohn’s disease and DNase I hypersensitive sites.The results show that with cell line specific functional annotations,the expected replication rate is improved,but only at modest level.     

Insights from the cDNA and EST analysis of Antrodia cinnamomea

It is of interest to document the insights gleaned from the cDNA and EST analysis of Antrodia cinnamomea (a fungal species). Hence a library of sequences was constructed and analysed using standard procedures to gain new insights. Therefore, 65 ESTs, with size ranging from 300-2000 bp, were constructed. This included 46 ESTs with definite annotation, 18 ESTs were hypothetical and 1 new protein derived from BLAST analysis. We assigned 227 Gene Ontology terms linked to cell composition, transport, catalytic activity, and regulation functions in these sequences. Moreover, 56 matching genes were found in 8 Kyoto Encyclopedia of Genes and Genomes pathways. Data also showed 271 SSRs from Antrodia cinnamomea ESTs with an occurrence frequency of 96.82%. The STRING data analysis showed 29 genes encoded enzymes highly involved in protein-to-protein interactions linked to expression of regulation function. Thus, we documented some insights from the cDNA and EST analysis of Antrodia cinnamomea for further data mining.     

Approaches to the molecular cloning of protein-tyrosine phosphatases in insulin-sensitive tissues

The intrinsic tyrosyl kinase activity of the insulin receptor is regulated by a balance between insulin-induced receptor autophosphorylation, which stimulates the receptor kinase, and enzymatic dephosphorylation of the receptor, which deactivates its kinase activity. The cellular protein-tyrosine phosphatase (PTPase) enzymes responsible for reversing the activated state of the insulin receptor have not been characterized. Our laboratory is interested in identifying and cloning the specific PTPase(s) that regulate the phosphorylation state of the insulin receptor. This chapter will summarize the design and results of our initial molecular cloning studies to identify specific PTPases in insulin-sensitive tissues that may have a potential physiological role in insulin action and clinical insulin resistance.     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

Genome-wide biochemical analysis of Arabidopsis protein phosphatase using a wheat cell-free system

Plant genome possesses over 100 protein phosphatase (PPase) genes that are key regulators of signal transduction via phosphorylation/dephosphorylation event. Here we report a comprehensive functional analysis of protein serine/threonine, dual-specificity and tyrosine phosphatases using recombinant PPases produced by wheat cell-free protein synthesis system. Eighty-two recombinant PPases were successfully produced using Arabidopsis full-length cDNA as templates. In vitro PPase assay was performed using phosphorylated myelin basic protein as substrate. Among the AtPPases examined, 26 serine/threonine, three dual-specificity and one tyrosine PPases exhibited catalytic activity, including 20 serine/threonine and one dual-specificity PPases that showed in vitro activities for the first time. Our study demonstrates genome-wide biochemical analysis of AtPPases using wheat cell-free system, and provides new information and insights on enzyme activities.

Structured summary of protein interactions

PTP1dephosphorylatesMBP by phosphatase assay (View interaction).AtPP2CdephosphorylatesMBP by phosphatase assay (View interaction).POLTEdephosphorylatesMBP by phosphatase assay (View interaction).TOPP8dephosphorylatesMBP by phosphatase assay (View interaction).HAB1dephosphorylatesMBP by phosphatase assay (View interaction).ABI2dephosphorylatesMBP by phosphatase assay (View interaction).At1g34750dephosphorylatesMBP by phosphatase assay (View interaction).At1g43900dephosphorylatesMBP by phosphatase assay (View interaction).At3g15260dephosphorylatesMBP by phosphatase assay (View interaction).At5g53140dephosphorylatesMBP by phosphatase assay (View interaction).At1g18030dephosphorylatesMBP by phosphatase assay (View interaction).At3g06270dephosphorylatesMBP by phosphatase assay (View interaction).At2g25070dephosphorylatesMBP by phosphatase assay (View interaction).At3g02750dephosphorylatesMBP by phosphatase assay (View interaction).At5g10740dephosphorylatesMBP by phosphatase assay (View interaction).at4g26080dephosphorylatesMBP by phosphatase assay (View interaction).At4g28400dephosphorylatesMBP by phosphatase assay (View interaction).At5g06750dephosphorylatesMBP by phosphatase assay (View interaction).At4g31860dephosphorylatesMBP by phosphatase assay (View interaction).At3g17250dephosphorylatesMBP by phosphatase assay (View interaction).At4g38520dephosphorylatesMBP by phosphatase assay (View interaction).At3g05640dephosphorylatesMBP by phosphatase assay (View interaction).At5g66080dephosphorylatesMBP by phosphatase assay (View interaction).At1g79630dephosphorylatesMBP by phosphatase assay (View interaction).At2g30170dephosphorylatesMBP by phosphatase assay (View interaction).At5g24940dephosphorylatesMBP by phosphatase assay (View interaction).     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.