Functional Annotation of Conserved Hypothetical Proteins from Haemophilus influenzae Rd KW20

Haemophilus influenzae is a Gram negative bacterium that belongs to the family Pasteurellaceae, causes bacteremia, pneumonia and acute bacterial meningitis in infants. The emergence of multi-drug resistance H. influenzae strain in clinical isolates demands the development of better/new drugs against this pathogen. Our study combines a number of bioinformatics tools for function predictions of previously not assigned proteins in the genome of H. influenzae. This genome was extensively analyzed and found 1,657 functional proteins in which function of 429 proteins are unknown, termed as hypothetical proteins (HPs). Amino acid sequences of all 429 HPs were extensively annotated and we successfully assigned the function to 296 HPs with high confidence. We also characterized the function of 124 HPs precisely, but with less confidence. We believed that sequence of a protein can be used as a framework to explain known functional properties. Here we have combined the latest versions of protein family databases, protein motifs, intrinsic features from the amino acid sequence, pathway and genome context methods to assign a precise function to hypothetical proteins for which no experimental information is available. We found these HPs belong to various classes of proteins such as enzymes, transporters, carriers, receptors, signal transducers, binding proteins, virulence and other proteins. The outcome of this work will be helpful for a better understanding of the mechanism of pathogenesis and in finding novel therapeutic targets for H. influenzae.     

集成改进KNN算法预测蛋白质亚细胞定位

基于Adaboost算法对多个相似性比对K最近邻(K-nearest neighbor,KNN)分类器集成实现蛋白质的亚细胞定位预测。相似性比对KNN算法分别以氨基酸组成、二肽、伪氨基酸组成为蛋白序列特征,在KNN的决策阶段使用Blast比对决定蛋白质的亚细胞定位。在Jackknife检验下,Adaboost集成分类算法提取3种蛋白序列特征,3种特征在数据集CH317和Gram1253的最高预测成功率分别为92.4%和93.1%。结果表明Adaboost集成改进KNN分类预测方法是一种有效的蛋白质亚细胞定位预测方法。     

Cysteine-rich secretory protein 3 inhibits hepatitis C virus at the initial phase of infection

Hepatitis C virus (HCV) affects 2–3% of the global population. Approximately one-quarter of acute infections cause chronic hepatitis that leads to liver cirrhosis or hepatocellular carcinoma. The major obstacle of current research is the extremely narrow host tropism of HCV. A single HCV strain can replicate in the Huh7 human hepatoma cell line. Huh7 cells can be adapted under selective pressure in vitro to identify host factors that influence viral replication. Here, we extended this strategy to the in vivo condition and generated a series of cell lines by multiple rounds of adaptation in immunocompromised mice. Adaptation increased the cellular resistance to HCV infection. Microarray analyses revealed that the expression levels of several genes were associated with HCV resistance. Notably, up-regulation of the mRNA encoding cysteine-rich secretory protein 3 (CRISP3), a glycoprotein with unknown function that is secreted from multiple exocrine glands, was correlated with HCV resistance. The presence of CRISP3 in the culture medium limited HCV replication at the early phase of infection.     

Molecular cloning and sequence analysis of prion protein gene in Xiji donkey in China

Prion diseases are a group of human and animal neurodegenerative disorders caused by the deposition of an abnormal isoform prion protein (PrP^Sc) encoded by a single copy prion protein gene (PRNP). Prion disease has been reported in many herbivores but not in Equus and the species barrier might be playing a role in resistance of these species to the disease. Therefore, analysis of genotype of prion protein (PrP) in these species may help understand the transmission of the disease. Xiji donkey is a rare species of Equus not widely reared in Ningxia, China, for service, food and medicine, but its PRNP has not been studied. Based on the reported PrP sequence in GenBank we designed primers and amplified, cloned and sequenced the PRNP of Xiji donkey. The sequence analysis showed that the Xiji donkey PRNP was consisted of an open reading frame of 768 nucleotides encoding 256 amino acids. Amino acid residues unique to donkey as compared with some Equus animals, mink, cow, sheep, human, dog, sika deer, rabbit and hamster were identified. The results showed that the amino acid sequence of Xiji donkey PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to the PrP of other Equus species in this study. Amino acid sequence analysis showed high identity within species and close relation to the PRNP of sika deer, sheep, dog, camel, cow, mink, rabbit and hamster with 83.1–99.7% identity. The results provided the PRNP data for an additional Equus species, which should be useful to the study of the prion disease pathogenesis, resistance and cross species transmission.     

High-level heterologous expression of fungal cytochrome P450s in Escherichia coli

A thorough understanding of the sequence–structure–function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5′ end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.     

in silico analyses of metabolic pathway and protein interaction network for identification of next gen therapeutic targets in Chlamydophila pneumoniae

Chlamydophila pneumoniae, the causative agent of chronic obstructive pulmonary disease (COPD), is presently the fifth mortality causing chronic disease in the world. The understanding of disease and treatment options are limited represents a severe concern and a need for better therapeutics. With the advancements in the field of complete genome sequencing and computational approaches development have lead to metabolic pathway analysis and protein-protein interaction network which provides vital evidence to the protein function and has been appropriate to the fields such as systems biology and drug discovery. Protein interaction network analysis allows us to predict the most potential drug targets among large number of the non-homologous proteins involved in the unique metabolic pathway. A computational comparative metabolic pathway analysis of the host H. sapiens and the pathogen C pneumoniae AR39 has been carried out at three level analyses. Firstly, metabolic pathway analysis was performed to identify unique metabolic pathways and non-homologous proteins were identified. Secondly, essentiality of the proteins was checked, where these proteins contribute to the growth and survival of the organism. Finally these proteins were further subjected to predict protein interaction networks. Among the total 65 pathways in the C pneumoniae AR39 genome 10 were identified as the unique metabolic pathways which were not found in the human host, 32 enzymes were predicted as essential and these proteins were considered for protein interaction analysis, later using various criteria''s we have narrowed down to prioritize ribonucleotide-diphosphate reductase subunit beta as a potential drug target which facilitate for the successful entry into drug designing.     

Novel Ca/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca²⁺/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca²⁺/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.     

Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms

Background

Polyunsaturated fatty acids (PUFAs) have antifungal properties, but the mode by which they induce their action is not always clear. The aim of the study was to investigate apoptosis as a mode of action of antifungal PUFAs (stearidonic acid, eicosapentaenoic acid and docosapentaenoic acid) which are inhibitory towards biofilm formation of C. albicans and C. dubliniensis.

Methods

Candida biofilms were grown in the absence or presence of 1 mM PUFAs (linoleic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid) for 48 h at 37 °C. The effect of these PUFAs on the membrane fatty acid profile and unsaturation index, oxidative stress, mitochondrial transmembrane potential and apoptosis was evaluated.

Results

When biofilms of C. albicans and C. dubliniensis were exposed to certain PUFAs there was an increase in unsaturation index of the cellular membranes and accumulation of intracellular reactive oxygen species (ROS). This resulted in apoptosis, evidenced by reduced mitochondrial membrane potential and nuclear condensation and fragmentation. The most effective PUFA was stearidonic acid.

Conclusions

The resultant cell death of both C. albicans and C. dubliniensis is due to apoptosis.

General significance

Due to the increase in drug resistance, alternative antifungal drugs are needed. A group of natural antifungal compounds is PUFAs. However, understanding their mechanisms of action is important for further use and development of these compounds as antifungal drugs. This paper provides insight into a possible mode of action of antifungal PUFAs.     

丙型肝炎病毒蛋白作用于细胞信号转导途径的研究进展

细胞信号转导异常往往与人类疾病的发生、发展密切相关。一些病毒致病和感染机制即为病毒抗原蛋白作用宿主细胞信号转导途径,导致宿主细胞内信号转导发生紊乱。丙型肝炎病毒（HCV）是引发慢性丙型肝炎,导致肝硬化和肝细胞癌发生的主要病原体,但目前HCV的致病机制与宿主内持续感染机制尚不清楚。HCV致病机制可能与HCV表达的蛋白质干扰宿主细胞信号转导途径而导致异常的细胞信号转导有关。研究HCV蛋白对宿主细胞信号转导途径的影响不仅有助于阐明其致病机制,还能为新药设计和寻找新的治疗方法提供新思路和新靶点。本文主要综述了近年来国内外有关HCV蛋白作用细胞信号转导途径的研究进展。     

The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.     

Analysis of expressed sequence tags from oil palm (Elaeis guineensis)

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm.     

从新冠病毒起源到BLAST工具的正确实践

基本局部比对搜索工具(basic local alignment search tool,BLAST)是核酸或蛋白质序列相似性分析最常用的工具之一.因为程序涉及参数较多,一些学生和研究者有时不根据实际情况也不阅读说明书就直接选择默认参数,可能会得出错误结论.BLAST可以进行核酸、蛋白质序列及其相互间的比对,一些低年级...     

Cloning,characterization, and expression analysis of acyl–acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis)

Acyl–acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl–ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61–73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75 days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30a vector and transformed into Escherichia coli BL21(DE3)△ FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~ 40.5 kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl–ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.     

Insight from γC1 protein model for implication in cotton leaf curl disease

DNA γ is approximately half of the size of Begomovirus DNA. It encodes a γC1 gene that is conserved in position and size. This gene has the capacity to encode a 13 to 14 kDa protein comprising 118 amino acid residues. It has been shown earlier that γC1 protein is necessary for inducing symptoms of cotton leaf curl disease. The structure for γC1 (CLCuDγ01-Pakistan) is still unknown. Therefore, a model of γC1 (CLCuDγ01-Pakistan) was developed using DoBo and I-TASSER servers followed by validation by PROCHECK and VERIFY 3D servers. The developed model provides an insight in a role for this multifunctional protein in causing Cotton Leaf Curl Disease (CLCuD). A possible function of this protein might be the suppression of RNAsilencing in cotton plants.     

Minority of mammalian orthologs can be regarded as physiologically closest genes

Orthologs are genes from different genomes that originate from a common ancestor gene by speciation event. They are most similar by the structure of encoded proteins and therefore should have a similar function. Here I apply the principle used for detection of structural orthology for a genome-wide analysis of gene expression. For this purpose, I determine the mutual similarity rank in all-by-all comparison of among-tissues expression patterns. The expression of most part of human–mouse orthologs in homologous tissues is poorly correlated (average mutual coexpression rank is only 4835 out of 18,092). Genes from evolutionarily labile gene families, which experience rapid turnover of family composition, are among those with the strongest expression change. However, the revealed phenomenon is not limited to them. There is no or very weak relationship between protein sequence divergence and mutual coexpression rank. Also, generally there is no relationship between the ratio of nonsynonymous to synonymous nucleotide substitutions and coexpression rank. This relationship is tangible only within evolutionarily labile gene families. These results indicate that despite of a similar biochemical function of orthologs reflected in the conserved protein sequence, the physiological (systemic) context of this function can be changed. Also, these results suggest that gene biochemical function and its physiological role in the organism can evolve independently.