The DING family of proteins: ubiquitous in eukaryotes,but where are the genes?

PstS and DING proteins are members of a superfamily of secreted, high‐affinity phosphate‐binding proteins. Whereas microbial PstS have a well‐defined role in phosphate ABC transporters, the physiological function of DING proteins, named after their DINGGG N termini, still needs to be determined. PstS and DING proteins co‐exist in some Pseudomonas strains, to which they confer a highly adhesive and virulent phenotype. More than 30 DING proteins have now been purified, mostly from eukaryotes. They are often associated with infections or with dysregulation of cell proliferation. Consequently, eukaryotic DING proteins could also be involved in cell–cell communication or adherence. The ubiquitous presence in eukaryotes of proteins structurally and functionally related to bacterial virulence factors is intriguing, as is the absence of eukaryotic genes encoding DING proteins in databases. DING proteins in eukaryotes could originate from unidentified commensal or symbiotic bacteria and could contribute to essential functions. Alternatively, DING proteins could be encoded by eukaryotic genes sharing special features that prevent their cloning. Both hypotheses are discussed.     

大肠杆菌基因中密码子前后碱基的使用与蛋白质结构

对一组E.coli基因中编码蛋白质各类二级结构（α-螺旋、β－折叠片、无规卷曲和回折）的密码子前后碱基的使用情况进行统计分析和比较,发现一些密码子前后碱基的使用有偏向,而且这些偏向与蛋白质的二级结构有关联,这同时亦表明,E.coli基因中同义密码子的选用与蛋白质的二级结构有一些关联。模型对于蛋白质结构预测算法的改进以及基因工程的研究有辅助作用。     

Compositional variation in bacterial genes and proteins with potential expression level

Usage of guanine and cytosine at three codon sites in eubacterial genes vary distinctly with potential expressivity, as predicted by Codon Adaptation Index (CAI). In bacteria with moderate/high GC-content, G(3) follows a biphasic relationship, while C(3) increases with CAI. In AT-rich bacteria, correlation of CAI is negative with G(3), but non-specific with C(3). Correlations of CAI with residues encoded by G-starting codons are positive, while with those by C-starting codons are usually negative/random. Average Size/Complexity Score and aromaticity of gene-products decrease with CAI, confirming general validity of cost-minimization principle in free-living eubacteria. Alcoholicity of bacterial gene-products usually decreases with expressivity.     

Cloning and characterization of a novel periplasmic heme-transport protein from the human pathogen <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Successful iron acquisition plays a crucial role in bacterial virulence. Numerous Gram-negative pathogenic bacteria have developed a novel heme-acquisition system to steal iron from hosts. This system involves a cell-surface heme receptor, a periplasmic heme-transport protein (HTP) and inner-membrane proteins typical for ATP binding cassette transporters. We have cloned the gene encoding a periplasmic HTP from Pseudomonas aeruginosa, overexpressed it in Escherichia coli and purified it as a 33-kDa His-tagged protein. Heme-staining and heme-content assays reveal that the isolated HTP contains approximately 50% heme-bound and apo forms. The heme is noncovalently attached and can be transferred to apomyoglobin in vitro. Electron paramagnetic resonance and UV-vis spectroscopies indicate a five-coordinate, high-spin, ferric heme in HTP. HTP is reduced by dithionite but not by either dithiothreitol or ascorbate. Fluorescence and circular dichroism spectroscopies indicate a well-ordered structure for the HTP and a conformational change upon heme binding to apo-HTP. This was confirmed by limited proteolysis assays. Apo-HTP binds heme or protoporphyrin IX at 1:1 ratio with high affinity (K (d) approximately 1.2 and 14 nM, respectively). A BLASTP search revealed approximately 52 putative bacterial periplasmic heme transporters, which can be grouped into six classes, most of which are associated with pathogenic bacteria. Multiple sequence alignment reveals that these HTPs share low sequence similarity and no conserved common binding motif for heme ligation. However, a tyrosine residue (Y71) is highly conserved in the HTP sequences, which is likely an axial heme ligand in HTPs. Mutagenesis studies support Y71-heme iron ligation in the recombinant HTP.     

Birth, life and death of nascent polypeptide chains

The journey of nascent polypeptides from synthesis at the peptidyl transferase center of the ribosome ("birth") to full function ("maturity") involves multiple interactions, constraints, modifications and folding events. Each step of this journey impacts the ultimate expression level and functional capacity of the translated protein. It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, and that this provides an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. Multiple quality control systems ensure that proteins achieve their native, functional form. Unproductive co-translational folding intermediates that arise during protein synthesis may undergo enhanced interaction with components of these systems, such as chaperones, and/or be subjects of co-translational degradation ("death"). This review provides an overview of our current understanding of the complex co-translational events that accompany the synthesis, maturation, folding and degradation of nascent polypeptide chains.     

铜绿假单胞菌分泌性蛋白的抗肿瘤效应

目的 探讨3株不同来源的铜绿假单胞菌分泌性蛋白的抗肿瘤活性。方法 将3株不同来源的铜绿假单胞菌经LB培养基对其过夜静置培养,离心获得上清液,采用硫酸铵盐析沉淀蛋白质,再经PBS透析除盐。然后将蛋白作用于人肝癌细胞Hep-G2、宫颈癌细胞HeLa、肺癌细胞A549和人永生化表皮细胞HaCaT,CCK-8检测其对细胞的毒性作用,吉姆萨染色观察凋亡细胞形态变化。结果 SDS-PAGE证实成功获得不同分子量的分泌蛋白,CCK-8结果显示混合蛋白对3种不同肿瘤细胞的生长均有不同程度的抑制作用,且其抑制作用存在一定的时间和浓度依赖性,但对人正常细胞无明显抑制作用。吉姆萨染色初步显示细胞形态为凋亡状态。结论 初步证实该3株铜绿假单胞菌产生的分泌性蛋白具有不同程度的抗肿瘤作用,为进一步分离纯化具有抗肿瘤活性的单一蛋白质及研究其抗肿瘤机制提供依据。     

Periplasmic proteins of Escherichia coli are highly resistant to aggregation: reappraisal for roles of molecular chaperones in periplasm

Periplasmic proteins of Gram-negative bacteria like Escherichia coli are subjected to immediate affect of environmental fluctuation that may unfold proteins, due to the permeability of the outer membrane to small molecules. They are thus supposedly protected by certain molecular chaperones. Nevertheless, no homologues of typical molecular chaperones have so far been found in periplasm, and the recently reported chaperone activities of periplasmic protein disulfide isomerase (PDI) and peptidyl prolyl isomerase (PPI) seem to be too weak to satisfy such assumed needs. In an attempt to reveal whether periplasmic proteins exhibit certain unusual properties, we discovered that such proteins as a whole are highly resistant to aggregation under a wide variety of denaturing conditions. Furthermore, in an effort to unveil the nature behind this phenomenon we purified and examined four prominent periplasmic proteins. Our results demonstrate that these proteins unfold at rather mild denaturing conditions and expose hydrophobic surfaces during such unfolding process, but hardly form complexes with a typical molecular chaperone. Based on these observations, we propose that the periplasmic proteins have been evolved to resist the formation of aggregates when subjected to various denaturing conditions and molecular chaperones may thus not be needed in periplasm.     

Analysis of the codon use frequency of AMPK family genes from different species

In mammals, AMP-activated protein kinase (AMPK) is involved in the regulation of cellular energy homeostasis and, on the whole animal level, in regulating energy balance and food intake. In this paper, the relative synonymous codon use frequency of 40 AMPK family genes from seven mammal species (Bos taurus, Homo sapiens, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Sus scrofa) were analyzed using correspondence analysis and hierarchical cluster method. The result suggests that gene function is the dominant factor that determines codon usage bias in AMPK family genes, while species is a minor factor that determines further difference in codon usage bias for genes with similar functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message^{for review see 1}. However, it''s now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates¹. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others². Codon bias is organism as well as tissue specific^2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs⁴. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes^5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein folding^{for review see 9}. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding^1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems^6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.     

Multiple precursor proteins bind individual Tat receptor complexes and are collectively transported

The thylakoid t win a rginine protein t ranslocation (Tat) system is thought to have a multivalent receptor complex with each cpTatC‐Hcf106 pair constituting a signal peptide‐binding unit. Conceptual models suggest that translocation of individual precursor proteins occurs upon assembly of a Tha4 oligomer with a precursor‐occupied cpTatC‐Hcf106. However, results reported here reveal that multiple precursor proteins bound to a single receptor complex can be transported together. Precursor proteins that contain one or two cysteine residues readily formed intermolecular disulphide bonds upon binding to the receptor complex, resulting in dimeric and tetrameric precursor proteins. Three lines of evidence indicate that all members of precursor oligomers were specifically bound to a receptor unit. Blue native–polyacrylamide gel electrophoresis analysis showed that oligomers were present on individual receptor complexes rather than bridging two or more receptor complexes. Upon energizing the membrane, the dimeric and tetrameric precursors were transported across the membrane with efficiencies comparable with that of monomeric precursors. These results imply a novel aspect of Tat systems, whereby multiple precursor‐binding sites can act in concert to transport an interlinked oligo‐precursor protein.     

Channel-forming properties and structural homology of major outer membrane proteins from Pseudomonas fluorescens MFO and OE 28.3

Abstract The major outer membrane proteins (OprF) from Pseudomonas fluorescens MFO and OE 28.3 were purified by a new method involving native electrophoresis in octyl-polyoxyethylene media. Both proteins, characterized by the same size, heat-modifiability and N-terminal sequence were re-incorporated in virtually solvent-free planar lipid bilayers. They displayed very similar channel-forming properties: the major conductance level was between 250 pS and 270 pS in l M NaCl. From experiments of zero-current potential, both porins were determined weakly cation selective. Amplification by PCR and sequencing of the oprF gene of strain MFO allowed to point out 94% identity between the amino acid sequences of these two OprFs isolated from ecological niches as different as milk (strain MFO) and soil (strain OE 28.3).     

Statistical analysis of pentose phosphate pathway genes from eubacteria and eukarya reveals translational selection as a major force in shaping codon usage pattern

Comparative analysis of metabolic pathways among widely diverse species provides an excellent opportunity to extract information about the functional relation of organisms and pentose phosphate pathway exemplifies one such pathway. A comparative codon usage analysis of the pentose phosphate pathway genes of a diverse group of organisms representing different niches and the related factors affecting codon usage with special reference to the major forces influencing codon usage patterns was carried out. It was observed that organism specific codon usage bias percolates into vital metabolic pathway genes irrespective of their near universality. A clear distinction in the codon usage pattern of gram positive and gram negative bacteria, which is a major classification criterion for bacteria, in terms of pentose phosphate pathway was an important observation of this study. The codon utilization scheme in all the organisms indicates the presence of translation selection as a major force in shaping codon usage. Another key observation was the segregation of the H. sapiens genes as a separate cluster by correspondence analysis, which is primarily attributed to the different codon usage pattern in this genus along with its longer gene lengths. We have also analyzed the amino acid distribution comparison of transketolase protein primary structures among all the organisms and found that there is a certain degree of predictability in the composition profile except in A. fumigatus and H. sapiens, where few exceptions are prominent. In A. fumigatus, a human pathogen responsible for invasive aspergillosis, a significantly different codon usage pattern, which finally translated into its amino acid composition model portraying a unique profile in a key pentose phosphate pathway enzyme transketolase was observed.     

Pseudomonas aeruginosa internalization by corneal epithelial cells involves MEK and ERK signal transduction proteins

Invasion of epithelial cells represents a potential pathogenic mechanism for Pseudomonas aeruginosa. We explored the role of mitogen-activated protein kinase kinases (MEK 1/2) and the extracellular signal-regulated kinases (ERK 1/2) in P. aeruginosa invasion. Treatment of corneal epithelial cells with MEK inhibitors, PD98059 (20 microM) or UO126 (100 microM), reduced P. aeruginosa invasion by approximately 60% without affecting bacterial association with the cells (P=0.0001). UO124, a negative control for UO126, had no effect on bacterial internalization. Infection of cells with an internalization-defective flhA mutant of P. aeruginosa was associated with less ERK 1/2 tyrosine phosphorylation than infection with wild-type invasive P. aeruginosa. An ERK-2 inhibitor, 5-iodotubercidin (20 microM), reduced P. aeruginosa invasion by approximately 40% (P=0.035). Together, these data suggest that P. aeruginosa internalization by epithelial cells involves a pathway(s) that includes MEK and ERK signaling proteins.     

Crystal structure at high resolution of ferric-pyochelin and its membrane receptor FptA from Pseudomonas aeruginosa

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. We describe at 2.0 A resolution the crystal structure of the pyochelin outer membrane receptor FptA bound to the iron-pyochelin isolated from Pseudomonas aeruginosa. One pyochelin molecule bound to iron is found in the protein structure, providing the first three-dimensional structure at the atomic level of this siderophore. The pyochelin molecule provides a tetra-dentate coordination of iron, while the remaining bi-dentate coordination is ensured by another molecule not specifically recognized by the protein. The overall structure of the pyochelin receptor is typical of the TonB-dependent transporter superfamily, which uses the proton motive force from the cytoplasmic membrane through the TonB-ExbB-ExbD energy transducing complex to transport ferric ions across the bacterial outer membrane: a transmembrane 22 beta-stranded barrel occluded by a N-terminal domain that contains a mixed four-stranded beta-sheet. The N-terminal TonB box is disordered in two crystal forms, and loop L8 is found to point towards the iron-pyochelin complex, suggesting that the receptor is in a transport-competent conformation.     

Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas

Two cytochromes c₅ (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c₅₅₁ (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.     

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.     

Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa

In Gram-negative bacteria, most of the sec-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of 12 Xcp proteins (XcpA and XcpP to XcpZ) organized as a multiproteic complex within the envelope. In this study, by a co-purification approach using a His-tagged XcpZ as a bait, XcpY and XcpZ were found associated together to constitute the most stable functional unit so far isolated from the P. aeruginosa secreton. This subcomplex was also found to interact with XcpR and XcpS to form a XcpRSYZ complex which was isolated under native conditions. Another component, XcpP was not found to be associated to the complex but the results suggest that it can transiently interact with the XcpYZ subcomplex in vivo.     

Endoplasmic reticulum-resident proteins are constitutively transported to vacuoles for degradation

Soluble endoplasmic reticulum (ER)-resident proteins have very long lives because of their ER residency. This residency depends largely on ER-retrieval signals at their C-terminus. We examined the long-term destiny of endogenous ER-resident proteins, a lumenal binding protein (BiP) and a protein disulfide isomerase (PDI), with cultured cells of Arabidopsis. ER residents, in contrast to vacuolar proteinases, were considerably degraded in cells at the stationary phase. A subcellular fractionation analysis suggested that ER residents were transported into the vacuoles, which accumulated the residents lacking the ER-retrieval signals. We showed that the PDI located in the vacuoles had high mannose glycans, but not complex glycans, which suggested that the ER resident was transported to the vacuoles independent of the medial/trans-Golgi complex. To visualize the pathway of transport of ER-resident proteins, tobacco BY-2 cells were transformed with a chimeric gene encoding an ER-targeted green fluorescent protein (30 kDa GFP-HDEL). In the transformed cells at the stationary phase, GFP fluorescence was observed in the vacuoles. A subcellular fractionation revealed that a trimmed form of 27 kDa GFP was localized in the vacuoles. Treatment with E-64d, an inhibitor of papain-type cysteine proteinases that inhibits the degradation of GFP in the vacuoles, resulted in a stable accumulation of 27 kDa GFP in the vacuoles, even in the logarithmic phase. Our results suggest that endogenous ER residents are transported constitutively to the vacuoles by bypassing the Golgi complex and are then degraded.