A computational analysis on the specificity of interactions between histidine kinases and response regulators

Bacteria process and transmit signals simultaneously through several two-component/phosphorelay networks using closely related proteins. Therefore discrimination against mismatches and discrete recognition between protein partners is an absolute requirement for producing the correct responses. We tried to address this issue by comparing and analyzing sequences from the helix-bundle regions of histidine kinases of Bacillus subtilis. Our analysis shows how conservation and variability in the sequences give rise to selective association and unique recognition. The observed pattern suggests that the chances for cross talk between non-partner proteins are extremely low, but cross talk could take place in special cases.     

Kinetic Buffering of Cross Talk between Bacterial Two-Component Sensors

Two-component systems are a class of sensors that enable bacteria to respond to environmental and cell-state signals. The canonical system consists of a membrane-bound sensor histidine kinase that autophosphorylates in response to a signal and transfers the phosphate to an intracellular response regulator. Bacteria typically have dozens of two-component systems. The key questions are whether these systems are linear and, if they are, how cross talk between systems is buffered. In this work, we studied the EnvZ/OmpR and CpxA/CpxR systems from Escherichia coli, which have been shown previously to exhibit slow cross talk in vitro. Using in vitro radiolabeling and a rapid quenched-flow apparatus, we experimentally measured 10 biochemical parameters capturing the cognate and non-cognate phosphotransfer reactions between the systems. These data were used to parameterize a mathematical model that was used to predict how cross talk is affected as different genes are knocked out. It was predicted that significant cross talk between EnvZ and CpxR only occurs for the triple mutant ΔompR ΔcpxA ΔactA-pta. All seven combinations of these knockouts were made to test this prediction and only the triple mutant demonstrated significant cross talk, where the cpxP promoter was induced 280-fold upon the activation of EnvZ. Furthermore, the behavior of the other knockouts agrees with the model predictions. These results support a kinetic model of buffering where both the cognate bifunctional phosphatase activity and the competition between regulator proteins for phosphate prevent cross talk in vivo.     

Molecular Modelling of Protein-Nucleic Acid Interactions

Abstract

Computer modeling techniques to study the interaction of proteins with nucleic acids are presented. The methods utilize information from genetic and chemical modification experiments and macromolecular structural constraints. These techniques, in addition to computer model building procedures and theoretical energy calculations, are illustrated for the study of the lac and cro repressor-operator systems. Our predicted interactions between lac and its operator agree with those recently reported for lac based upon sequence alignment with the cro repressor. Several molecular models of the putative helical segment of cro interacting with its OR3 operator are presented. These models are reflective of intermediate conformations experienced by the repressor in recognition of the operator sequence. The results of our studies are further discussed in terms of the design of short peptides interacting with nucleic acid sequences and the evolutionary requirements in establishing these repressor interactions.     

Membrane traffic and synaptic cross-talk during host cell entry byTrypanosoma cruzi

It is widely accepted that Trypanosoma cruzi can exploit the natural exocytic response of the host to cell damage, utilizing host cell lysosomes as important effectors. It is, though, increasingly clear that the parasite also exploits endocytic mechanisms which allow for incorporation of plasma membrane into the parasitophorous vacuole. Further, that these endocytic mechanisms are involved in cross‐talk with the exocytic machinery, in the recycling of vesicles and in the manipulation of the cytoskeleton. Here we review the mechanisms by which T. cruzi exploits features of the exocytic and endocytic pathways in epithelial and endothelial cells and the evidence for cross‐talk between these pathways.     

双向电泳和质谱技术分析樟芝无性孢子萌发相关蛋白

【目的】采用双向电泳(2DE)、质谱技术和实时荧光定量PCR(RT-q PCR)技术分析樟芝无性孢子萌发相关蛋白。【方法】分别提取培养0 h和24 h的樟芝孢子总蛋白并进行双向电泳分离,再用PDQuest软件进行差异蛋白分析,并用MALDI-TOF-MS技术对差异蛋白进行鉴定;其次将鉴定成功的蛋白与孢子萌发相关蛋白的本地数据库进行比对,获得樟芝中的孢子萌发相关蛋白信息,最后用RT-qPCR技术对相关基因的转录水平进行分析。【结果】两组样品共有32个差异蛋白点,其中在24 h表达量上调的蛋白25个,下调的蛋白7个。将32个差异蛋白点挖取鉴定,成功鉴定24个。其中,与孢子萌发相关的蛋白有10个,分别为GerO、Ubc1、Cat-1、Snf1、Cas2、SfaD、Chaperonin、Fad5、Tyrosine-P和ChiA。【结论】该研究结果为进一步解析樟芝无性孢子萌发的分子机制提供了理论依据。     

Cross‐compartment proteostasis regulation during redox imbalance induced ER stress

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle‐specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ‐based LC–MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross‐talk between ER‐ and mitochondrial proteostasis exemplified by an Ire1p‐dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross‐talk during stress.     

The Predicted Presence of Large Helical Structural Variation in Yeast HIS4 Upstream Region is Correlated with General Amino Acid Control on the CYC1 Gene

Abstract

A series of CYC1 constructions in which the upstream promoter portion has been replaced by a variety of HIS4 synthetic fragments has demonstrated that the 5′ TGACTC 3′ repeat is crucial for conferring amino acid general control. Efficient regulation, however, is obtained only with fragments containing both the repeat and flanking sequences. Analysis of the flanks shows the presence of a 16 nucleotide long sequence composed of alternations of two purines and two pyrimidines between the upstream and downstream repeats. Such a sequence has very large twist angle variations. Homologous sequences are observed in HIS1, HIS3, and in TRP5 upstream regions between copies of the repeat. Sequences which confer special structural characteristics may aid in protein recognition of the promoter region.     

Immunoproteomic analysis of Clostridium botulinum type B secretome for identification of immunogenic proteins against botulism

Objectives

To identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis.

Results

In the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome.

Conclusions

Identified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism.

     

Preliminary molecular analysis of Pelecanoides georgicus (Procellariiformes: Pelecanoididae) on Whenua Hou (Codfish Island): Implications for its taxonomic status

Abstract

Using sequences from the central and 3' regions of the mitchondrial small subunit (12S) ri‐bosomal RNA gene for six procellariiform species, this study confirms that the population of Pelecanoides georgicus on Whenua Hou (Codfish Island), New Zealand, is most closely related to P. georgicus from the southern Indian Ocean. The level of variation between sequences for this conserved gene suggests that the isolated Whenua Hou P. georgicus population split off from populations in the Indian Ocean several hundred thousand years ago. Although P. georgicus is globally numerous, the Whenua Hou population is endangered. As our preliminary sequence data suggest that the Whenua Hou population may have diverged from its parent population, it may deserve recognition in its own right.     

Talk among yourselves: RNA sponges mediate cross talk between functionally related messenger RNAs

When Francis Crick first proposed the central dogma, he predicted that genetic information flows from DNA to RNA and finally to proteins. By this classical concept, the sole purpose of mRNA is to serve as a template for translation. Recent work has expanded our understanding of the function of mRNA well beyond this singular definition. A paper published in this issue sheds more light on the myriad roles mRNAs can play in genetic regulation. Miyakoshi et al ( 2015a ) report an intriguing scenario in Salmonella where a small RNA molecule derived from a larger polycistronic mRNA promotes cross talk between physically unlinked mRNAs via controlling turnover of a global small RNA repressor.     

The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.     

The Escherichia coli metal-binding chaperone SlyD interacts with the large subunit of [NiFe]-hydrogenase 3

The multi-step biosynthesis of the [NiFe]-hydrogenase enzyme involves a variety of accessory proteins. To further understand this process, a Strep-tag II variant of the large subunit of Escherichia coli hydrogenase 3, HycE, was constructed to enable isolation of protein complexes. A complex with SlyD, a chaperone protein implicated in hydrogenase production through association with the nickel-binding accessory protein HypB, was observed. A SlyD–HycE interaction preceding both iron and nickel insertion to the enzyme was detected, mediated by the chaperone domain of SlyD, and independent of HypB. These results support a model of several roles for SlyD during hydrogenase maturation.

Structured summary

HycEphysically interacts with HypA, HypB and SlyD by cross linking study (view interaction)HycEphysically interacts with DnaK and GroEL by cross linking study (view interaction)HypBphysically interacts with SlyD by cross linking study (view interaction)HycEphysically interacts with SlyD by cross linking study (view interaction 1, 2)     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Comparative analyses of three legume species reveals conserved and unique root extracellular proteins

Increasing evidence suggests that root extracellular proteins are involved in interactions between roots and their soil environment. In the present study, exudates released by 6‐day‐old roots of the three legume species white lupin (Lupinus albus), soybean (Glycine max), and cowpea (Vigna sinensis) were collected under axenic conditions, and their constitutively secreted proteomes were analyzed. Between 42 and 93 unique root extracellular proteins with 2 or more different peptide fragments per protein were identified by LC‐MS/MS. Functional annotation of these proteins classified them into 14–16 different functional categories. Among those 14 homologous proteins were identified in at least two legume species. Among the unique proteins, 58 in white lupin, 85 in soybean, and 31 in cowpea were specific for each plant species, and many of them were classified in the same functional categories. Interestingly, in contrast to soybean and cowpea, two protein bands of approximately 16 and 30 kDa were present on the SDS‐PAGE gel of white lupin. The identification of these bands revealed a class III chitinase and a thaumatin‐like protein. Both belong to the class of pathogenesis‐related proteins. The results imply that root extracellular proteins play important roles in the cross‐talk between plant roots and the rhizosphere.     

NB‐3 signaling mediates the cross‐talk between post‐traumatic spinal axons and scar‐forming cells

Little is known about the molecules mediating the cross‐talk between post‐traumatic axons and scar‐forming cells after spinal cord injury. We found that a sustained NB‐3 induction was simultaneously present in the terminations of post‐traumatic corticospinal axons and scar‐forming cells at the spinal lesion site, where they were in direct contact when axons tried to penetrate the glial scar. The regrowth of corticospinal axons was enhanced in vivo with NB‐3 deficiency or interruption of NB‐3 trans‐homophilic interactions. Biochemical, in vitro and in vivo evidence demonstrated that NB‐3 homophilically interacted in trans to initiate a growth inhibitory signal transduction from scar‐forming cells to neurons by modulating mTOR activity via CHL1 and PTPσ. NB‐3 deficiency promoted BMS scores, electrophysiological transmission, and synapse reformation between regenerative axons and neurons. Our findings demonstrate that NB‐3 trans‐homophilic interactions mediate the cross‐talk between post‐traumatic axons and scar‐forming cells and impair the intrinsic growth ability of injured axons.     

Synthesis of Protected 8-Substituted Deoxyribonucleosides and Its Helix Stability in Oligodeoxyribonucleotides Containing the Eco RI Recognition Site

Abstract

Octadeoxyribonucleotides with the sequences d(GGA?ATTCC), d(GGAA?TTCC), and d(GG?AATTCC) have been prepared by solid phase synthesis using the H-phosphonate units containing modified base moieties. These oligomers which have an isosterically altered recognition sequence of the restriction endodeoxyribonuclease Eco RI. The oligomers, with replacement to deoxy-7,8-dihyroadenosine-8-one (dA^OH), 8-methoxydeoxyadenosine (dA_OMe) and 8-methoxydeoxyguanosine (dG^OMe) from deoxyadenosine or deoxyguanosine were used for studying recognition phenomena at the functional group level. From thermodyamic data of these alternating octamers it was shown that the oligomer containing 8-methoxydeoxyadenosine in the center of the recognition sequence destabilizes such duplexes less strongly than the oligomers containing other 8-substituted nucleosides in the 5′-side of the recognition sequences. Further, the hydrolysis by Eco RI of the modified oligomers perfectly resisted compared to d(GGAATTCC).     

Identification of efflux proteins using efficient radial basis function networks with position‐specific scoring matrices and biochemical properties

Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25–30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C‐terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78 and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using fivefold cross‐validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7 and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/～sachen/EFFLUXpredict/Efflux‐RBF.php . We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences.Proteins 2013. © 2013 Wiley Periodicals, Inc.