Combining evolutionary and structural information for local protein structure prediction

We study the effects of various factors in representing and combining evolutionary and structural information for local protein structural prediction based on fragment selection. We prepare databases of fragments from a set of non-redundant protein domains. For each fragment, evolutionary information is derived from homologous sequences and represented as estimated effective counts and frequencies of amino acids (evolutionary frequencies) at each position. Position-specific amino acid preferences called structural frequencies are derived from statistical analysis of discrete local structural environments in database structures. Our method for local structure prediction is based on ranking and selecting database fragments that are most similar to a target fragment. Using secondary structure type as a local structural property, we test our method in a number of settings. The major findings are: (1) the COMPASS-type scoring function for fragment similarity comparison gives better prediction accuracy than three other tested scoring functions for profile-profile comparison. We show that the COMPASS-type scoring function can be derived both in the probabilistic framework and in the framework of statistical potentials. (2) Using the evolutionary frequencies of database fragments gives better prediction accuracy than using structural frequencies. (3) Finer definition of local environments, such as including more side-chain solvent accessibility classes and considering the backbone conformations of neighboring residues, gives increasingly better prediction accuracy using structural frequencies. (4) Combining evolutionary and structural frequencies of database fragments, either in a linear fashion or using a pseudocount mixture formula, results in improvement of prediction accuracy. Combination at the log-odds score level is not as effective as combination at the frequency level. This suggests that there might be better ways of combining sequence and structural information than the commonly used linear combination of log-odds scores. Our method of fragment selection and frequency combination gives reasonable results of secondary structure prediction tested on 56 CASP5 targets (average SOV score 0.77), suggesting that it is a valid method for local protein structure prediction. Mixture of predicted structural frequencies and evolutionary frequencies improve the quality of local profile-to-profile alignment by COMPASS.     

Protein-Protein Interactions in the β-Oxidation Part of the Phenylacetate Utilization Pathway: CRYSTAL STRUCTURE OF THE PaaF-PaaG HYDRATASE-ISOMERASE COMPLEX*

Microbial anaerobic and so-called hybrid pathways for degradation of aromatic compounds contain β-oxidation-like steps. These reactions convert the product of the opening of the aromatic ring to common metabolites. The hybrid phenylacetate degradation pathway is encoded in Escherichia coli by the paa operon containing genes for 10 enzymes. Previously, we have analyzed protein-protein interactions among the enzymes catalyzing the initial oxidation steps in the paa pathway (Grishin, A. M., Ajamian, E., Tao, L., Zhang, L., Menard, R., and Cygler, M. (2011) J. Biol. Chem. 286, 10735–10743). Here we report characterization of interactions between the remaining enzymes of this pathway and show another stable complex, PaaFG, an enoyl-CoA hydratase and enoyl-Coa isomerase, both belonging to the crotonase superfamily. These steps are biochemically similar to the well studied fatty acid β-oxidation, which can be catalyzed by individual monofunctional enzymes, multifunctional enzymes comprising several domains, or enzymatic complexes such as the bacterial fatty acid β-oxidation complex. We have determined the structure of the PaaFG complex and determined that although individually PaaF and PaaG are similar to enzymes from the fatty acid β-oxidation pathway, the structure of the complex is dissimilar from bacterial fatty acid β-oxidation complexes. The PaaFG complex has a four-layered structure composed of homotrimeric discs of PaaF and PaaG. The active sites of PaaF and PaaG are adapted to accept the intermediary components of the Paa pathway, different from those of the fatty acid β-oxidation. The association of PaaF and PaaG into a stable complex might serve to speed up the steps of the pathway following the conversion of phenylacetyl-CoA to a toxic and unstable epoxide-CoA by PaaABCE monooxygenase.     

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.     

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications.     

Tetramerisation of alpha-latrotoxin by divalent cations is responsible for toxin-induced non-vesicular release and contributes to the Ca(2+)-dependent vesicular exocytosis from synaptosomes

A novel procedure of alpha-latrotoxin (alpha LTX) purification has been developed. Pure alpha LTX has been demonstrated to exist as a very stable homodimer. Such dimers further assemble into tetramers, and Ca(2+), Mg(2+) or higher toxin concentrations facilitate this process. However, when the venom is treated with EDTA, purified alpha LTX loses the ability to tetramerise spontaneously; the addition of Mg(2+) or Ca(2+) restores this ability. This suggests that alphaLTX has some intrinsically bound divalent cation(s) that normally support its tetramerisation. Single-particle cryoelectron microscopy and statistical image analysis have shown that: 1) the toxin has a non-compact, branching structure; 2) the alpha LTX dimers are asymmetric; and 3) the tetramers are symmetric and have a 25 A-diameter channel in the centre. Both alpha LTX oligomers bind to the same receptors in synaptosomes and rat brain sections. To study the effects of the dimers and tetramers on norepinephrine release from rat cerebrocortical synaptosomes, we used the EDTA-treated and untreated toxin preparations. The number of tetramers present in a preparation correlates with alpha LTX pore formation, suggesting that the tetramers are the pore-forming species of alpha LTX. The toxin actions mediated by the pore include: 1) Ca(2+) entry from the extracellular milieu; and 2) passive efflux of neurotransmitters via the pore that occurs independently of Ca(2+). The Ca(2+)-dependent alpha LTX-stimulated secretion conforms to all criteria of vesicular exocytosis but also depends upon intact intracellular Ca(2+) stores and functional phospholipase C (PLC). The Ca(2+)-dependent effect of the toxin is stronger when dimeric alpha LTX is used, indicating that higher receptor occupancy leads to its stronger activation, which contributes to stimulation of neuroexocytosis. In contrast, the Ca(2+)-independent release measured biochemically represents leakage of neurotransmitters through the toxin pore. These results are discussed in relation to the previously published observations.     

Presynaptic effects of arachidonic acid and prostaglandin E2 in the frog neuromuscular synapse

Arachidonic acid and prostaglandin E2 decreased the frequency of miniature endplate currents without changing their amplitude-temporary parameters. They also reduced the evoked transmitter release and the amplitude of the 3rd phase of nerve ending response corresponding to the voltage-dependent K(+)-current. Using perineural recording, It was shown that arachidonic acid and prostaglandin E2 decreased the Ca2+ currents of nerve endings. Indometacin: inhibitor of cyclooxygenase, enhanced the evoked transmitter release and decreased the 3rd phase of nerve ending response. Indometacin prevented the effects of arachidonic acid on evoked transmitter release, whereas the effects of arachidonic acid on the 3rd phase was preserved. Prostaglandin E2 seems to mediate the effects of arachidonic acid on spontaneous and evoked transmitter release, Ca(2+)- and Ca(2+)-activated K(+)-currents. Moreover, the arachidonic acid and prostaglandin E2 exerted their own effects upon voltage-dependent potassium current of motor nerve ending.     

Purinergic Membrane Receptors as Targets for the Effect of Purotoxin 1, a Component of Venom of Spiders from the <Emphasis Type="Italic">Geolycosa</Emphasis> Genus

We examined effects of purotoxin 1 (PT1), a component of the venom of Geolycosa spiders, on a few voltageand ligand-operated ion channels present in the plasma membrane of sensory neurons from the rat dorsal root ganglia (DRGs). Purotoxin 1 in a 100 nM concentration evoked no changes in ion currents through voltage-operated sodium, potassium, and calcium channels in the membranes of isolated sensory neurons. This agent was also found to be ineffective with respect to capsaicin-sensitive receptor-channel complexes (TRPV1). Testing of the effects of PT1 on purinergic receptor-channel complexes P2X3, P2X2, and P2X2/3 showed that this toxin is a highly selective blocker of exclusively P2X3 receptors. The selectivity of action of PT1 demonstrated in our experiments shows that it is a unique agent, which opens up new prospects in the studies of structural/functional peculiarities of receptor-channel complexes P2X3 as a peripheral link of the nociception system.     

The ABC transporters in Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram‐negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter‐related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In‐depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid‐like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

COMPASS: a tool for comparison of multiple protein alignments with assessment of statistical significance

We present a novel method for the comparison of multiple protein alignments with assessment of statistical significance (COMPASS). The method derives numerical profiles from alignments, constructs optimal local profile-profile alignments and analytically estimates E-values for the detected similarities. The scoring system and E-value calculation are based on a generalization of the PSI-BLAST approach to profile-sequence comparison, which is adapted for the profile-profile case. Tested along with existing methods for profile-sequence (PSI-BLAST) and profile-profile (prof_sim) comparison, COMPASS shows increased abilities for sensitive and selective detection of remote sequence similarities, as well as improved quality of local alignments. The method allows prediction of relationships between protein families in the PFAM database beyond the range of conventional methods. Two predicted relations with high significance are similarities between various Rossmann-type folds and between various helix-turn-helix-containing families. The potential value of COMPASS for structure/function predictions is illustrated by the detection of an intricate homology between the DNA-binding domain of the CTF/NFI family and the MH1 domain of the Smad family.     

Alterations in the creatine kinase system in the myocardium of cardiomyopathic hamsters

Immunochemical and biochemical methods were used to assess quantitatively the changes in the heart creatine kinase system in the myopathic Syrian hamsters, line CHF I46. Cardiomyopathy in I75-200 day old animals was characterized by decreased content of mitochondria and lower total creatine kinase activity. In isolated mitochondria only the creatine kinase activity was decreased while cytochromes aa3 content and respiration rate were unchanged. The share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to I8% and that of BB form was elevated from 5% in control to 20%, at unchanged relative level of MM. Immunoassay showed decreased amount of the mitochondrial creatine kinase in the tissue and its decreased ratio to cytochromes aa3. The results show altered expression of creatine kinase isoenzymes in cardiomyopathy.