Molecular tools to support metabolic and immune function research in the Guinea Fowl (Numida meleagris).

BACKGROUND: Guinea fowl (Numidia meleagris) production as an alternative source of meat and poultry has shown potential for economic viability. However, there has been little progress in characterizing the transcriptome of the guinea fowl. In this study RNA-sequencing and de novo transcriptome assembly of several Guinea fowl tissues (pancreas, hypothalamus, liver, bone marrow and bursa) which play key roles in regulating feed intake, satiety, and immune function was performed using Illumina's Hi-Seq 2000. RESULTS: 74 million sequences were generated and assembled into 96,492 contigs using the Trinity software suite. Over 39,000 of these transcripts were found to have in silico translated protein sequences that are homologous to chicken protein sequences. Gene ontology analysis uncovered 416 transcripts with metabolic functions and 703 with immune function. CONCLUSION: The transcriptome information presented here will support the development of molecular approaches to improve production efficiency of the guinea fowl and other avian species.

The Class D beta-lactamase family: residues governing the maintenance and diversity of function.

Class D ß-lactamases, a major source of bacterial resistance to ß-lactam antibiotic therapies, represent a distinct subset of the ß-lactamase superfamily. They share a serine hydrolase mechanism with Classes A/C vs. Class B. Further understanding of their sequence-structure-function relationships would benefit efforts to design a new generation of antibiotics as well as to predict evolutionary mechanisms in response to such therapies. Here we describe analyses based on our high-resolution multiple sequence alignment and phylogenetic tree of ∼80 Class D ß-lactamases that leverage several 3D structures of these enzymes. We observe several sequence clusters on the phylogenetic tree, some that are species specific while others include several species from α-, ß- and γ-proteobacteria. Residues characteristic of a specific cluster were identified and shown to be located just outside the active site, possibly modulating the function of the catalytic residues to facilitate reactions with specific types of ß-lactams. Most significant was the discovery of a likely disulfide bond in a large group composed of α-, ß- and γ-proteobacteria that would contribute to enzyme stability and hence bacterial viability under antibiotic assault. A network of co-evolving residues was identified which suggested the importance of maintaining a surface for binding a highly conserved Phe69.

A Mechanism for Evolving Novel Plant Sesquiterpene Synthase Function.

Plant sesquiterpene synthases, a subset of the terpene synthase superfamily, are a mechanistically diverse family of enzymes capable of synthesizing hundreds of complex compounds with high regio- and stereospecificity and are of biological importance due to their role in plant defense mechanisms. In the current report we describe a large-scale, high-resolution phylogenetic analysis of ∼200 plant sesquiterpene synthases integrated with structural and experimental data that address these issues. We observe that all sequences that cluster together on the phylogenetic tree into well-defined groups share at least the first reaction in the catalytic mechanism subsequent to the initial ionization step and many share steps beyond this down to proton transfers between the enzyme and substrate. Most significant is the previously unreported high conservation of an Asp-Tyr-Asp triad. Due to its high conservation, patterns in the phylogenetic tree as well as experimental and modeling results, we suggest that this Asp-Tyr-Asp triad is an important functional element responsible for many proton transfers to and from the substrate and intermediates along the plant sesquiterpene synthase catalytic cycle and whose position can be tuned by residues outside the active site that can lead to the evolution of novel enzyme function.

MPI-PHYLIP: parallelizing computationally intensive phylogenetic analysis routines for the analysis of large protein families.

BACKGROUND: Phylogenetic study of protein sequences provides unique and valuable insights into the molecular and genetic basis of important medical and epidemiological problems as well as insights about the origins and development of physiological features in present day organisms. Consensus phylogenies based on the bootstrap and other resampling methods play a crucial part in analyzing the robustness of the trees produced for these analyses. METHODOLOGY: Our focus was to increase the number of bootstrap replications that can be performed on large protein datasets using the maximum parsimony, distance matrix, and maximum likelihood methods. We have modified the PHYLIP package using MPI to enable large-scale phylogenetic study of protein sequences, using a statistically robust number of bootstrapped datasets, to be performed in a moderate amount of time. This paper discusses the methodology used to parallelize the PHYLIP programs and reports the performance of the parallel PHYLIP programs that are relevant to the study of protein evolution on several protein datasets. CONCLUSIONS: Calculations that currently take a few days on a state of the art desktop workstation are reduced to calculations that can be performed over lunchtime on a modern parallel computer. Of the three protein methods tested, the maximum likelihood method scales the best, followed by the distance method, and then the maximum parsimony method. However, the maximum likelihood method requires significant memory resources, which limits its application to more moderately sized protein datasets.

Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease.

Post-translational sulfation of tyrosines affects the affinity and binding of at least some chemokine receptors to their ligand(s) and has been hypothesized to be a feature in all chemokine receptors. This binding initiates downstream signaling cascades. By this mechanism, tyrosine sulfation can influence the cells involved in acute and chronic events of cellular immunity. These events include leukocyte trafficking and airway inflammation important in asthma and chronic obstructive pulmonary disease (COPD). We are using computational methods to convert the poorly defined hypothesis of more widespread sulfation of chemokine receptors to more specific assessments of how closely the sequence environment of each tyrosine residue resembles the sequence environment of tyrosine residues proven to be sulfated. Thus, we provide specific and readily tested hypotheses about the tyrosine residues in all of the chemokine receptors. Tyrosine sulfation was predicted with high scores in the N-terminus domain of 13 out of 18 human chemokine receptor proteins using a position-specific scoring matrix, which was determined to be 94.2% accurate based on Receiver Operating Characteristic analysis. The remaining chemokine receptors have sites exhibiting features of tyrosine sulfation. These putative sites demonstrate clustering in a manner consistent with known tyrosine sulfation sites and conservation both within the chemokine receptor family and across mammalian species. Human chemokine receptors important in asthma and COPD, such as CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR4, CCR5, and CCR8, contain at least one known or predicted tyrosine sulfation site. Recognition that tyrosine sulfation is found in most clinically relevant chemokine receptors could help the development of specific receptor-ligand antagonists to modulate events important in airway diseases.

Crucial role of position 40 for interactions of CCK-58 revealed by sequence of cat CCK-58.

Evidence suggests that amino terminal extensions of CCK-8 affect the carboxyl terminal bioactive region of CCK. Cat CCK-58 was purified by low pressure reverse phase and ion-exchange chromatography steps and several reverse phase HPLC steps. The purified peptide and its tryptic fragments were characterized by mass spectral analysis and microsequence analysis. The structure of cat CCK-58 is: AVQKVDGEPRAHLGALLARYIQQARKAPSGRMSVIKNLQSLDPSHRISDRDY(SO3) MGWMDF-amide. Cat and dog CCK-58 are identical except for position 40 which is serine in cat and asparagine in dog. Radioimmunoassay detected cat CCK-58 about 1/10th as well as dog CCK-58, indicating a marked effect on C-terminal immunoreactivity. Cat CCK-58 with a serine at position 40, the same residue found in pig, mouse, cow and rabbit CCK-58, can be used as a unique bioprobe for defining how amino terminal amino acids influence the structure and bioactivity of the carboxyl terminal region of CCK.

Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation.

Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near-attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM-Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM-GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step-wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process.

Mathematically complete nucleotide and protein sequence searching using Ssearch.

In this unit a protocol is described for predicting the structure of simple transmembrane a-helical bundles. The protocol is based on a global molecular dynamics search (GMDS) of the configuration space of the helical bundle, yielding several candidate structures. The correct structure among these candidates is selected using information from silent amino acid substitutions, employing the premise that only the correct structure must (by definition) accept all of the silent amino acid substitutions. Thus, the correct structure is found by repeating the GMDS for several close homologs and selecting the structure that persists in all of the trials.

Prediction of tyrosine sulfation sites in animal viruses.

Post-translational modification of proteins by tyrosine sulfation enhances the affinity of extracellular ligand-receptor interactions important in the immune response and other biological processes in animals. For example, sulfated tyrosines in polyomavirus and varicella-zoster virus may help modulate host cell recognition and facilitate viral attachment and entry. Using a Position-Specific-Scoring-Matrix with an accuracy of 96.43%, we analyzed the possibility of tyrosine sulfation in all 1517 animal viruses available in the Swiss-Prot database. From a total of 97,729 tyrosines, we predicted 5091 sulfated tyrosine sites from 1024 viruses. Our site predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus strongly suggest an important link between tyrosine sulfation and viral disease mechanisms. In each of these three viral proteins, we observed highly conserved amino acid sequences surrounding predicted sulfated tyrosine sites. Tyrosine sulfation appears to be much more common in animal viruses than is currently recognized.

An algorithm for identification and ranking of family-specific residues, applied to the ALDH3 family.

An algorithm for detecting amino acid residues characteristic of individual protein families from within aligned collections of paralogous sequences, and its application to the ALDH3 family versus the rest of the ALDH extended family is described. Residues illuminated by this analysis include a key intramolecular tether, a lysine that makes an intersubunit contact at the dimer interface, three residues in close association with the substrate-binding funnel, and a pair of residues suggested to participate in proton relay during the catalytic cycle.

Initial catalytic events in class 3 aldehyde dehydrogenase: MM and QM/MM simulations.

A novel enzyme mechanism has been predicted by computer simulations for formation of the thiohemiacetal intermediate in the rat ALDH3A1 enzyme. We used molecular mechanics simulations to study the atomic details of substrate binding and quantum mechanical/molecular mechanical methods to study the Cys-243 thiolate attack on benzaldehyde (BA) substrate. BA was found to produce more reactive conformers when aligned for formation of the tetrahedral thiohemiacetal in the R-configuration. In addition, the sulfhydryl proton was seen to be important for initial binding of the substrate. Finally, the free energy differences between forming a thiohemiacetal oxyanion intermediate versus forming a neutral thiohemiacetal intermediate where a proton is donated to the intermediate from the surroundings strongly favor the latter. Our results suggest that the proton donor is the amide proton from the Cys-243 backbone supported by interactions with Lys-235.

Strategies for multiple sequence alignment.

We present an overview of multiple sequence alignments to outline the practical consequences for the choices among different techniques and parameters. We begin with a discussion of the scoring methods for quantifying the quality of a multiple sequence alignment, followed by a discussion of the algorithms implemented within a variety of multiple sequence alignment programs. We also discuss additional alignment details such as gap penalty and distance metrics. The paper concludes with a discussion on how to improve alignment quality and the limitations of the techniques described in this paper

Prediction of tyrosine sulfation in seven-transmembrane peptide receptors.

Posttranslational modification by tyrosine sulfation regulates many important protein protein interactions and modulates the binding affinity and specificity of seventransmembrane peptide receptors. We developed a log-odds position-specific-scoring-matrix (PSSM) to accurately predict tyrosine sulfation using 62 tyrosine sites known to be sulfated and 421 tyrosine sites known not to be sulfated. We predict that 49 tyrosines of 32 seven-transmembrane peptide receptors are sulfated. Although we did not incorporate characteristics of confirmed sulfation sites such as clustering and conservation across species into our PSSM, our predicted sites nevertheless exhibited these characteristics. The observed conservation suggests that there are strong evolutionary pressures to preserve selected biological activity of seven-transmembrane receptors. The predicted tyrosine sulfation sites predominantly occur in the extracellular tail and extracellular loop 2, regions consistent with their association with binding pockets of the receptor.