Structural characterization of intermolecular self-association in a T-cell specific kinase

This dissertation structurally examines the intermolecular self-association and quaternary structure of interleukin-2 tyrosine kinase (Itk) and draws conclusions about its relationship to the regulation and signaling of this immunologically important protein. Found primarily in haematopoietic cells, Itk is a member of the Tec family, the second largest family of non-receptor tyrosine kinases. All Tec family members share an SH3, SH2, and catalytic kinase domain structure. The activation of Itk occurs following T-cell receptor response to antigen stimulation. The exact mechanism of regulation in Tec family kinases is unclear. However, intermolecular self-association is emerging as a common characteristic among many members of the Tec family. To explore the potentially functionally significant self-association in Itk, high-resolution NMR solution structures were solved for the Itk SH3 domain, the Itk SH2 domain, and the Itk SH3/SH2 complex. The non-classical interaction between the Itk SH3 and SH2 domains mediates, in part, the self-association of full length Itk. The structure of the SH3/SH2 complex provides insight on how isomerization of a proline imide bond acts as an intrinsic molecular switch that preorganizes the CD loop of the SH2 domain for a non-classical interaction with the SH3 domain. Additionally, the oligomeric state of Itk self-association is characterized and the SH3/SH2 domain complex is used as a starting point to generate a structural model of the Itk SH3-SH2 fragment self-association that accounts for the oligomerization seen in native gel analysis. The same SH3/SH2 interaction is mutually exclusive with a quaternary structural rearrangement that supports autophosphorylation. Therefore, a structural model is described for Itk autophosphorylation that was generated using a previous point mutational analysis of the Itk SH2 domain coupled with covalent bond restraints found in the linkers of the SH3-SH2-kinase fragment. These studies bring us closer to understanding the structural mechanism behind Itk self-association and describe a model for one of many quaternary structural conformations in which Itk is likely to exist

Murine Itk SH3 domain

Interleukin-2 tyrosine kinase (Itk) is a non-receptor tyrosine kinase of the Tec family that is activated upon antigen binding to the T cell receptor (Schwartzberg et al. 2005; Berg 2007). Itk is comprised of four regulatory domains: PH (Pleckstrin homology), TH (Tec homology), SH3, SH2 and the catalytic kinase domain. SH3 domains share a common fold consisting of five anti-parallel β strands that form a β barrel and bind canonical proline rich ligands as well as a variety of noncanonical ligands (Agrawal and Kishan 2002)

Comprehensive Analysis of Non Redundant Protein Database

Background: Scientists around the world use NCBI’s non-redundant (NR) database to identify the taxonomic origin and functional annotation of their favorite protein sequences using BLAST. Unfortunately, due to the exponential growth of this database, many scientists do not have a good understanding of the contents of the NR database. There is a need for tools to explore the contents of large biological datasets, such as NR, to better understand the assumptions and limitations of the data they contain. Results: Protein sequence data, protein functional annotation, and taxonomic assignment from NCBI’s NR database were placed into a BoaG database, a domain-specific language and shared data science infrastructure for genomics, along with a CD-HIT clustering of all these protein sequences at different sequence similarity levels. We show that BoaG can efficiently perform queries on this large dataset to determine the average length of protein sequences and identify the most common taxonomic assignments and functional annotations. Using the clustering information, we also show that the non-redundant (NR) database has a considerable amount of annotation redundancy at the 95% similarity level. Conclusions: We implemented BoaG and provided a web-based interface to BoaG’s infrastructure that will help researchers to explore the dataset further. Researchers can submit queries and download the results or share them with others. Availability and implementation: The web-interface of the BoaG infrastructure can be accessed here: http://boa.cs.iastate.edu/boag. Please use user = boag and password = boag to login. Source code and other documentation are also provided as a GitHub repository: https://github.com/boalang/NR_Dataset

Changes in Twelve Homoeologous Genomic Regions in Soybean following Three Rounds of Polyploidy

With the advent of high-throughput sequencing, the availability of genomic sequence for comparative genomics is increasing exponentially. Numerous completed plant genome sequences enable characterization of patterns of the retention and evolution of genes within gene families due to multiple polyploidy events, gene loss and fractionation, and differential evolutionary pressures over time and across different gene families. In this report, we trace the changes that have occurred in 12 surviving homoeologous genomic regions from three rounds of polyploidy that contributed to the current Glycine max genome: a genome triplication before the origin of the rosids (;130 to 240 million years ago), a genome duplication early in the legumes (;58 million years ago), and a duplication in the Glycine lineage (;13 million years ago). Patterns of gene retention following the genome triplication event generally support predictions of the Gene Balance Hypothesis. Finally, we find that genes in networks with a high level of connectivity are more strongly conserved than those with low connectivity and that the enrichment of these highly connected genes in the 12 highly conserved homoeologous segments may in part explain their retention over more than 100 million years and repeated polyploidy events

Transcriptome profiling of soybean (Glycine max) roots challenged with pathogenic and non-pathogenic isolates of Fusarium oxysporum

Background Fusarium oxysporum is one of the most common fungal pathogens causing soybean root rot and seedling blight in U.S.A. In a recent study, significant variation in aggressiveness was observed among isolates of F. oxysporum collected from roots in Iowa, ranging from highly pathogenic to weakly or non-pathogenic isolates. Results We used RNA-seq analysis to investigate the molecular aspects of the interactions of a partially resistant soybean genotype with non-pathogenic/pathogenic isolates of F. oxysporum at 72 and 96 h post inoculation (hpi). Markedly different gene expression profiles were observed in response to the two isolates. A peak of highly differentially expressed genes (HDEGs) was triggered at 72 hpi in soybean roots and the number of HDEGs was about eight times higher in response to the pathogenic isolate compared to the non-pathogenic one (1,659 vs. 203 HDEGs, respectively). Furthermore, the magnitude of induction was much greater in response to the pathogenic isolate. This response included a stronger activation of defense-related genes, transcription factors, and genes involved in ethylene biosynthesis, secondary and sugar metabolism. Conclusions The obtained data provide an important insight into the transcriptional responses of soybean-F. oxysporum interactions and illustrate the more drastic changes in the host transcriptome in response to the pathogenic isolate. These results may be useful in the developing new methods of broadening resistance of soybean to F. oxysporum, including the over-expression of key soybean genes

SH2-Dependent Autophosphorylation within the Tec Family Kinase Itk

The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the βD strand. These results are extended into Btk (Bruton\u27s tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation

Proline isomerization preorganizes the Itk SH2 domain for binding the Itk SH3 domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires

A Comparative Analysis of Methylome Profiles of Campylobacter jejuni Sheep Abortion Isolate and Gastroenteric Strains Using PacBio Data

Campylobacter jejuni is a leading cause of human gastrointestinal disease and small ruminant abortions in the United States. The recent emergence of a highly virulent, tetracycline-resistant C. jejuni subsp. jejunisheep abortion clone (clone SA) in the United States, and that strain\u27s association with human disease, has resulted in a heightened awareness of the zoonotic potential of this organism. Pacific Biosciences\u27 Single Molecule, Real-Time sequencing technology was used to explore the variation in the genome-wide methylation patterns of the abortifacient clone SA (IA3902) and phenotypically distinct gastrointestinal-specific C. jejuni strains (NCTC 11168 and 81-176). Several notable differences were discovered that distinguished the methylome of IA3902 from that of 11168 and 81-176: identification of motifs novel to IA3902, genome-specific hypo- and hypermethylated regions, strain level variability in genes methylated, and differences in the types of methylation motifs present in each strain. These observations suggest a possible role of methylation in the contrasting disease presentations of these three C. jejuni strains. In addition, the methylation profiles between IA3902 and a luxS mutant were explored to determine if variations in methylation patterns could be identified that might explain the role of LuxS-dependent methyl recycling in IA3902 abortifacient potential

NCBI-BLAST programs optimization on XSEDE resources for sustainable aquaculture

The development of genomic resources of non-model organisms is now becoming commonplace as the cost of sequencing continues to decrease. The Genome Informatics Facility in collaboration with the Southwest Fisheries Science Center (SWFSC), NOAA is creating these resources for sustainable aquaculture in Seriola lalandi. Gene prediction and annotation are common steps in the pipeline to generate genomic resources, which are computationally intense and time consuming. In our steps to create genomic resources for Seriola lalandi, we found BLAST to be one of our most rate limiting steps. Therefore, we took advantage of our XSEDE Extended Collaborative Support Services (ECSS) to reduce the amount of time required to process our transcriptome data by 300 percent. In this paper, we describe an optimized method for the BLAST tool on the Stampede cluster, which works with any existing datasets or database, without any modification. At modest core counts, our results are similar to the MPI-enabled BLAST algorithm (mpiBLAST), but also allow the much needed and improved flexibility of output formats that the latest versions of BLAST provide. Reducing this time-consuming bottleneck in BLAST will be broadly applicable to the annotation of large sequencing datasets for any organism

Genome, transcriptome and methylome sequencing of a primitively eusocial wasp reveal a greatly reduced DNA methylation system in a social insect

Comparative genomics of social insects has been intensely pursued in recent years with the goal of providing insights into the evolution of social behaviour and its underlying genomic and epigenomic basis. However, the comparative approach has been hampered by a paucity of data on some of the most informative social forms (e.g. incipiently and primitively social) and taxa (especially members of the wasp family Vespidae) for studying social evolution. Here, we provide a draft genome of the primitively eusocial model insect Polistes dominula, accompanied by analysis of caste-related transcriptome and methylome sequence data for adult queens and workers. Polistes dominula possesses a fairly typical hymenopteran genome, but shows very low genomewide GC content and some evidence of reduced genome size. We found numerous caste-related differences in gene expression, with evidence that both conserved and novel genes are related to caste differences. Most strikingly, these –omics data reveal a major reduction in one of the major epigenetic mechanisms that has been previously suggested to be important for caste differences in social insects: DNA methylation. Along with a conspicuous loss of a key gene associated with environmentally responsive DNA methylation (the de novo DNA methyltransferase Dnmt3), these wasps have greatly reduced genomewide methylation to almost zero. In addition to providing a valuable resource for comparative analysis of social insect evolution, our integrative –omics data for this important behavioural and evolutionary model system call into question the general importance of DNA methylation in caste differences and evolution in social insects