Molecular Evolution of Aryl Hydrocarbon Receptor Signaling Pathway Genes.

The Aryl hydrocarbon receptor is an ancient transcriptional factor originally discovered as a sensor of dioxin. In addition to its function as a receptor of environmental toxicants, it plays an important role in development. Although a significant amount of research has been carried out to understand the AHR signal transduction pathway and its involvement in species' susceptibility to environmental toxicants, none of them to date has comprehensively studied its evolutionary origins. Studying the evolutionary origins of molecules can inform ancestral relationships of genes. The vertebrate genome has been shaped by two rounds of whole-genome duplications (WGD) at the base of vertebrate evolution approximately 600 million years ago, followed by lineage-specific gene losses, which often complicate the assignment of orthology. It is crucial to understand the evolutionary origins of this transcription factor and its partners, to distinguish orthologs from ancient non-orthologous homologs. In this study, we have investigated the evolutionary origins of proteins involved in the AHR pathway. Our results provide evidence of gene loss and duplications, crucial for understanding the functional connectivity of humans and model species. Multiple studies have shown that 2R-ohnologs (genes and proteins that have survived from the 2R-WGD) are enriched in signaling components relevant to developmental disorders and cancer. Our findings provide a link between the AHR pathway's evolutionary trajectory and its potential mechanistic involvement in pathogenesis.

The complete genome of 2,6-dichlorobenzamide (BAM) degrader Aminobacter sp. MSH1 suggests a polyploid chromosome, phylogenetic reassignment, and functions of plasmids.

Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.

Integrated Multi-Omics Analysis of Mechanisms Underlying Yeast Ethanol Tolerance.

For yeast cells, tolerance to high levels of ethanol is vital both in their natural environment and in industrially relevant conditions. We recently genotyped experimentally evolved yeast strains adapted to high levels of ethanol and identified mutations linked to ethanol tolerance. In this study, by integrating genomic sequencing data with quantitative proteomics profiles from six evolved strains (data set identifier PXD006631) and construction of protein interaction networks, we elucidate exactly how the genotype and phenotype are related at the molecular level. Our multi-omics approach points to the rewiring of numerous metabolic pathways affected by genomic and proteomic level changes, from energy-producing and lipid pathways to differential regulation of transposons and proteins involved in cell cycle progression. One of the key differences is found in the energy-producing metabolism, where the ancestral yeast strain responds to ethanol by switching to respiration and employing the mitochondrial electron transport chain. In contrast, the ethanol-adapted strains appear to have returned back to energy production mainly via glycolysis and ethanol fermentation, as supported by genomic and proteomic level changes. This work is relevant for synthetic biology where systems need to function under stressful conditions, as well as for industry and in cancer biology, where it is important to understand how the genotype relates to the phenotype.

Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung.

A suppressor of axillary meristem maturation promotes longevity in flowering plants.

Post-embryonic development and longevity of flowering plants are, for a large part, determined by the activity and maturation state of stem cell niches formed in the axils of leaves, the so-called axillary meristems (AMs)1,2. The genes that are associated with AM maturation and underlie the differences between monocarpic (reproduce once and die) annual and the longer-lived polycarpic (reproduce more than once) perennial plants are still largely unknown. Here we identify a new role for the Arabidopsis AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15) gene as a suppressor of AM maturation. Loss of AHL15 function accelerates AM maturation, whereas ectopic expression of AHL15 suppresses AM maturation and promotes longevity in monocarpic Arabidopsis and tobacco. Accordingly, in Arabidopsis grown under longevity-promoting short-day conditions, or in polycarpic Arabidopsis lyrata, expression of AHL15 is upregulated in AMs. Together, our results indicate that AHL15 and other AHL clade-A genes play an important role, directly downstream of flowering genes (SOC1, FUL) and upstream of the flowering-promoting hormone gibberellic acid, in suppressing AM maturation and extending the plant's lifespan.

Large-scale docking predicts that sORF-encoded peptides may function through protein-peptide interactions in Arabidopsis thaliana.

Several recent studies indicate that small Open Reading Frames (sORFs) embedded within multiple eukaryotic non-coding RNAs can be translated into bioactive peptides of up to 100 amino acids in size. However, the functional roles of the 607 Stress Induced Peptides (SIPs) previously identified from 189 Transcriptionally Active Regions (TARs) in Arabidopsis thaliana remain unclear. To provide a starting point for functional annotation of these plant-derived peptides, we performed a large-scale prediction of peptide binding sites on protein surfaces using coarse-grained peptide docking. The docked models were subjected to further atomistic refinement and binding energy calculations. A total of 530 peptide-protein pairs were successfully docked. In cases where a peptide encoded by a TAR is predicted to bind at a known ligand or cofactor-binding site within the protein, it can be assumed that the peptide modulates the ligand or cofactor-binding. Moreover, we predict that several peptides bind at protein-protein interfaces, which could therefore regulate the formation of the respective complexes. Protein-peptide binding analysis further revealed that peptides employ both their backbone and side chain atoms when binding to the protein, forming predominantly hydrophobic interactions and hydrogen bonds. In this study, we have generated novel predictions on the potential protein-peptide interactions in A. thaliana, which will help in further experimental validation.

Correlated duplications and losses in the evolution of palmitoylation writer and eraser families.

BACKGROUND: Protein post-translational modifications (PTMs) change protein properties. Each PTM type is associated with domain families that apply the modification (writers), remove the modification (erasers) and bind to the modified sites (readers) together called toolkit domains. The evolutionary origin and diversification remains largely understudied, except for tyrosine phosphorylation. Protein palmitoylation entails the addition of a palmitoyl fatty acid to a cysteine residue. This PTM functions as a membrane anchor and is involved in a range of cellular processes. One writer family and two erasers families are known for protein palmitoylation. RESULTS: In this work we unravel the evolutionary history of these writer and eraser families. We constructed a high-quality profile hidden Markov model (HMM) of each family, searched for protein family members in fully sequenced genomes and subsequently constructed phylogenetic distributions of the families. We constructed Maximum Likelihood phylogenetic trees and using gene tree rearrangement and tree reconciliation inferred their evolutionary histories in terms of duplication and loss events. We identified lineages where the families expanded or contracted and found that the evolutionary histories of the families are correlated. The results show that the erasers were invented first, before the origin of the eukaryotes. The writers first arose in the eukaryotic ancestor. The writers and erasers show co-expansions in several eukaryotic ancestral lineages. These expansions often seem to be followed by contractions in some or all of the lineages further in evolution. CONCLUSIONS: A general pattern of correlated evolution appears between writer and eraser domains. These co-evolution patterns could be used in new methods for interaction prediction based on phylogenies.

ARA-PEPs: a repository of putative sORF-encoded peptides in Arabidopsis thaliana.

BACKGROUND: Many eukaryotic RNAs have been considered non-coding as they only contain short open reading frames (sORFs). However, there is increasing evidence for the translation of these sORFs into bioactive peptides with potent signaling, antimicrobial, developmental, antioxidant roles etc. Yet only a few peptides encoded by sORFs are annotated in the model organism Arabidopsis thaliana. RESULTS: To aid the functional annotation of these peptides, we have developed ARA-PEPs (available at http://www.biw.kuleuven.be/CSB/ARA-PEPs ), a repository of putative peptides encoded by sORFs in the A. thaliana genome starting from in-house Tiling arrays, RNA-seq data and other publicly available datasets. ARA-PEPs currently lists 13,748 sORF-encoded peptides with transcriptional evidence. In addition to existing data, we have identified 100 novel transcriptionally active regions (TARs) that might encode 341 novel stress-induced peptides (SIPs). To aid in identification of bioactivity, we add functional annotation and sequence conservation to predicted peptides. CONCLUSION: To our knowledge, this is the largest repository of plant peptides encoded by sORFs with transcript evidence, publicly available and this resource will help scientists to effortlessly navigate the list of experimentally studied peptides, the experimental and computational evidence supporting the activity of these peptides and gain new perspectives for peptide discovery.

Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains.

Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which-including some found in human cancers-induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains.

Discovery and validation of the antimetastatic activity of citalopram in colorectal cancer.

Inverse gene expression profiling was recently shown to help drug repositioning. We showed that this approach works best for cancer and predicted novel drug candidates that may reduce metastasis in colorectal cancer. Antimetastatic activity of our predicted candidate, citalopram, was validated in an orthotopic mouse model of metastatic colorectal cancer.

Novel drug candidates for the treatment of metastatic colorectal cancer through global inverse gene-expression profiling.

Drug-induced gene-expression profiles that invert disease profiles have recently been illustrated to be a starting point for drug repositioning. In this study, we validate this approach and focus on prediction of novel drugs for colorectal cancer, for which there is a pressing need to find novel antimetastatic compounds. We computationally predicted three novel and still unknown compounds against colorectal cancer: citalopram (an antidepressant), troglitazone (an antidiabetic), and enilconazole (a fungicide). We verified the compounds by in vitro assays of clonogenic survival, proliferation, and migration and in a subcutaneous mouse model. We found evidence that the mode of action of these compounds may be through inhibition of TGFß signaling. Furthermore, one compound, citalopram, reduced tumor size as well as the number of circulating tumor cells and metastases in an orthotopic mouse model of colorectal cancer. This study proposes citalopram as a potential therapeutic option for patients with colorectal cancer, illustrating the potential of systems pharmacology.

Spike-in SILAC proteomic approach reveals the vitronectin as an early molecular signature of liver fibrosis in hepatitis C infections with hepatic iron overload.

Hepatitis C virus (HCV)-induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal-restricted histological distribution of pathological iron deposits has hampered the attempt to perform large-scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload-induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike-in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron-associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV-infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV-infected patients without iron overload.

Characterization of drug-induced transcriptional modules: towards drug repositioning and functional understanding.

In pharmacology, it is crucial to understand the complex biological responses that drugs elicit in the human organism and how well they can be inferred from model organisms. We therefore identified a large set of drug-induced transcriptional modules from genome-wide microarray data of drug-treated human cell lines and rat liver, and first characterized their conservation. Over 70% of these modules were common for multiple cell lines and 15% were conserved between the human in vitro and the rat in vivo system. We then illustrate the utility of conserved and cell-type-specific drug-induced modules by predicting and experimentally validating (i) gene functions, e.g., 10 novel regulators of cellular cholesterol homeostasis and (ii) new mechanisms of action for existing drugs, thereby providing a starting point for drug repositioning, e.g., novel cell cycle inhibitors and new modulators of α-adrenergic receptor, peroxisome proliferator-activated receptor and estrogen receptor. Taken together, the identified modules reveal the conservation of transcriptional responses towards drugs across cell types and organisms, and improve our understanding of both the molecular basis of drug action and human biology.

Drug-induced regulation of target expression.

Drug perturbations of human cells lead to complex responses upon target binding. One of the known mechanisms is a (positive or negative) feedback loop that adjusts the expression level of the respective target protein. To quantify this mechanism systems-wide in an unbiased way, drug-induced differential expression of drug target mRNA was examined in three cell lines using the Connectivity Map. To overcome various biases in this valuable resource, we have developed a computational normalization and scoring procedure that is applicable to gene expression recording upon heterogeneous drug treatments. In 1290 drug-target relations, corresponding to 466 drugs acting on 167 drug targets studied, 8% of the targets are subject to regulation at the mRNA level. We confirmed systematically that in particular G-protein coupled receptors, when serving as known targets, are regulated upon drug treatment. We further newly identified drug-induced differential regulation of Lanosterol 14-alpha demethylase, Endoplasmin, DNA topoisomerase 2-alpha and Calmodulin 1. The feedback regulation in these and other targets is likely to be relevant for the success or failure of the molecular intervention.

Impact of genome reduction on bacterial metabolism and its regulation.

To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and 13C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.

Bioinformatic analysis of functional differences between the immunoproteasome and the constitutive proteasome.

Intracellular proteins are degraded largely by proteasomes. In cells stimulated with gamma interferon, the active proteasome subunits are replaced by "immuno" subunits that form immunoproteasomes. Phylogenetic analysis of the immunosubunits has revealed that they evolve faster than their constitutive counterparts. This suggests that the immunoproteasome has evolved a function that differs from that of the constitutive proteasome. Accumulating experimental degradation data demonstrate, indeed, that the specificity of the immunoproteasome and the constitutive proteasome differs. However, it has not yet been quantified how different the specificity of two forms of the proteasome are. The main question, which still lacks direct evidence, is whether the immunoproteasome generates more MHC ligands. Here we use bioinformatics tools to quantify these differences and show that the immunoproteasome is a more specific enzyme than the constitutive proteasome. Additionally, we predict the degradation of pathogen proteomes and find that the immunoproteasome generates peptides that are better ligands for MHC binding than peptides generated by the constitutive proteasome. Thus, our analysis provides evidence that the immunoproteasome has co-evolved with the major histocompatibility complex to optimize antigen presentation in vertebrate cells.