Phylointeractomics reconstructs functional evolution of protein binding
Abstract
Molecular phylogenomics investigates evolutionary relationships based on genomic data and has been substantially advanced in recent years by the increasing availability of high-throughput DNA sequencing. Despite genomic sequence conservation, changes in protein interactions can advance rapidly and with strong functional diversification. To help investigate functional evolution, we here combine the power of phylogenomics with that of interaction proteomics. In ‘phylointeractomics’, a bait is interrogated with the proteome of evolutionary diverse organisms using a DNA pull-down assay combined with label-free quantitative proteomics. We demonstrate the concept by investigating the molecular evolution of the shelterin complex, which protects telomeres from being recognized as DNA double strand breaks, across 16 vertebrate species from zebrafish to humans covering 450 million years of divergent evolution, but still all sharing the same TTAGGG telomeric repeat motif. Our phylointeractomics screen discovers previously unknown telomere-associated proteins and reveals how homologous proteins undergo functional evolution. For instance, TRF1, a well-characterized member of the shelterin complex, evolved as a telomere-binding protein in the common stem lineage of marsupial and placental mammals. Despite being present in the entire vertebrate lineage, TRF1 does not show TTAGGG-binding capacity in non-mammalian vertebrates or monotremata, whereas it consistently binds to telomeric repeats in all marsupials and placental mammals tested. By selected single amino acid exchanges, we identify the necessary gain-of-function mutations for this evolutionary switch and thus approximate key changes enabling TRF1 to directly bind telomeres. Our findings establish that simple presence of gene homologs in different species does not necessarily equate to functional conservation and recapitulate how TRF1 became a telomere-binding protein. Beyond the specific example, our phylointeractomics screen unveils previously unknown telomere-associated proteins that are consistently identified in several vertebrate species. Here, we will exemplify the role of a novel direct telomere-binding protein by investigating its function with a multi-layer omics approach of ChIP-seq, RNA-seq and label-free quantitative proteomics expression profiling, illustrating how this cross-species comparison represents a valuable resource for novel factors with putative roles in telomere homeostasis. Overall, phylointeractomics is a versatile and scalable approach to investigate evolutionary changes in protein function and interactions, e.g. protein complexes, RNA folds and DNA elements. It can provide experimental evidence for phylogenomic relationships and helps to extrapolate the results obtained in model organisms.
Gachon College of Pharmacy, Gachon University, Korea
Title
Development of tissue-based proteome map of Canis (beagle)
Abstract
The genome sequence of domestic dog (Canis familiaris) was released in 2005. The effort to complete the annotation of protein-coding and non-protein-coding gene sets is still going on to fully exploit this model organism. As a veterinary research, the canis proteome resources are considered for finding early diagnostic disease marker, because of high similarities for various diseases and co-experiences of environment conditions. To investigate the similarities, it is necessary to resolve the proteome variation in the different tissues and organs of the Canis familiaris.
In this talk, we present proteome profiling of 12 organ tissue of male and female beagle, including mammary gland, cerebrum, cerebellum, liver, lung, ovary, stomach, kidney, colon, pancreas, testis, and prostate gland. To analyze the proteomes, we carried out high pH reversed-phase chromatographic fractionation, and LC-MS/MS analysis using high-resolution orbitrap mass spectrometry.
Synaptic pathology of Alzheimer's disease: genomics and proteomics studies
Abstract
The interaction between β-amyloid (Aβ) and tau pathologies is critical for the pathogenesis of Alzheimer’s disease (AD) but still little understood. We characterized the synaptic phosphoproteome of APP/PS1 mice by label-free quantitative mass spectrometry, and found that amyloid pathology induced hyperphosphorylation on a subset of 40 synaptic proteins, with tau being one of the most affected. Proteins associated with cytoskeletons and involved in calmodulin signaling pathways were primary hyperphosphorylation targets. Tau hyperphosphorylation occurred at proline-directed kinase (PDK) sites (S199, S202, S396, S404) and non-proline directed sites (S400, S416). The PDK sites were prominently associated with the pathological features of tau in AD subjects, validated using patient-derived synaptosomes. Global analysis of hyperphosphorylation motifs suggested cyclin-dependent kinase 5 (CDK5) as the major synaptic PDK upregulated in amyloid-induced tauopathy, while casein kinase II was also hyperactive. Our proteomics data established the synaptic terminal as an important site of pathological interactions between Aβ and tau. On the other hand, we have developed new methods to sort individual synaptic terminals by fluorescence-activated cell sorter, which allowed us to collect about 1 million highly-purified synaptosomes. From these sorted synaptosomes we have performed RNA extraction and cDNA synthesis. This will allow us to conduct RNA deep-sequencing to understand the transcriptomics changes at Aβ-affected synapses. By combining transcriptomics and proteomics appraoches, we can better understand how Aβ triggers the dysfunction of synaptic terminals.
Prediction of Protein Variants by RNA Editomes in a Companion Dog
Abstract
Affiliations: 1Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, South Korea, 2Department of Nanobiomedical Science, BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea, 3Department of Microbiology, College of Natural Sciences, Dankook University, Cheonan 31116, Republic of Korea.
Abstract: RNA editing refers to a phenomenon that some nucleotides of RNA are altered by specific enzymes after transcription by RNA polymerase. In mammals, the main type of RNA editing is the conversion of adenosine to inosine (A-to-I RNA editing) which is translated as if it were guanosine. This reaction is catalyzed by the ADAR enzymes. In this study, we identified a large number of RNA editing sites in a companion dog (Beagle) using next-generation sequencing. To this end, RNA editing sites detected by RNA-seq were compared with DNA variants (SNPs) after the identification of RNA and DNA variants using the Genome Analysis ToolKit (GATK) method. Interestingly, more than 20,000 RNA editing sites were identified in 13 different tissues including brain, liver, and lung. Up to 10% of the RNA editing sites can potentially cause changes in protein-coding regions. These editing events were observed more frequently than randomly expected. In addition, a number of tissues-specific RNA editing sites were also identified. Overall, this study shows the importance of RNA editing events that could potentially affect the function of proteins.
The Walter and Eliza Hall Institute of Medical Research(WEHI), Australia
Title
CIS is a potent checkpoint in NK cell-mediated tumor immunity
Abstract
The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. Here we have identified CIS (encoded by Cish) as the critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15 and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity towards tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. A mass spectrometry–based approach quantifying changes in active JAK levels was used to confirm the elevated JAK1 activity and to examine the selectivity of the CIS-deficient effects. Cish−/− mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. This study has uncovered a potent intracellular checkpoint in NK cell-mediated tumor immunity and holds promise for novel cancer immunotherapies directed at blocking CIS function.
Efforts are now underway to map the precise interactions between CIS and JAK1 in order to better understand how the complex can be targeted by small molecules to improve the NK cells' response to the growth factor and help patients fight cancer with their own immune system. We are using cross linking mass spectrometry to define the CIS/JAK1 binding interface.
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