Project description:The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, two different probes were evaluated, and the probe with the best performance was employed to further study the tyrosine residues in the human proteome. Then, tagged tyrosine-containing peptides were selectively enriched using bioorthogonal chemistry, and after the cleavage, a small tag on the peptides perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5000 tyrosine sites in MCF7 cells, and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.
Project description:Platelet reactivity (PR) in cardiovascular (CV) patients is variable between individuals and modulates clinical outcome. However, the determinants of platelet reactivity are largely unknown. Integration of data derived from high-throughput omics technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study was to identify candidate genes modulating platelet reactivity in aspirin-treated cardiovascular patients PR was assessed in 110 CV patients treated with aspirin 100mg/d by aggregometry using several agonists. 12 CV patients with extreme high or low PR were selected for transcriptomics, proteomics and miRNA analysis. 2 extreme patient groups, 6 samples per group
Project description:Platelet reactivity (PR) in cardiovascular (CV) patients is variable between individuals and modulates clinical outcome. However, the determinants of platelet reactivity are largely unknown. Integration of data derived from high-throughput omics technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study was to identify candidate genes modulating platelet reactivity in aspirin-treated cardiovascular patients PR was assessed in 110 CV patients treated with aspirin 100mg/d by aggregometry using several agonists. 12 CV patients with extreme high or low PR were selected for transcriptomics, proteomics and miRNA analysis. 2 extreme patient groups, 6 samples per group
Project description:The type III receptor tyrosine kinase, FLT3 is frequently mutated in acute myeloid leukemia. Phosphorylation of FLT3 on tyrosine residues is a critical event for downstream signaling. Here we studied the role of Y842 residue of FLT3, which is located in the activation loop.
Project description:Genetically encoded unnatural amino acids (Uaas) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These Uaas, including a series of alkyl bromide-based Uaas, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based Uaa, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.
Project description:Platelet reactivity (PR) in cardiovascular (CV) patients is variable between individuals and modulates clinical outcome. However, the determinants of platelet reactivity are largely unknown. Integration of data derived from high-throughput omics technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study was to identify candidate genes modulating platelet reactivity in aspirin-treated cardiovascular patients PR was assessed in 110 CV patients treated with aspirin 100mg/d by aggregometry using several agonists. 12 CV patients with extreme high or low PR were selected for transcriptomics, proteomics and miRNA analysis.
Project description:Platelet reactivity (PR) in cardiovascular (CV) patients is variable between individuals and modulates clinical outcome. However, the determinants of platelet reactivity are largely unknown. Integration of data derived from high-throughput omics technologies may yield novel insights into the molecular mechanisms that govern platelet reactivity. The aim of this study was to identify candidate genes modulating platelet reactivity in aspirin-treated cardiovascular patients PR was assessed in 110 CV patients treated with aspirin 100mg/d by aggregometry using several agonists. 12 CV patients with extreme high or low PR were selected for transcriptomics, proteomics and miRNA analysis.
Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].
Project description:Chemokines are small secreted proteins with important roles in immune responses. They consist of a conserved three-dimensional (3D) structure, so-called IL8-like chemokine fold, which is supported by disulfide bridges characteristic of this protein family. Sequence- and profile-based computational methods have been proficient in discovering novel chemokines by making use of their sequence-conserved cysteine patterns. However, it has been recently shown that some chemokines escaped annotation by these methods due to low sequence similarity to known chemokines and to different arrangement of cysteines in sequence and in 3D. Innovative methods overcoming the limitations of current techniques may allow the discovery of new remote homologs in the still functionally uncharacterized fraction of the human genome. We report a novel computational approach for proteome-wide identification of remote homologs of the chemokine family that uses fold recognition techniques in combination with a scaffold-based automatic mapping of disulfide bonds to define a 3D profile of the chemokine protein family. By applying our methodology to all currently uncharacterized human protein sequences, we have discovered two novel proteins that, without having significant sequence similarity to known chemokines or characteristic cysteine patterns, show strong structural resemblance to known anti-HIV chemokines. Detailed computational analysis and experimental structural investigations based on mass spectrometry and circular dichroism support our structural predictions and highlight several other chemokine-like features. The results obtained support their functional annotation as putative novel chemokines and encourage further experimental characterization. The identification of remote homologs of human chemokines may provide new insights into the molecular mechanisms causing pathologies such as cancer or AIDS, and may contribute to the development of novel treatments. Besides, the genome-wide applicability of our methodology based on 3D protein family profiles may open up new possibilities for improving and accelerating protein function annotation processes.
Project description:Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino- p -cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino- p -cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.