Project description:In order to identify the binding proteins of VHL and BICD2, we enriched their binding proteins by TurboID-based proximity labeling technology and Flag pull-down assay, and performed mass spectrum analysis.
Project description:Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain-enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress-specific kinase for eIF2α, leading to stress-induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase-1 cleavage and Il18 expression corroborate microglia-associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.
Project description:Beta- and gamma-herpesviruses transcribe their late genes in a manner distinct from host transcription. This process is directed by a complex of viral transcriptional activator proteins that hijack cellular RNA polymerase II, and an unknown set of additional factors. We employed proximity labeling to identify the ensemble of viral and cellular proteins dynamically associated with the KSHV late gene transcriptional complex late during infection.
Project description:In the related study, to determine whether DUSP2 definitively served as a phosphatase for STAT3, an in vitro phosphatase assay was used. Using S-tag beads, p-STAT3 was pulled down from IL-6 stimulated HEK293T cells transfected with STAT3-S-Tag. DUSP2 or control protein, which was purified from HEK293T cells transfected with DUSP2-FLAG or empty vector through extraction with anti-FLAG beads and elution with FLAG peptides, were incubated with p-STAT3-S-Tag-beads in phosphatase buffer. Then bound proteins were eluted and subjected to MS analysis. When compared with the control, incubation of STAT3 and DUSP2 led to dephosphorylation of two STAT3 residues that have been reported to promote its activity, namely tyrosine 705 and serine 727.
Project description:Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF-7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes demonstrated distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer.
Project description:Inhibitors directed towards PARP1 and PARP2 are approved agents for the treatment of BRCA-related cancers. Other members of the PARP family have also been implicated in cancer and are being assessed as therapeutic targets in cancer and other diseases. In fact, an inhibitor of PARP7 (RBN-2397) has now reached early-stage human clinical trials. Here, we performed a genome-wide CRISPR screen for genes that modify the response of cells to RBN-2397. We identify the polycyclic aromatic hydrocarbon receptor AHR and multiple components of the cohesin complex as determinants of resistance to this agent. Activators and inhibitors of AHR modulate the cellular response to PARP7 inhibition, suggesting potential combination therapy approaches.
Project description:Proximity labeling (PL) was recently developed to detect protein–protein interactions (PPIs) and members of subcellular multiprotein structures in living cells. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity, such as biotin ligase, to a protein of interest (bait protein) to biotinylate adjacent proteins. The biotinylated protein can be purified by strep-tavidin beads, and identified by mass spectrometry (MS). TurboID is an engineered biotin ligase with high catalytic efficiency, which is used for proximity labeling. Although TurboID-based proximity labeling technology has been successfully established in mammals, its application in plant systems is limited. Here, we report the usage of TurboID for proximity labeling of FIP37, a core member of m6A methyltransferase complex, to identify FIP37 interacting proteins in Ara-bidopsis thaliana. By analyzing the MS data, we found 214 proteins biotinylated by GFP-TurboID-FIP37 fusion, including five components of m6A methyltransferase complex that have been previously confirmed. Therefore, the identified proteins may include potential proteins directly involved in the m6A pathway or functionally related to m6A-coupled mRNA processing due to spatial proximity. Moreover, we demonstrated the feasibility of proximity labeling tech-nology in plant epitranscriptomics studies, thereby expanding the application of this technology to more subjects of plant research.
Project description:Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry, with tunable reactivity. We prepared sulfamate-based inhibitors for BTK and Pin1 displaying high potency, low intrinsic reactivity and high stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of ‘turn-on’ probes as well as for traceless ligand-directed site-specific labeling of proteins. Using alkyne-modified sulfamate-based probes, we performed proteomics studies with samples enriched with probe-bound proteins, and the sulfamate probes displayed comparable or improved selectivity compared to the parent compounds. Further characterization of off-target using IsoDTB-ABPP confirmed this result. This submission contains the pull down data.