Project description:GFI is a DNA binding transcriptional repressor that regulates myeloid differentiation. Here, we show that GFI1 interacts with the chromodomain helicase CHD4 and other components of the nucleosome remodeling and deacetylase (NuRD) complex. Our data demonstrated that GFI1 and GFI1/CHD4 complexes occupy sites of open chromatin enriched for histone marks associated with active transcription or different sets of genes that are either enriched for IRF1 or SPI-1 consensus binding sites. In addition, our study provided evidence that GFI1 affects the chromatin remodeling activity of the NuRD complex. Overall, our results indicate that GFI1/CHD4 complexes control chromatin openness and histone modifications differentially to regulate target genes, which govern the immune response, nucleosome organization, or metabolic processes.
Project description:Centrosomes ensure accurate chromosome segregation during cell division. Although the regulation of centrosome number is well-established, less is known about the suppression of non-centrosomal Microtubule-Organising Centres (ncMTOCs). The E3 ligase TRIM37, implicated in Mulibrey nanism and 17q23-amplified cancers, has emerged as a key regulator of both centrosomes and ncMTOCs. Yet, the mechanism by which TRIM37 achieves enzymatic activation to target these mesoscale structures had thus far remained unknown. Here, we elucidate the activation process of TRIM37, unveiling a process that initiates with TRAF domain-directed substrate recognition, followed by B-box domain-mediated oligomerisation, and culminates in RING domain dimerisation. Using optogenetics, we demonstrate that the E3 activity of TRIM37 is directly coupled to the assembly state of its substrates, being activated only when centrosomal proteins cluster into higher-order assemblies resembling MTOCs. This regulatory framework provides a mechanistic basis for understanding TRIM37-driven pathologies and echoes the restriction of the HIV capsid by TRIM5, thus unveiling a conserved activation blueprint among TRIM proteins to control turnover of complexes assembled at the mesoscale level.
Project description:Deciphering the intricate dynamic events governing type I interferon (IFN) signaling is critical to unravel key regulatory mechanisms in host antiviral defense. Here, we leveraged TurboID-based proximity labeling coupled with affinity purification-mass spectrometry to comprehensively map temporal changes to the proximal human proteomes of all seven canonical type I IFN signaling cascade members following IFN stimulation. This established a network of 108 proteins in close proximity to the core members IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, and validated several known protein assemblies, while also revealing novel, transient associations between key signaling molecules.
Project description:This study evaluates liquid-chromatography operated at 50 ul/min using 1 mm diameter columns coupled online to tandem mass spectrometry (LC-MS/MS) for proteome analysis. Data from >1,000 human cell line, tissue, body fluid, affinity- and phospho-proteomes demonstrate excellent chromatographic (<0.3 % CV) and quantitative (<7.5% CV) reproducibility. Combined with sample multiplexing by tandem mass tags, the system allows profiling of 11 proteomes in 16 hours to a depth of 8,000 proteins.
Project description:Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry and topological organisation of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs on a global level.
Project description:During the purification of recombinantely expressed MRAS and MRAS mutants, there were additional proteins which appeared to co-precipitate and were detected by Coomassie staining. Since the apparent molecular weight was within the range for small GTPase proteins, we sought to determine the identity of these proteins.
Project description:Hepatocyte nuclear factor 4 (HNF4) is a transcription factor that acts as a master regulator of genes in several endodermal-derived tissues, including the intestine in which it plays a central role during development and tumorigenesis. To better delineate the mechanisms by which HNF4 can interfere during these processes, we combined stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein (GFP) as well as with proximity-dependent purification by the biotin ligase BirA, both fused to HNF4. Surprisingly, these analyses identified a significant enrichment of proteins falling into the DNA repair gene ontology term, a so far unidentified biological feature of this transcription factor. Several of these proteins including PARP1, RAD50 and DNA-PKcs were confirmed to interact with HNF4in colorectal cancer cell lines. During DNA damage response, HNF4 localized to double strand DNA breaks in these cells. HNF4was able to interfere functionally during non-homologous end-joining (NHEJ). Overall, these observations identify an unsuspected role for this transcription factor during the DNA damage response.
Project description:Ubash3b, also known as suppressor of T-cell receptor signaling or Sts-1, is an ill-studied atypical tyrosine phosphatase with ubiquitin binding ability. In our previous study, we hypothesized that Ubash3b plays an inhibitory role in BCR-ABL signaling through binding and dephosphorylating BCR-ABL and its interactors. The Hantschel lab recently solved the crystal structures of the p210 PH and DH domains, which are absent in the p190 variant, and demonstrated that loss-of-function mutations in the PH domain altered BCR-ABL localization, thereby reducing the interaction between Ubash3b and p2104. Taken together, this suggests differential subcellular localization of Ubash3b as a mechanism by which it interacts more strongly with p201 as compared to p190. To better understand the global impact Ubash3b has on p210, its direct kinase substrates and proteins in its phosphotyrosine signaling network, we have taken an integrative approach by combining global phosphotyrosine profiling, proximity-dependent biotinylation (BioID) and total protein analysis to investigate p210 signaling upon Ubash3b knockdown (KD). The BioID system was used to characterize Ubash3b function in p210 signaling by examining its interactome. Importantly, in all of our BioID experiments, we employed a newly technique that we have recently developed, Biotinylation Site Identification Technology (BioSITe), which directly identifies biotinylated peptides thereby increasing the reliability of the identified interactors. Here, we additionally used short hairpin RNA (shRNA) interference and generated Ubash3b knock-down (KD) and non-targeting control shRNA lines in Ba/F3 BirA*-p210 cells. Ubash3b expression was reduced >90 % in the KD cells and had a substantial effect on global tyrosine phosphorylation and on the interactome of p210. Of the 1,421 unique tyrosine phosphorylation sites identified from 830 proteins, 379 sites (from 286 proteins) exhibited a substantial increase (≥2-fold) in tyrosine phosphorylation upon Ubash3b KD cells compared to control cells. To date, the interactome of Ubash3b has not been extensively investigated, however, some examination of Ubash3b in the context p210 signaling has been undertaken. We designed constructs of C-terminal BirA* tagged full length Ubash3b and a deletion mutant lacking the UBA and SH3 domains leaving only the phosphatase domain tethered to BirA*. A comparative analysis of the core interactors of p210 from previous studies and Ubash3b interactome from the current study revealed 36 proteins that interact with both p210 and Ubash3b.
Project description:Endocytic recycling controls the return of internalised cargos to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargos to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of well characterised and new cargos and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock sideways relocalisation approach we were further able to confirm novel links between Rab11/25 and the ESCPE-1 and retromer multiprotein sorting complexes and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulate cancer cell migration in 3D-matrix.
Project description:The nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor activates cytoprotective and metabolic gene expression in response to various electrophilic stressors. In disease, constitutive NRF2 activity promotes cancer progression while decreased NRF2 function contributes to neurodegenerative diseases. In contrast to the regulation of NRF2 protein stability in the cytoplasm, co-complexed proteins that govern NRF2 activity on chromatin are less clear. Using biotin proximity proteomics, we report networks for NRF2 and its family members NRF1, NRF3 and the NRF2 heterodimer MAFG. We found that the Parkinson’s disease zinc finger transcription factor ZNF746 (PARIS) physically associated with NRF2 and MAFG, resulting in suppression of NRF2-driven transcription. ZNF746 expression increased oxidative stress and apoptosis, phenotypes that were reversed by chemical and genetic hyperactivation of NRF2. This study presents a functionally annotated proximity network for NRF2 and suggests that ZNF746 overexpression in Parkinson’s disease directly inhibits NRF2-driven neuroprotection