MI-AEG-TKK-YL-JSB_8LFQ_ ARL5_ARMH3
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Juan Bonifacino
PROVIDER: MSV000094677 | MassIVE | Sun May 05 10:40:00 BST 2024
SECONDARY ACCESSION(S): PXD051993
REPOSITORIES: MassIVE
Similar Datasets
Project description:Label free mass spectrometry analysis to identify potential effectors of the ARL5A and ARL5B GTPases. During the MitoID proximity biotinylation experiment, bait constructs were made by fusing sequences encoding a mitochondrial-targeting sequence (MTS) from the outer mitochondrial membrane protein TOM20, along with the biotin ligase BioID2, to the N-terminus of constitutively active (Q70L) or inactive (T30N) forms of human ARL5A or ARL5B lacking the N-terminal amphipathic helix. MTS-BioID2 was used as a negative control.
2024-05-04 | MSV000094675 | MassIVE
Project description:The subject of the current study is the finding of possible molecular partners of Drosophila EcR receptor. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. All fused proteins were expressed using the Act5C promoter in Drosophila S2 cells. To ensure functionality of the generated proteins, we verified their ability to bind EcR and Usp sites in the Drosophila genome with the ChIP-Seq. Our results demonstrate that EcR and Usp fusions can be recruited to genomic sites endogenous for the EcR/Usp proteins. Conversely, unfused BioID2 and APEX2 enzymes do not bind to EcR/Usp sites. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. ChIP-Seq experiments revealed an enrichment of CP190 binding on transcription start and end sites of 20E-dependent genes but not at EcR/Usp-bound enhancers.
2020-03-17 | GSE139316 | GEO
Project description:To investigate the effect of depletion of Integrator complex subunits IntS4, IntS6, mts, or Pp2A-29B on gene expression
2023-11-10 | GSE223974 | GEO
Project description:Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in the sensory ataxia, dystonia musculorum (dt). While much investigation has focused on understanding dt pathology, divergent functions of dystonin isoforms are still unknown. Here, we highlight a novel function of the dystonin-a2 isoform in mediating microtubule (MT) stability, golgi organization and flux through the secretory pathway. Using dystonin-null mice combined with isoform-specific loss of function analysis, we find dystonin-a2 is bound to MAP1B in the area surrounding the centrosome, where it maintains MT acetylation. In dt, the absence of the MAP1B-dystonin-A2 interaction results in a loss of MAP1B perinuclear localization, leading to MT deacetylation and instability. Deacetylated MTs lead to golgi fragmentation and prevent anterograde trafficking of motor proteins. Maintenance of MT acetylation through TSA administration or MAP1B overexperssion in vitro, mitigates the observed defect. These aberrations are apparent in pre-phenotype dorsal root ganglia (DRG) and primary sensory neurons, suggesting they are causal in the dt disorder. P4 dorsal root ganglia (DRG) tissue from 3 WT and 3 dt27J animals from 2 separate litters was subjected to RNA extraction and analyzed.
2012-02-23 | E-GEOD-30960 | biostudies-arrayexpress
Project description:Identifying protein-protein interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity ligation method that uses a promiscuous biotin ligase to detect protein-protein interactions in cells in a highly reproducible manner. Recent advancements in proximity ligation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2 to create a smaller, highly active, biotin ligase that we named MicroID. Truncation of the c-terminal of BioID2 and mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller, highly active construct with lower baseline labeling. Several additional point mutations improved the function of our modified MicroID construct compared to BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID is the smallest biotin ligase (180 AA for microID vs. 257 AA for miniTurbo and 338 AA for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurboID. MicroID also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we developed a MicroID mutant that has lower labeling efficiency, but significantly reduced biotin scavenging compared to BioID2. Finally, we demonstrate utility of MicroID in mass spectrometry experiments by localizing MicroID constructs to subcellular organelles and measuring protein-protein interactions.
2022-08-12 | PXD031146 | Pride
Project description:Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in the sensory ataxia, dystonia musculorum (dt). While much investigation has focused on understanding dt pathology, divergent functions of dystonin isoforms are still unknown. Here, we highlight a novel function of the dystonin-a2 isoform in mediating microtubule (MT) stability, golgi organization and flux through the secretory pathway. Using dystonin-null mice combined with isoform-specific loss of function analysis, we find dystonin-a2 is bound to MAP1B in the area surrounding the centrosome, where it maintains MT acetylation. In dt, the absence of the MAP1B-dystonin-A2 interaction results in a loss of MAP1B perinuclear localization, leading to MT deacetylation and instability. Deacetylated MTs lead to golgi fragmentation and prevent anterograde trafficking of motor proteins. Maintenance of MT acetylation through TSA administration or MAP1B overexperssion in vitro, mitigates the observed defect. These aberrations are apparent in pre-phenotype dorsal root ganglia (DRG) and primary sensory neurons, suggesting they are causal in the dt disorder.
2012-02-23 | GSE30960 | GEO
Project description:Endoplasmic reticulum exit sites (ERESs) are ER subdomains where coat protein complex II carriers are assembled for ER-to-Golgi protein transport. In plants, ERES cycles Golgi-associated and Golgi-free states. A plant-specific ER transmembrane protein, MAIGO3, stabilizes Golgi-associated ERESs in Arabidopsis. To identify proteins that interact with MAIGO3, we conducted coimmunoprecipitation coupled to mass spectrometry analysis of GFP-MAIGO3.
2025-01-06 | PXD051419 | JPOST Repository
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including: 1) Cleavage Under Targets and Tagmentation (CUT&Tag); 2) chromatin accessibility characterization using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq); 3) transcriptomics (RNA-Seq); 4) label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility, and 5) MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. Total coverage comprised over 21,000 chromatin regions, 18,000 transcripts, 6,000 proteins and 400 metabolic features. Pair-wise comparison using univariate and supervised multivariate statistical methods revealed significant differences between constructs in each omic level. Furthermore, effector pathway analysis combining omics data revealed distinctive reprogramming effects for each EZH2 mutant.
2023-07-17 | GSE235013 | GEO
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including: 1) Cleavage Under Targets and Tagmentation (CUT&Tag); 2) chromatin accessibility characterization using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq); 3) transcriptomics (RNA-Seq); 4) label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility, and 5) MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. Total coverage comprised over 21,000 chromatin regions, 18,000 transcripts, 6,000 proteins and 400 metabolic features. Pair-wise comparison using univariate and supervised multivariate statistical methods revealed significant differences between constructs in each omic level. Furthermore, effector pathway analysis combining omics data revealed distinctive reprogramming effects for each EZH2 mutant.
2023-07-17 | GSE235011 | GEO
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including: 1) Cleavage Under Targets and Tagmentation (CUT&Tag); 2) chromatin accessibility characterization using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq); 3) transcriptomics (RNA-Seq); 4) label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility, and 5) MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. Total coverage comprised over 21,000 chromatin regions, 18,000 transcripts, 6,000 proteins and 400 metabolic features. Pair-wise comparison using univariate and supervised multivariate statistical methods revealed significant differences between constructs in each omic level. Furthermore, effector pathway analysis combining omics data revealed distinctive reprogramming effects for each EZH2 mutant.
2023-07-17 | GSE235010 | GEO