LAGE3-TurboID to screen the interacting proteins
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Xinxing Lyu
PROVIDER: PXD066518 | iProX | Thu Jul 24 00:00:00 BST 2025
REPOSITORIES: iProX
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Project description:dentification of LAGE3-Interacting Proteins Under DNA Replication Stress via Flag-LAGE3 Pull-Down Coupled with Mass Spectrometry Analysis
2025-07-24 | PXD066520 |
Project description:We employed anti-HA or FLAG immunoprecipitation (IP) combined with LC-MS/MS to identify interacting partners for endogenously C-terminal 6xHA-tagged mtAP2-(1/2/3) and endogenously C-terminal 3xFLAG-tagged mitochondrial ribosomal proteins (TguL4m, TgbL27m, and TguS5m). Additionally, we detected the protein abundance of LSU upon conditional knockdown of mtAP2-2 and mtAP2-3. Furthermore, we utilized the TurboID technology to identify the proximal proteins of three mtAP2s (mtAP2-1/2/3).
2023-11-17 | PXD047073 |
Project description:The cohesin complex is a plieotropic regulator of genome structure and function. It is known to play a role in sister chromatin cohesin, DNA replication, DNA repair, and regulation of gene expression. Cohesin has several known interacting proteins that aid in the various roles within a nucleus, yet a comprehensive cohesin interactome has yet to be established. In this study, we utilized the biotin proximity ligation assay TurboID followed by LC-MS/MS in NIH-3T3 mouse fibroblast cells to identify novel proteins that interact with the cohesin complex.
2025-06-02 | PXD060289 | Pride
Project description:Although intensive studies have been performed to identify and characterize host factors involved in viral replication complex formation, the high dynamism of this process and the insoluble properties of the complex make it difficult to be analyzed comprehensively using traditional affinity purification methods. TurboID-based proximity labeling emerges as a robust tool for probing molecular interactions in planta, yet there is no report on the application of TurboID on the study of plant virus replication. Here, we used Beet black scorch virus (BBSV), an endoplasmic reticulum-replicating virus, as a model, and systematically investigated the composition of BBSV virus replication complex (VRC) by fusing the TurboID enzyme to viral replicase protein p23. Among the 185 identified p23-proximal proteins, reticulon family proteins showed high reproducibility in the mass spectrometry datasets. We focused on the reticulon-like protein B2 (RTNLB2) and demonstrated its pro-viral functions in BBSV replication. Mechanistic analysis revealed that RTNLB2 binds to p23, induces the ER membrane curvature and constricts ER tubules to facilitate the assembly of BBSV VRC. Our comprehensive proximal interactome analysis of BBSV VRC provides a resource for understanding of plant viral replication and offers new insights into the formation of membrane scaffolds for viral RNA synthesis.
2023-02-12 | PXD040073 |
Project description:While Lck has been widely recognized to play a pivotal role in the initiation of the T cell receptor (TCR) signaling pathway, an understanding of the precise regulation of Lck in T cells upon TCR activation remains elusive. Investigation of protein-protein interaction (PPI) using proximity labeling techniques such as TurboID has the potential to provide valuable molecular insights into Lck regulatory networks. By expressing Lck-TurboID in Jurkat T cells, we have uncovered a dynamic, short-range Lck protein interaction network upon 30 minutes of TCR stimulation. In this novel application of TurboID, we detected 27 TCR stimulation-induced Lck-proximal interactors in living T cells, including both bona fide and uncanonical Lck interactors, validating the discovery power of this tool. Our results revealed previously unappreciated Lck PPI which may be associated with cytoskeletal rearrangement, ubiquitination of TCR signaling proteins, activation of the MAPK cascade, coalescence of the LAT signalosome, and formation of the immunological synapse. In this study, we demonstrated for the first time in immune cells and for the kinase Lck that TurboID can be utilized to unveil PPI dynamics in living cells at a time scale consistent with TCR signaling.
2020-11-12 | PXD020759 | Pride
Project description:DNA replication stress is a threat to genome integrity. The large SNF2-family of ATPases participates in preventing and mitigating DNA replication stress by employing their ATP-driven motor to remodel DNA or DNA-bound proteins. To understand the contribution of these ATPases in genome maintenance, we undertook CRISPR-based synthetic lethality screens with three SNF2-type ATPases: SMARCAL1, ZRANB3 and HLTF. Here we show that SMARCAL1 displays a profound synthetic lethal interaction with FANCM, another ATP-dependent translocase involved in DNA replication and genome stability. Their combined loss causes severe genome instability that we link to chromosome breakage at loci enriched in simple repeats, which are known to challenge replication fork progression. Our findings illuminate a critical genetic buffering mechanism that provides an essential function for maintaining genome integrity.
2024-12-24 | GSE276441 | GEO
Project description:BackgroundProtein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes.ResultsThe FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification.ConclusionThe TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.
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Project description:Polo-like kinase 1 (Plk1) is an important protein kinase for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is present in S phase, little is known about its localization and function during unperturbed DNA replication. Using Xenopus laevis egg extracts, mimicking early embryonic replication, we developed a Plk1 chromatin immunoprecipitation approach followed by a proteomic shotgun analysis (XChIP-MS) to identify Plk1 interactions with chromatin and with specific nuclear proteins. We purified and cross-linked nuclei after pre-RC assembly before the start of DNA replication, or later during DNA replication. The DNA was fractionated by sonication and immunoprecipited using anti Plk1 or control antibodies then proteins were analyzed by nanoLC/MS/MS.
2021-08-25 | PXD022440 | Pride
Project description:Through the APEX2 technique, we identified MCM2-interacting proteins associated with the replication initiation complex. After excluding proteins from the negative controls, we detected a substantial number of interacting proteins in the positive groups of both cell types. This research establishes a basis for further study on the component proteins of the conserved DNA replication initiation complex and the transient regulatory network involving its proximal proteins.
2025-05-07 | PXD054939 | Pride
Project description:Proximal proteins to S1PR1-WT and S1PR1-TM4 (a swap mutant of the transmembrane domain 4 with that of S1PR3) were obtained in human umbilical vein endothelial cells by a TurboID-catalyzed proximity biotin labeling technique.
2023-05-21 | PXD039461 | JPOST Repository