Anti-Flag AP-MS of cytoplasmic 3×Flag-importin α1 complexes in HeLa S3 cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell
DISEASE(S): Endocervical Adenocarcinoma
SUBMITTER: 
Yutaka Ogawa
LAB HEAD: Naoko Imamoto
PROVIDER: PXD076745 | Pride | 2026-04-22
REPOSITORIES: Pride
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Project description:Importin-beta family proteins are nucleocytoplasmic transport receptors (NTRs), and they transport most nuclear proteins as their cargoes into and out of the nuclei. Human cells have 20 species of importin-beta family NTRs, of which 10 (importin-beta, transportin-1, -2, -SR, importin-4, -5, -7, -8, -9, and -11) are nuclear import receptors and 2 (importin-13, and exportin-4) are bi-directional receptors. Here, we identified the import cargo proteins of these 12 NTRs by a method called SILAC-Tp, which employs stable isotope labeling with amino acids in cell culture (SILAC), an in vitro reconstituted transport system, and LC-MS/MS.
2017-01-20 | PXD004655 | Pride
Project description:To test whether nuclear importin alpha2 can regulate transcription, we sought to examine gene expression changes in cells with nuclear accumulation of importin alpha2 by performing microarray analysis. We designed an experiment in which EGFP-fused full-length importin alpha2 was transfected into HeLa cells. To exclude the possibility that exogenous, full-length importin alpha2 protein enhances nuclear transport of karyophilic proteins such as transcription factors and thereby directly influences gene expression, a mutant of importin alpha2 which is mutated in the C-terminal CAS-binding domain was also transfected, so that it is never recycled to the cytoplasm and shows complete nuclear localization. We undertook to investigate whether there were changes in gene expression common to cells expressing the full-length importin alpha2 and the C-terminal mutant (C-mutant) isoform. EGFP vs. EGFP-importin alpha2 full length, or EGFP vs EGFP-importin alpha2 CAS-binding mutant. One replicate each. EGFP-expressing cells are used as a control.
2010-11-24 | E-GEOD-25303 | biostudies-arrayexpress
Project description:Importin 13 is a bidirectional transport receptor that mediates both nuclear import and export of proteins. To expand the range of human importin 13 cargoes, we performed importin 13 binding experiments in combination with quantitative stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry. Apart from known importin 13 interaction partners, such as Ubc9 and eIF1A, more than 300 potential importin 13 cargoes were identified, several of which could be validated using cellular and biochemical assays.
2018-04-20 | PXD008459 | Pride
Project description:Liprin-α1 is a widely expressed scaffolding protein that regulates cellular processes such as cell motility and synaptic transmission through assembly of localized higher-order molecular complexes. The dynamic regulation of these complexes remains poorly understood. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins into cellular nanodomains, facilitating efficient spatiotemporal signaling. Whether liprin-α1 undergoes regulated LLPS remains unclear. MS-based interactomics identified PPP2R5D, the regulatory B56δ subunit of PP2A, as a liprin-α1 interaction partner through a canonical short linear motif (SLiM) in its N-terminal dimerization domain. Mutation of SLiM4 nearly abolished liprin-α1 interaction with PP2A holoenzyme and resulted in a significant increase in GFP-liprin-α1 LLPS in HEK293 cells. Consistently, GFP-liprin-α1 exhibited increased droplet formation in PPP2R5D knockout HEK293 cells. Phospho-analysis of liprin-α1 SLiM4 mutant via MS revealed increased phosphorylation of multiple Ser/Thr sites, including S763, as validated by a novel phospho-specific antibody. A liprin-α1 S763E phospho-mimetic mutant appeared sufficient to drive LLPS. Expression of the PPP2R5D missense variant E420K, recurrently found in Houge-Janssens Syndrome Type 1 compromised suppression of liprin-α1 LLPS, correlating with increased liprin-α1 S763 phosphorylation. Mechanistically, a liprin-α1 E942A mutant unable to bind liprin-β1 underwent increased LLPS, despite preserved PPP2R5D holoenzyme binding. Furthermore, liprin-α1/β1 heterodimerization significantly decreased under conditions where liprin-α1 LLPS was promoted, i.e. upon SLiM4 or S763E mutation in wild type cells, or in PPP2R5D knockout and PPP2R5D E420K knock-in cells. Our findings identify both liprin-β1 and PPP2R5D-PP2A as potent inhibitors of liprin-α1 LLPS, with PP2A contributing to liprin-α1/β1 heterodimerization via phosphorylation of at least liprin-α1 S763.
2025-06-23 | PXD063716 | Pride
Project description:Background: Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Methods: Using an unbiased, discovery and quantitative proteomic approach, we discovered the differentially expressed proteins binding to Na, K-ATPase α1 between LPS-A549 cells and Control-A549 cells. These Na, K-ATPase α1 interacting proteins were screened by co-immunoprecipitation (Co-IP) technology. Among them, some of the differentially expressed proteins with significant performance were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network was constructed by the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Several differentially expressed proteins were validated by Western blot. Results: Of identified 1598 proteins, 89 were differentially expressed proteins between LPS-A549 cells and Control-A549 cells. Intriguingly, protein-protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. The related GO and KEGG analysis found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Conclusions: In conclusion, our proteomic approach revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS.
2022-08-12 | PXD032209 | Pride
Project description:During early embryonic cell divisions, the total nuclear volume doubles with each cycle. At a critical volume ratio between the nucleus and cytoplasm, embryos start transcribing their genome in a reproducible and sequential manner. However, the underlying molecular mechanism for these well-timed events remains unclear. Previous studies proposed that titration of a regulatory factor by the exponentially increasing DNA content of the system triggers the onset of the first transcripts. Nevertheless, a systematic investigation of nuclear proteome changes during development is still missing. In this study, we perform this investigation and find that differential nuclear import timing can largely explain the sequential onset of transcription. First, we investigated nuclear proteome changes in early development. We found that nuclear proteins sequentially titrate into the embryonic nuclei. Interestingly, transcription factors and RNA polymerases enter the nucleus at different times, which correspond to the activation of their nuclear functions. Notably, we observed a high correlation between when transcription factors enter the nucleus and the order in which they activate downstream transcription. Time-lapse imaging of protein import into nuclei encapsulated in droplets of cytoplasmic extract validated our discovery of the sequential nuclear import by mass spectrometry. Lastly, we investigated the underlying molecular mechanism responsible for the differential timing of protein entry into embryonic nuclei. To this end, we quantified the relative affinity of substrates to importin and observe that high-affinity substrates tend to enter the nucleus earlier than low-affinity substrates. Notably, we can explain > 36% of the observed time-variation of when proteins enter the nucleus in the embryo with this simple affinity assay. Together, our results suggest that embryos control protein entry into the nucleus by modulating importin affinities, which then determines the timing of essential nuclear processes in early development.
2022-08-30 | PXD036403 | Pride
Project description:During early embryonic cell divisions, the total nuclear volume doubles with each cycle. At a critical volume ratio between the nucleus and cytoplasm, embryos start transcribing their genome in a reproducible and sequential manner. However, the underlying molecular mechanism for these well-timed events remains unclear. Previous studies proposed that titration of a regulatory factor by the exponentially increasing DNA content of the system triggers the onset of the first transcripts. Nevertheless, a systematic investigation of nuclear proteome changes during development is still missing. In this study, we perform this investigation and find that differential nuclear import timing can largely explain the sequential onset of transcription. First, we investigated nuclear proteome changes in early development. We found that nuclear proteins sequentially titrate into the embryonic nuclei. Interestingly, transcription factors and RNA polymerases enter the nucleus at different times, which correspond to the activation of their nuclear functions. Notably, we observed a high correlation between when transcription factors enter the nucleus and the order in which they activate downstream transcription. Time-lapse imaging of protein import into nuclei encapsulated in droplets of cytoplasmic extract validated our discovery of the sequential nuclear import by mass spectrometry. Lastly, we investigated the underlying molecular mechanism responsible for the differential timing of protein entry into embryonic nuclei. To this end, we quantified the relative affinity of substrates to importin and observe that high-affinity substrates tend to enter the nucleus earlier than low-affinity substrates. Notably, we can explain > 36% of the observed time-variation of when proteins enter the nucleus in the embryo with this simple affinity assay. Together, our results suggest that embryos control protein entry into the nucleus by modulating importin affinities, which then determines the timing of essential nuclear processes in early development.
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Project description:Accumulating evidence suggests that insulin secretion in pancreatic β cells is regulated by mechanisms analogous to synaptic vesicle release at the neuronal presynaptic active zone (AZ). Neurotransmitter release is tightly regulated in magnitude, space and time by a presynaptic macromolecular complex including scaffold proteins liprin, ELKS, RIM and RIM-BP, to ensure precise control of synaptic responses. This presynaptic complex tethers, docks and locally controls the fusion of synaptic vesicles at the AZ. β-cells express many of the same neuronal presynaptic scaffold proteins including liprin-α1, however, no examination into the existence of a β cell presynaptic-like complex has been done. Here, we performed the first comprehensive interactome analysis of liprin-α1 in MIN6 cells using co-immunoprecipitation coupled to mass spectrometry-based proteome analysis to identify liprin-α1 binding partners and probe the existence of a wider β cell presynaptic-like complex. We identified a broad range of liprin-α1 interacting proteins in both basal (low glucose) and stimulated (high glucose) conditions. Key among these were the synaptic proteins liprin-α1,2, liprin-β1, GIT1 and LAR, supporting a β cell presynaptic-like complex. Other proteins of interest included β2 syntrophin, a regulator of insulin granule mobility, the microtubule organising proteins Mtcl1 and Mtcl3, as well as protein phosphatase 2A complexes and the functionally related 14-3-3 isoforms involved in phospho-protein dependent binding. Together, these data indicate liprin-α1 is central to a wider presynaptic-like complex that is the focus for phosphorylation and structurally links insulin granule positioning and cytoskeletal dynamics.
2026-02-09 | PXD049219 | Pride
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2015-03-31 | E-GEOD-58466 | biostudies-arrayexpress
Project description:We examine the role of Klf6 in oligodendrocyte progenitor cells and determine that Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), a key controller of nuclear trafficking in oligodendrocytes. Examination of expression profiles of 2 different cell stages exposed to siRNA vs. control
2016-04-01 | E-GEOD-79244 | biostudies-arrayexpress