- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Measuring the interaction of transcription factor Nrf2 with its negative regulator Keap1 in single live cells by an improved FRET/FLIM analysis
摘要： Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. Detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualise and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured Instrument Response Function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.
关键词： FRET,live cell imaging,fluorescence lifetime,FLIM,sfGFP,protein-protein interaction,global binning,Keap1,Instrument Response Function,Nrf2
更新于2025-11-21 11:08:12
-
Systems toxicology assessment revealed the impact of graphenea??based materials on cell cycle regulators
摘要： Understanding the cellular and molecular toxicity of graphene and its derivatives is essential for their biomedical applications. Herein, gene expression profile of graphene-exposed cells was retrieved from the GEO database. Differentially expressed genes and their functional roles were then investigated through the pathway, protein-protein interaction network, and module analysis. High degree (hub) and high betweenness centrality (bottleneck) nodes were subsequently identified. The functional analysis of central genes indicated that these graphene-gene interactions could be of great value for further investigation. Accordingly, we also followed the expression of five hub-bottleneck genes in graphene-treated murine peritoneal macrophages and human breast cancer cell line by real-time PCR. The five hub-bottleneck genes related to graphene cytotoxicity; CDK1, CCNB1, PLK1, TOP2A, and CCNA2 were identified through network analysis, which were highly correlated with regulation of cell cycle processes. The module analysis indicated the cell cycle pathway to be the predominant one. Gene expression evaluation showed down-regulation of these genes in the macrophages and cancer cells treated with graphene. These results provided some new intuitions concerning the graphene-cell interactions and unveiled targeting critical cell cycle regulators. The present study indicated some toxic effects of graphene-based materials through systems toxicology assessment. Integrating gene expression and protein-protein interaction network may help explaining biological responses of graphene and lead to beneficial impacts in nanomedicine.
关键词： Cell cycle,Graphene,Protein-protein interaction network,Gene expression,Systems toxicology
更新于2025-09-23 15:19:57
-
Near-infrared light remotely up-regulate autophagy with spatiotemporal precision via upconversion optogenetic nanosystem
摘要： In vivo noninvasively manipulating biological functions by the mediation of biosafe near infrared (NIR) light is becoming increasingly popular. For these applications, upconversion rare-earth nanomaterial holds great promise as a novel photonic element, and has been widely adopted in optogenetics. In this article, an upconversion optogenetic nanosystem that was promised to achieve autophagy up-regulation with spatiotemporal precision was designed. The biocompatible system worked via two separated parts: blue light-receptor optogenetics-autophagy upregulation plasmids for import; and upconversion rods-encapsulated flexible capsule for converting tissue-penetrative NIR light into local visible blue light. Results validated that this system could achieve up-regulation of autophagy in vitro (in both HeLa and 293T cell lines) and remotely penetrate tissue (~3.5mm) in vivo. Since autophagy serves at a central position in intracellular signalling pathways, which is correlative with diverse pathologies, we expect that this method could establish an upconversion material-based autophagy up-regulation strategy for fundamental and clinical applications.
关键词： optogenetics,autophagy,upconversion materials,protein-protein interaction (PPI),near-infrared (NIR) light
更新于2025-09-19 17:15:36
-
Photocaged Quinone Methide Cross-linkers for Light-controlled Chemical Cross-linking of Protein-protein and Protein-DNA Complexes
摘要： Small molecule cross-linkers are invaluable for probing biomolecular interactions and for cross-linking mass spectrometry (CXMS) in addressing large protein complexes and intrinsically disordered proteins. Existing chemical cross-linkers target only a small selection of amino acid residues, limiting the number and type of cross-links, while conventional photocross-linkers target virtually all residues non-selectively, complicating data analysis. Here we report photocaged quinone methide (PQM)-based cross-linkers that are able to multitarget nine nucleophilic residues through specific Michael addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM cross-linkers notably cross-linked Gln, Arg, and Asn hitherto untargetable by existing chemical cross-linkers, markedly increasing the number of residues targetable with a single cross-linker. Such multiplicity of cross-links will increase the abundance of cross-linked peptides for CXMS identification and afford ample constraints to facilitate structural deciphering. PQM cross-linkers were used in vitro, in E. coli, and in mammalian cells to cross-link dimeric proteins and endogenous membrane receptors. The cross-linker NHQM could directly cross-link proteins to DNA, for which few cross-linkers exist. The photoactivatable and multitargeting reactivity of these PQM cross-linkers will substantially enhance chemical cross-linking based technologies for studies of protein-protein and protein-DNA networks and for structural biology.
关键词： DNA-protein interaction,cross-linker,protein-protein interaction,photo-cross-linking,quinone methide
更新于2025-09-19 17:13:59
-
Photocaged Quinone Methide Cross-linkers for Light-controlled Chemical Cross-linking of Protein-protein and Protein-DNA Complexes
摘要： Small molecule cross-linkers are invaluable for probing biomolecular interactions and for cross-linking mass spectrometry (CXMS) in addressing large protein complexes and intrinsically disordered proteins. Existing chemical cross-linkers target only a small selection of amino acid residues, limiting the number and type of cross-links, while conventional photocross-linkers target virtually all residues non-selectively, complicating data analysis. Here we report photocaged quinone methide (PQM)-based cross-linkers that are able to multitarget nine nucleophilic residues through specific Michael addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM cross-linkers notably cross-linked Gln, Arg, and Asn hitherto untargetable by existing chemical cross-linkers, markedly increasing the number of residues targetable with a single cross-linker. Such multiplicity of cross-links will increase the abundance of cross-linked peptides for CXMS identification and afford ample constraints to facilitate structural deciphering. PQM cross-linkers were used in vitro, in E. coli, and in mammalian cells to cross-link dimeric proteins and endogenous membrane receptors. The cross-linker NHQM could directly cross-link proteins to DNA, for which few cross-linkers exist. The photoactivatable and multitargeting reactivity of these PQM cross-linkers will substantially enhance chemical cross-linking based technologies for studies of protein-protein and protein-DNA networks and for structural biology.
关键词： quinone methide,photo-cross-linking,DNA-protein interaction,cross-linker,protein-protein interaction
更新于2025-09-11 14:15:04
-
Er:YAG Laser and Cyclosporin A Effect on Cell Cycle Regulation of Human Gingival Fibroblast Cells
摘要： Introduction: Periodontitis is a set of inflammatory disorders characterized by periodontal attachment loss and alveolar bone resorption. Because of deficiency in periodontitis mechanical therapy, this study was aimed to explore the molecular influence of the erbium-doped: yttrium aluminum garnet (Er:YAG) laser and cyclosporin A (CsA) on human gingival fibroblasts (HGFs) for improvement in periodontal diseases therapy. Methods: We focused on articles that studied the proteome profiles of HGFs after treatment with laser irradiation and application of CsA. The topological features of differentially expressed proteins were analyzed using Cytoscape Version 3.4.0 followed by module selection from the protein-protein interaction (PPI) network using Cluster ONE plugin. In addition, we performed gene ontology (GO) enrichment analysis for the densely connected region and key proteins in both PPI networks. Results: Analysis of PPI network of Er:YAG laser irradiation on HGFs lead to introducing YWHAZ, VCP, HNRNPU, YWHAE, UBA52, CLTC, FUS and IGHG1 as key proteins while similar analysis revealed that ACAT1, CTSD, ALDOA, ANXA2, PRDX1, LGALS3, ARHGDI and EEF1A1 are the crucial proteins related to the effect of drug. GO enrichment analysis of hub-bottleneck proteins of the 2 networks showed the different significant biological processes and cellular components. The functional enrichments of module of Er:YAG laser network are included as fatty acid transmembrane transport, cytokinesis, regulation of RNA splicing and asymmetric protein localization. There are not any significant clusters in network of HGF treated by CsA. Conclusion: The results indicate that there are 2 separate biomarker panels for the 2 treatment methods.
关键词： cyclosporin A (CsA),human fibroblast cell,Er:YAG laser,Protein-protein interaction (PPI) network analysis,Gene ontology
更新于2025-09-11 14:15:04
-
Detection of soluble expression and in vivo interactions of the inner membrane protein OppC using green fluorescent protein
摘要： In this study, the in vivo interaction system of oligopeptide permease (Opp) proteins was analyzed, and a high expression system of inner membrane protein OppC was constructed by flexible usage of the green fluorescent protein (GFP). The Escherichia coli OppC gene, which encodes a transmembrane component of oligopeptide transporter, was cloned into different vectors. Recombinant plasmids were transformed into different E. coli strains, and the expression conditions were optimized. The effect of plasmids and expression strains on OppC production was evaluated by in-gel and western blot analyses. OppC produced by the pWaldo-GFPe vector, harboring the GFP reporter gene, transformed into E. coli C43(DE3) provided sufficient functional protein for biochemical and biophysical studies. In vivo protein-protein interactions were detected among oligopeptide permease proteins using a GFP fragment reassembly protocol. The substrate binding protein OppA showed no interaction with the other components, while the ATP-binding component OppD did not interact with OppF. OppD and OppF interacted with the transmembrane components OppB and OppC. OppB also showed direct interaction with OppC. In vivo OppC functionality was determined by constructing an OppC gene deletion strain. OppC was shown to be essential for peptide uptake, and non-essential for cell viability. These results could help in elucidating the oligopeptide transport mechanism in bacteria.
关键词： Oligopeptide permease,Protein-protein interaction,Inner membrane protein,Green fluorescent protein
更新于2025-09-10 09:29:36
-
Photolytic Labeling and Its Applications in Protein Drug Discovery and Development
摘要： In this mini-review, the major types of photolytic labeling reagents are presented together with their reaction mechanisms. The applications of photolytic labeling in protein drug discovery and development are then discussed; these have expanded from studies of protein-protein interactions in vivo to protein-matrix interactions in lyophilized solids. The mini-review concludes with recommendations for further development of the approach, which include the need for new and more chemically diverse photo-reactive reagents and better understanding of the mechanisms of photolytic labeling reactions in various media.
关键词： protein drug,diazirine,lyophilized solid,photolytic labeling,carbene,protein-protein interaction,benzophenone,phenyl azide
更新于2025-09-09 09:28:46