Tumor suppressor p53 is a key player in the cell response to DNA damage that suffers by frequent inactivating aberrations. Some of them disturb p53 oligomerization and influence cell decision between proliferation, growth arrest and apoptosis. Active p53 resides mostly in the nucleus, degradation occurs in the cytoplasm. Acute myeloid leukemia (AML)-related mutation of NPM (NPMmut) induces massive mislocalization of p53 to the cytoplasm, which might be related to leukemia initiation. Since both proteins interact and execute their function as oligomers, we investigated the role of perturbed p53 oligomerization in the p53 mislocalization process in live cells by FLIM (fluorescence lifetime imaging microscopy), fluorescence anisotropy imaging (FAIM), fluorescence cross-correlation spectroscopy (FCCS) and immunochemical methods. On a set of fluorescently labeled p53 variants, monomeric R337G and L344P, dimeric L344A, and multimeric D352G and A353S, we correlated their cellular localization, oligomerization and interaction with NPMmut. Interplay between nuclear export signal (NES) and nuclear localization signal (NLS) of p53 was investigated as well. While NLS was found critical for the nuclear p53 localization, NES plays less significant role. We observed cytoplasmic translocation only for multimeric A353S variant with sufficient stability and strong interaction with NPMmut. Less stable multimer D352G and L344A dimer were not translocated, monomeric p53 variants always resided in the nucleus independently of the presence of NPMmut and NES intactness. Oligomeric state of NPMmut is not required for p53 translocation, which happens also in the presence of the nonoligomerizing NPMmut variant. The prominent structural and functional role of the R337 residue is shown.
- MeSH
- akutní myeloidní leukemie * genetika metabolismus MeSH
- buněčné jádro metabolismus MeSH
- cytoplazma metabolismus MeSH
- jaderné lokalizační signály metabolismus MeSH
- jaderné proteiny * genetika metabolismus MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- mutace * MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 * metabolismus genetika chemie MeSH
- nukleofosmin MeSH
- signály pro jaderný export MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations.
- Publikační typ
- časopisecké články MeSH
Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1. We observe joint oligomers formed from the fluorescently labeled NPM. Having validated the in vivo methods, we study an effect of substitutions in cysteine 21 (Cys21) of the NPM N-terminus on the oligomerization to demonstrate applicability of the methods. Inhibitory effect of mutations of the Cys21 to nonpolar Ala or to aromatic Phe on the oligomerization was reported in literature using in vitro semi-native electrophoresis. However, we do not detect any break-up of the joint NPM oligomers due to the Cys21 mutations in live cells. In vivo microscopy observations are supported by an in vitro method, the GFP-Trap immunoprecipitation assay. Our results therefore show importance of utilizing several methods for detection of biologically relevant protein aggregates. In vivo monitoring of the NPM oligomerization, a potential cancer therapy target, by the presented methods offers a new way to monitor effects of drugs that are tested as NPM oligomerization inhibitors directly in live cells.
- MeSH
- jaderné proteiny metabolismus MeSH
- lidé MeSH
- proteinové agregáty fyziologie MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Proteoliposomes carrying reconstituted yeast plasma membrane H(+)-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase. Its Deltapsi response can be calibrated by the K(+)/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Deltapsi and DeltapH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.
- MeSH
- arylsulfonany chemie MeSH
- biologické modely MeSH
- buněčná membrána fyziologie chemie MeSH
- elektrochemie MeSH
- financování organizované MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční spektrometrie MeSH
- isoxazoly chemie MeSH
- koncentrace vodíkových iontů MeSH
- membránové potenciály MeSH
- proteolipidy chemie MeSH
- protonové ATPasy chemie MeSH
- protony MeSH
