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

Somatic JAK2 mutations are the main molecular cause of the vast majority of polycythemia vera (PV) cases. According to a recent structural model, the prevalent acquired V617F mutation improves the stability of the JAK2 dimer, thereby enhancing the constitutive JAK2 kinase activity. Germline JAK2 mutations usually do not largely alter JAK2 signaling, although they may modulate the impact of V617F. We found an unusual germline JAK2 mutation L604F in homozygous form in a young PV patient, along with a low allele burden JAK2 V617F mutation, and in her apparently healthy sister. Their father with a PV-like disease had L604F in a heterozygous state, without V617F. The functional consequences of JAK2 L604Fmutation were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells were transfected with plasmids for exogenous JAK2-GFP expression, and (ii) endogenous JAK2 modifications were introduced into HeLa cells using CRISPR/Cas9. Both mutations significantly increased JAK2 constitutive activity in transfected HEK293T cells. In the second model, JAK2 modification resulted in reduced total JAK2 protein levels. An important difference was also detected: as described previously, the effect of V617F on JAK2 kinase activity was abrogated in the absence of the aromatic residue F595. In contrast, JAK2 hyperactivation by L604F was only partially inhibited by the F595 change to alanine. We propose that the L604F mutation increases the probability of spontaneous JAK2 dimer formation, which is physiologically mediated by F595. In addition, L604F may contribute to dimer stabilization similarly to V617F.

• MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• Janus kinasa 2 genetika   MeSH
• lidé MeSH
• mutace MeSH
• zárodečné buňky * MeSH
• zárodečné mutace * MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví 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

• Publikační typ
• abstrakt z konference MeSH