FOXO4 Dotaz Zobrazit nápovědu
Transcription factor p53 protects cells against tumorigenesis when subjected to various cellular stresses. Under these conditions, p53 interacts with transcription factor Forkhead box O (FOXO) 4, thereby inducing cellular senescence by upregulating the transcription of senescence-associated protein p21. However, the structural details of this interaction remain unclear. Here, we characterize the interaction between p53 and FOXO4 by NMR, chemical cross-linking, and analytical ultracentrifugation. Our results reveal that the interaction between p53 TAD and the FOXO4 Forkhead domain is essential for the overall stability of the p53:FOXO4 complex. Furthermore, contacts involving the N-terminal segment of FOXO4, the C-terminal negative regulatory domain of p53 and the DNA-binding domains of both proteins stabilize the complex, whose formation blocks p53 binding to DNA but without affecting the DNA-binding properties of FOXO4. Therefore, our structural findings may help to understand the intertwined functions of p53 and FOXO4 in cellular homeostasis, longevity, and stress response.
The FOXO forkhead transcription factors are potent transcriptional activators involved in a wide range of key biological processes. In this work, the real-time kinetics of the interaction between the FOXO4-DNA binding domain (FOXO4-DBD) and the DNA was studied by using surface plasmon resonance (SPR). SPR analysis revealed that the interaction between FOXO4-DBD and the double stranded DNA containing either the insulin-responsive or the Daf-16 family member-binding element is preferably described by using a conformational change model which suggests a structural change of FOXO4-DBD upon binding to the DNA. This was further confirmed by using the time-resolved tryptophan fluorescence anisotropy decay measurements which revealed profound reduction of segmental dynamics of FOXO4-DBD upon the complex formation. Alanine scanning of amino acid residues engaged in polar contacts with the DNA showed that certain non-specific contacts with the DNA backbone are very important for both the binding affinity and the binding specificity of FOXO4-DBD.
The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS). Masking or obscuring of NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equilibrium of FoxO localization toward the cytoplasm. According to our best knowledge, there is no experimental evidence showing a direct interaction between the 14-3-3 protein and NLS of FoxO. Therefore, the main goal of this work was to investigate whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FoxO4 NLS. We have used site-directed labeling of FoxO4 NLS with the extrinsic fluorophore 1,5-IAEDANS in conjunction with steady-state and time-resolved fluorescence spectroscopy to study conformational changes of FoxO4 NLS in vitro. Our data show that the 14-3-3 protein binding significantly changes the environment around AEDANS-labeled NLS and reduces its flexibility. On the other hand, the phosphorylation itself and the binding of double-stranded DNA have a small effect on the structure of this region. Our results also suggest that the DNA-binding domain of FoxO4 remains relatively mobile while bound to the 14-3-3 protein.
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- financování organizované MeSH
- fluorescenční spektrometrie MeSH
- fosforylace MeSH
- kinetika MeSH
- klonování DNA MeSH
- konzervovaná sekvence MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- proteiny 14-3-3 metabolismus MeSH
- rekombinantní proteiny chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- transkripční faktory chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
FoxO4 belongs to the "O" subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel beta-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors. In this report, we investigated the role of the N-terminal loop (portion located upstream of first helix H1) and the C-terminal region (loop known as wing W2) of the forkhead domain of transcription factor FoxO4 in DNA binding. Although the deletion of either portion partly reduces the FoxO4-DBD binding to the DNA, the simultaneous deletion of both regions inhibits DNA binding significantly. Förster resonance energy transfer measurements and molecular dynamics simulations suggest that both studied N- and C-terminal regions of FoxO4-DBD directly interact with DNA. In the presence of the N-terminal loop the protein kinase B-induced phosphorylation of wing W2 by itself has negligible effect on DNA binding. On the other hand, in the absence of this loop the phosphorylation of wing W2 significantly inhibits the FoxO4-DBD binding to the DNA. The binding of the 14-3-3 protein efficiently reduces DNA-binding potential of phosphorylated FoxO4-DBD regardless of the presence of the N-terminal loop. Our results show that both N- and C-terminal regions of forkhead domain are important for stability of the FoxO4-DBD.DNA complex
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- anizotropie MeSH
- delece genu MeSH
- DNA chemie MeSH
- financování organizované MeSH
- fosforylace MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- proteiny 14-3-3 chemie MeSH
- protoonkogenní proteiny c-akt chemie MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- terciární struktura proteinů MeSH
- transkripční faktory chemie metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
FOXO4 is a member of the FOXO subgroup of forkhead transcription factors that constitute key components of a conserved signalling pathway that connects growth and stress signals to transcriptional control. Here, the 1.9 Å resolution crystal structure of the DNA-binding domain of human FOXO4 (FOXO4-DBD) bound to a 13 bp DNA duplex containing a FOXO consensus binding sequence is reported. The structure shows a similar recognition of the core sequence as has been shown for two other FOXO proteins. Helix H3 is docked into the major groove and provides all of the base-specific contacts, while the N-terminus and wing W1 make additional contacts with the phosphate groups of DNA. In contrast to other FOXO-DBD-DNA structures, the loop between helices H2 and H3 has a different conformation and participates in DNA binding. In addition, the structure of the FOXO4-DBD-DNA complex suggests that both direct water-DNA base contacts and the unique water-network interactions contribute to FOXO-DBD binding to the DNA in a sequence-specific manner.
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- DNA chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- strukturní homologie proteinů MeSH
- terciární struktura proteinů MeSH
- transkripční faktory chemie metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.
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- DNA vazebné proteiny chemie metabolismus MeSH
- DNA chemie metabolismus MeSH
- hmotnostní spektrometrie MeSH
- molekulární struktura MeSH
- responzivní elementy MeSH
- transkripční faktory chemie metabolismus MeSH
- vodík-deuteriová výměna MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
FOXO transcription factors regulate cellular homeostasis, longevity and response to stress. FOXO1 (also known as FKHR) is a key regulator of hepatic glucose production and lipid metabolism, and its specific inhibition may have beneficial effects on diabetic hyperglycemia by reducing hepatic glucose production. Moreover, all FOXO proteins are considered potential drug targets for drug resistance prevention in cancer therapy. However, the development of specific FOXO inhibitors requires a detailed understanding of structural differences between individual FOXO DNA-binding domains. The high-resolution structure of the DNA-binding domain of FOXO1 reported in this study and its comparison with structures of other FOXO proteins revealed differences in both their conformation and flexibility. These differences are encoded by variations in protein sequences and account for the distinct functions of FOXO proteins. In particular, the positions of the helices H1, H2 and H3, whose interface form the hydrophobic core of the Forkhead domain, and the interactions between hydrophobic residues located on the interface between the N-terminal segment, the H2-H3 loop, and the recognition helix H3 differ among apo FOXO1, FOXO3 and FOXO4 proteins. Therefore, the availability of apo structures of DNA-binding domains of all three major FOXO proteins will support the development of FOXO-type-specific inhibitors.
- MeSH
- forkhead box protein O1 chemie genetika metabolismus MeSH
- forkhead transkripční faktory chemie genetika metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární modely MeSH
- myši MeSH
- protein FOXO3 chemie genetika metabolismus MeSH
- proteinové domény MeSH
- sekundární struktura proteinů MeSH
- sekvenční analýza proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
GLI1 fusions involving ACTB, MALAT1, PTCH1 and FOXO4 genes have been reported in a subset of malignant mesenchymal tumors with a characteristic nested epithelioid morphology and frequent S100 positivity. Typically, these multilobulated tumors consist of uniform epithelioid cells with bland nuclei and are organized into distinct nests and cords with conspicuously rich vasculature. We herein expand earlier findings by reporting a case of a 34-year-old female with an epithelioid mesenchymal tumor of the palate. The neoplastic cells stained positive for S100 protein and D2-40, whereas multiple other markers were negative. Genetic alterations were investigated by targeted RNA sequencing, and a PTCH1-GLI1 fusion was detected. Epithelioid mesenchymal tumors harboring a PTCH1-GLI1 fusion are vanishingly rare with only three cases reported so far. Due to the unique location in the mucosa of the soft palate adjacent to minor salivary glands, multilobulated growth, nested epithelioid morphology, focal clearing of the cytoplasm, and immunopositivity for S100 protein and D2-40, the differential diagnoses include primary salivary gland epithelial tumors, in particular myoepithelioma and myoepithelial carcinoma. Another differential diagnostic possibility is the ectomesenchymal chondromyxoid tumor. Useful diagnostic clues for tumors with a GLI1 rearrangement include a rich vascular network between the nests of neoplastic cells, tumor tissue bulging into vascular spaces, and absence of SOX10, GFAP and cytokeratin immunopositivity. Identifying areas with features of GLI1-rearranged tumors should trigger subsequent molecular confirmation. This is important for appropriate treatment measures as PTCH1-GLI1 positive mesenchymal epithelioid neoplasms have a propensity for locoregional lymph node and distant lung metastases.
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- dospělí MeSH
- lidé MeSH
- myoepiteliální nádor * patologie MeSH
- nádorové biomarkery genetika metabolismus MeSH
- nádory měkkých tkání * patologie MeSH
- nádory slinných žláz * MeSH
- patro měkké patologie MeSH
- protein Gli1 genetika metabolismus MeSH
- proteiny S100 MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- přehledy MeSH
