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1.
Cell ; 152(1-2): 132-43, 2013 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-23332751

RESUMO

The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways.


Assuntos
Fator de Ligação a CCAAT/química , Sequência de Aminoácidos , Animais , Fator de Ligação a CCAAT/metabolismo , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Choque Térmico HSP72/genética , Histonas/química , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Ubiquitinação
2.
Genomics ; 114(4): 110390, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35589059

RESUMO

NF-Y is a trimeric pioneer Transcription Factor (TF) whose target sequence -the CCAAT box- is present in ~25% of mammalian promoters. We reconstruct the phylogenetic history of the regulatory NF-YA subunit in vertebrates. We find that in addition to the remarkable conservation of the subunits-interaction and DNA-binding parts, the Transcriptional Activation Domain (TAD) is also conserved (>90% identity among bony vertebrates). We infer the phylogeny of the alternatively spliced exon-3 and partial splicing events of exon-7 -7N and 7C- revealing independent clade-specific losses of these regions. These isoforms shape the TAD. Absence of exon-3 in basal deuterostomes, cartilaginous fishes and hagfish, but not in lampreys, suggests that the "short" isoform is primordial, with emergence of exon-3 in chordates. Exon 7N was present in the vertebrate common ancestor, while 7C is a molecular innovation of teleost fishes. RNA-seq analysis in several species confirms expression of all these isoforms. We identify 3 blocks of amino acids in the TAD shared across deuterostomes, yet structural predictions and sequence analyses suggest an evolutionary drive for maintenance of an Intrinsically Disordered Region -IDR- within the TAD. Overall, these data help reconstruct the logic for alternative splicing of this essential eukaryotic TF.


Assuntos
Fatores de Transcrição , Vertebrados , Processamento Alternativo , Animais , Evolução Molecular , Peixes/metabolismo , Mamíferos , Filogenia , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/genética , Vertebrados/genética
3.
Plant J ; 105(1): 49-61, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33098724

RESUMO

NF-Y transcription factor comprises three subunits: NF-YA, NF-YB and NF-YC. NF-YB and NF-YC dimerize through their histone fold domain (HFD), which can bind DNA in a non-sequence-specific fashion while serving as a scaffold for NF-YA trimerization. Upon trimerization, NF-YA specifically recognizes the CCAAT box sequence on promoters and enhancers. In plants, each NF-Y subunit is encoded by several genes giving rise to hundreds of potential heterotrimeric combinations. In addition, plant NF-YBs and NF-YCs interact with other protein partners to recognize a plethora of genomic motifs, as the CCT protein family that binds CORE sites. The NF-Y subunit organization and its DNA-binding properties, together with the NF-Y HFD capacity to adapt different protein modules, represent plant-specific features that play a key role in development, growth and reproduction. Despite their relevance, these features are still poorly understood at the molecular level. Here, we present the structures of Arabidopsis and rice NF-YB/NF-YC dimers, and of an Arabidopsis NF-Y trimer in complex with the FT CCAAT box, together with biochemical data on NF-Y mutants. The dimeric structures identify the key residues for NF-Y HFD stabilization. The NF-Y/DNA structure and the mutation experiments shed light on HFD trimerization interface properties and the NF-YA sequence appetite for the bases flanking the CCAAT motif. These data explain the logic of plant NF-Y gene expansion: the trimerization adaptability and the flexible DNA-binding rules serve the scopes of accommodating the large number of NF-YAs, CCTs and possibly other NF-Y HFD binding partners and a diverse audience of genomic motifs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fator de Ligação a CCAAT/metabolismo , DNA de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Fator de Ligação a CCAAT/química , Fator de Ligação a CCAAT/genética , DNA de Plantas/química , Dimerização , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína
4.
Plant Cell ; 29(6): 1516-1532, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28526714

RESUMO

Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence specificity. NF-YA shares a conserved domain with the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) proteins. We show that CONSTANS (CO/B-BOX PROTEIN1 BBX1), a master flowering regulator, forms a trimer with Arabidopsis thaliana NF-YB2/NF-YC3 to efficiently bind the CORE element of the FLOWERING LOCUS T promoter. We term this complex NF-CO. Using saturation mutagenesis, electrophoretic mobility shift assays, and RNA-sequencing profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site. CO physically interacts with the same HFD surface required for NF-YA association, as determined by mutations in NF-YB2 and NF-YC9, and tested in vitro and in vivo. The co-7 mutation in the CCT domain, corresponding to an NF-YA arginine directly involved in CCAAT recognition, abolishes NF-CO binding to DNA. In summary, a unifying molecular mechanism of CO function relates it to the NF-YA paradigm, as part of a trimeric complex imparting sequence specificity to HFD/DNA interactions. It is likely that members of the large CCT family participate in similar complexes with At-NF-YB and At-NF-YC, broadening HFD combinatorial possibilities in terms of trimerization, DNA binding specificities, and transcriptional regulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , DNA de Plantas/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Monóxido de Carbono/metabolismo , Proteínas de Ligação a DNA/genética , Ligação Proteica , Fatores de Transcrição/genética
5.
FASEB J ; 33(4): 4790-4801, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30589568

RESUMO

Nuclear factor Y (NF-Y) is a transcription factor trimer binding to the functionally important CCAAT box, present in promoters of growth-promoting and cell cycle-regulated genes. The regulatory nuclear factor YA (NF-YA) subunit confers sequence-specificity to the histone-like nuclear factor YB/YC dimer. NF-YA harbors 2 serines-Ser320 and Ser326-shown to be phosphorylated by cyclin-dependent kinase 2. High-throughput proteomics data indicate that they are phosphorylated in vivo. Specifically, Ser320 makes structural contacts with the DNA phosphate backbone; Ser320-P is the major NF-YA phosphorylation isoform following overexpression in HeLa cells, increasing upon mitotic arrest. EMSA with recombinant Ala and Glu mutants confirm a role of Ser320, but not Ser326, in stabilization of DNA binding. Transactivation assays of the CCAAT-dependent MDR1 and RHOB promoters show loss in transcription function for Ser320Glu and Ser320Ala NF-YA mutants. Phylogenetic analysis of NF-YA proteins indicates that Ser320 is indeed evolutionarily conserved. We conclude that phosphorylation of this residue belongs to the core mechanisms of DNA-binding control, possibly driven by the necessity to unfasten binding of or to evict NF-Y from CCAAT sites under specific conditions of growth regulation.-Bernardini, A., Lorenzo, M., Nardini, M., Mantovani, R., Gnesutta, N. The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Serina/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Western Blotting , Fator de Ligação a CCAAT/genética , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína rhoB de Ligação ao GTP/metabolismo
6.
PLoS Genet ; 13(1): e1006530, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28068345

RESUMO

Rice flowering is controlled by changes in the photoperiod that promote the transition to the reproductive phase as days become shorter. Natural genetic variation for flowering time has been largely documented and has been instrumental to define the genetics of the photoperiodic pathway, as well as providing valuable material for artificial selection of varieties better adapted to local environments. We mined genetic variation in a collection of rice varieties highly adapted to European regions and isolated distinct variants of the long day repressor HEADING DATE 1 (Hd1) that perturb its expression or protein function. Specific variants allowed us to define novel features of the photoperiodic flowering pathway. We demonstrate that a histone fold domain scaffold formed by GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 8 (Ghd8) and several NF-YC subunits can accommodate distinct proteins, including Hd1 and PSEUDO RESPONSE REGULATOR 37 (PRR37), and that the resulting OsNF-Y complex containing Hd1 can bind a specific sequence in the promoter of HEADING DATE 3A (Hd3a). Artificial selection has locally favored an Hd1 variant unable to assemble in such heterotrimeric complex. The causal polymorphism was defined as a single conserved lysine in the CCT domain of the Hd1 protein. Our results indicate how genetic variation can be stratified and explored at multiple levels, and how its description can contribute to the molecular understanding of basic developmental processes.


Assuntos
Aclimatação/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Histonas/genética , Histonas/metabolismo , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
PLoS Genet ; 12(12): e1006496, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27977687

RESUMO

Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal "CORE" elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fator de Ligação a CCAAT/genética , Flores/genética , Sequência de Aminoácidos/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/biossíntese , Fator de Ligação a CCAAT/biossíntese , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Ligação Proteica
8.
Hum Mol Genet ; 24(15): 4185-97, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25911675

RESUMO

The p63 transcription factor, homolog to the p53 tumor suppressor gene, plays a crucial role in epidermal and limb development, as its mutations are associated to human congenital syndromes characterized by skin, craniofacial and limb defects. While limb and skin-specific p63 transcriptional targets are being discovered, little is known of the post-translation modifications controlling ΔNp63α functions. Here we show that the p300 acetyl-transferase physically interacts in vivo with ΔNp63α and catalyzes its acetylation on lysine 193 (K193) inducing ΔNp63α stabilization and activating specific transcriptional functions. Furthermore we show that Fibroblast Growth Factor-8 (FGF8), a morphogenetic signaling molecule essential for embryonic limb development, increases the binding of ΔNp63α to the tyrosine kinase c-Abl as well as the levels of ΔNp63α acetylation. Notably, the natural mutant ΔNp63α-K193E, associated to the Split-Hand/Foot Malformation-IV syndrome, cannot be acetylated by this pathway. This mutant ΔNp63α protein displays promoter-specific loss of DNA binding activity and consequent altered expression of development-associated ΔNp63α target genes. Our results link FGF8, c-Abl and p300 in a regulatory pathway that controls ΔNp63α protein stability and transcriptional activity. Hence, limb malformation-causing p63 mutations, such as the K193E mutation, are likely to result in aberrant limb development via the combined action of altered protein stability and altered promoter occupancy.


Assuntos
Anormalidades Congênitas/genética , Fator 8 de Crescimento de Fibroblasto/genética , Proteínas Proto-Oncogênicas c-abl/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Fatores de Transcrição de p300-CBP/genética , Animais , Linhagem Celular , Anormalidades Congênitas/embriologia , Anormalidades Congênitas/patologia , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Fator 8 de Crescimento de Fibroblasto/biossíntese , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Camundongos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/biossíntese , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/metabolismo , Fatores de Transcrição de p300-CBP/biossíntese , Fatores de Transcrição de p300-CBP/metabolismo
9.
Plant Cell ; 26(3): 1009-17, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24610724

RESUMO

For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fator de Ligação a CCAAT/genética , Cromatina/genética , Flores , Regiões Promotoras Genéticas
10.
Plant Cell ; 24(12): 4777-92, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23275578

RESUMO

The CCAAT box is one of the most common cis-elements present in eukaryotic promoters and is bound by the transcription factor NUCLEAR FACTOR Y (NF-Y). NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC. Unlike animals and fungi, plants have significantly expanded the number of genes encoding NF-Y subunits. We provide a comprehensive classification of NF-Y genes, with a separation of closely related, but distinct, histone fold domain proteins. We additionally review recent experiments that have placed NF-Y at the center of many developmental stress-responsive processes in the plant lineage.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteínas de Plantas/metabolismo , Fator de Ligação a CCAAT/química , Fator de Ligação a CCAAT/classificação , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/classificação
11.
Biochim Biophys Acta Rev Cancer ; 1879(2): 189082, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38309445

RESUMO

NF-Y is a Transcription Factor (TF) targeting the CCAAT box regulatory element. It consists of the NF-YB/NF-YC heterodimer, each containing an Histone Fold Domain (HFD), and the sequence-specific subunit NF-YA. NF-YA expression is associated with cell proliferation and absent in some post-mitotic cells. The review summarizes recent findings impacting on cancer development. The logic of the NF-Y regulome points to pro-growth, oncogenic genes in the cell-cycle, metabolism and transcriptional regulation routes. NF-YA is involved in growth/differentiation decisions upon cell-cycle re-entry after mitosis and it is widely overexpressed in tumors, the HFD subunits in some tumor types or subtypes. Overexpression of NF-Y -mostly NF-YA- is oncogenic and decreases sensitivity to anti-neoplastic drugs. The specific roles of NF-YA and NF-YC isoforms generated by alternative splicing -AS- are discussed, including the prognostic value of their levels, although the specific molecular mechanisms of activity are still to be deciphered.


Assuntos
Fator de Ligação a CCAAT , Neoplasias , Humanos , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Fatores de Transcrição/genética , Neoplasias/genética , Isoformas de Proteínas/genética , Regulação da Expressão Gênica
12.
Biochim Biophys Acta ; 1825(2): 131-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22138487

RESUMO

NF-Y is a sequence-specific transcription factor - TF - targeting the common CCAAT promoter element. p53 is a master TF controlling the response to stress signals endangering genome integrity, often mutated in human cancers. The NF-Y/p53 - and p63, p73 - interaction results in transcriptional repression of a subset of genes within the vast NF-Y regulome under DNA-damage conditions. Recent data shows that NF-Y is also involved in pro-apoptotic activities, either directly, by mediating p53 transcriptional activation, or indirectly, by being targeted by a non coding RNA, PANDA. The picture is subverted in cells carrying Gain-of-function mutant p53, through interactions with TopBP1, a protein also involved in DNA repair and replication. In summary, the connection between p53 and NF-Y is crucial in determining cell survival or death.


Assuntos
Fator de Ligação a CCAAT/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Animais , Fator de Ligação a CCAAT/metabolismo , Humanos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo
13.
J Mol Biol ; 433(18): 167119, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34181981

RESUMO

The E2F1 transcription factor is a master regulator of cell-cycle progression whose uncontrolled activation contributes to tumor cells growth. E2F1 binds DNA as a heterodimer with DP partners, resulting in a multi-domain quaternary-structure complex composed of DNA binding domains, a coiled coil domain and a marked box domain separated by short linkers. Building on the 3D knowledge of the single domains of E2F and DPs, we characterized the structure and dynamics of the complete E2F1/DP1/DNA complex by a combination of small-angle X-ray scattering and molecular dynamics simulations. It shows an asymmetric contribution of the dynamics of the two proteins. Namely, the coiled-coil domain leans toward the DP1 side of the complex; the DP1 loop between α2 and α3 of the DBD partially populates a helical structure leaning far from the DNA and in the same direction of the coiled-coil domain; and the N-terminal disordered region of DP1, rich in basic residues, contributes to DNA binding stabilization. Intriguingly, tumor mutations in the flexible regions of the complex suggest that perturbation of protein dynamics could affect protein function in a context-dependent way. Our data suggest fundamental contributions of DP proteins in distinct aspects of E2F biology.


Assuntos
DNA/química , DNA/metabolismo , Fator de Transcrição E2F1/química , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição DP1/química , Fator de Transcrição DP1/metabolismo , Ciclo Celular , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Conformação Proteica
14.
Int J Biol Macromol ; 193(Pt A): 401-413, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34673109

RESUMO

The trimeric CCAAT-binding NF-Y is a "pioneer" Transcription Factor -TF- known to cooperate with neighboring TFs to regulate gene expression. Genome-wide analyses detected a precise stereo-alignment -10/12 bp- of CCAAT with E-box elements and corresponding colocalization of NF-Y with basic-Helix-Loop-Helix (bHLH) TFs. We dissected here NF-Y interactions with USF1 and MAX. USF1, but not MAX, cooperates in DNA binding with NF-Y. NF-Y and USF1 synergize to activate target promoters. Reconstruction of complexes by structural means shows independent DNA binding of MAX, whereas USF1 has extended contacts with NF-Y, involving the USR, a USF-specific amino acid sequence stretch required for trans-activation. The USR is an intrinsically disordered domain and adopts different conformations based on E-box-CCAAT distances. Deletion of the USR abolishes cooperative DNA binding with NF-Y. Our data indicate that the functionality of certain unstructured domains involves adapting to small variation in stereo-alignments of the multimeric TFs sites.


Assuntos
DNA/metabolismo , Fatores Estimuladores Upstream/metabolismo , Regulação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos
15.
J Cell Physiol ; 224(3): 722-33, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20578242

RESUMO

The serine/threonine kinase PAK4 is a Rho GTPases effector protein implicated in many critical biological processes, including regulation of cell morphology and motility, embryonic development, cell survival, response to infection, and oncogenic transformation. Consistently with its pro-oncogenic features, PAK4 was found to be overexpressed in many cancer cell lines and tissues, and to be necessary to promote activation of survival pathways. PAK4, like other Paks, is now considered a promising target for specific therapy. Little is known on its modes of regulation, molecular partners, and substrates. Because the N-terminal regulatory moiety plays important roles in PAK4 activity and functions, even independently of GTPase interactions, in this study we employed an affinity chromatography approach to identify N-terminal domain binding partners. Within this protein region we identified a novel interaction domain involved in association with ribonucleoprotein (RNP) complexes, suggesting PAK4 implications in translational regulation. Indeed, we found that active PAK4 can affect (cap-independent) translation from specific IRES sequences in vivo, and that the N-terminal domain is critical for this regulation. Further, we could establish that within the RNP interacting sequence PAK4 regulatory domain contains targeting elements that drive cytoplasmic localization and act as nuclear export signal. Functional implication of endogenous PAK4 protein, which was found in both cytoplasmic and nuclear fractions, in IRES-mediated translation further underlines the significance of the reported findings. Our data reveal novel means for PAK4 regulation of gene expression, and provide new elements to understand the molecular mechanisms that determine PAK4 cellular localization and functions.


Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/química , Quinases Ativadas por p21/metabolismo , Linhagem Celular , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Frações Subcelulares/metabolismo , Quinases Ativadas por p21/genética
16.
Cells ; 9(11)2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33138093

RESUMO

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo "metabolic rewiring" through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.


Assuntos
Fator de Ligação a CCAAT/antagonistas & inibidores , Fator de Ligação a CCAAT/química , Suramina/química , Suramina/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Sequência de Aminoácidos , Fenômenos Biofísicos , DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Multimerização Proteica , Relação Estrutura-Atividade
17.
J Mol Neurosci ; 37(3): 212-24, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18607774

RESUMO

The brain-specific Ras guanine nucleotide exchange factor RasGRF1 is a protein harbouring a complex array of structural motifs. It contains a pleckstrin homology (PH1) domain, a coiled coil region (CC) and an ilimaquinone (IQ) one in addition to the catalytic Ras and Rac exchange factor domains. In this study, we used the recombinant N-terminal PH1, CC and IQ region (PHCCIQ) fused to the chitin-binding domain to isolate interacting proteins from mouse brain extracts. The use of an advanced software tool, the Pep-Miner, allowed clustering similar spectra from multiple mass spectrometry analysis, simplifying and improving the analysis of the complex peptide mixture. The most representative classes of RasGRF1-interacting proteins were ribosomal and other RNA-binding proteins, cytoskeletal proteins and proteins involved in vesicular trafficking. We confirmed the interaction of some of the identified proteins using different experimental approaches. We also demonstrated an RNA-dependent association of the PHCCIQ moiety of RasGRF1 with ribosomal protein S6 and Ras-GTPase-activating protein SH3-domain binding protein 2. In addition, we found that purified total RNA binds to the PHCCIQ fusion protein and the recombinant protein associates with poly(A)-sepharose. These data indicate that RasGRF1 can interact with different protein categories and exhibits a potential RNA-binding property.


Assuntos
Mapeamento de Interação de Proteínas , Proteoma/análise , ras-GRF1/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Análise em Microsséries , Dados de Sequência Molecular , Ligação Proteica , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , ras-GRF1/genética
18.
Plants (Basel) ; 8(10)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658622

RESUMO

Nuclear Factor Y (NF-Y) is an evolutionarily conserved trimer formed by a Histone-Fold Domain (HFD) heterodimeric module shared by core histones, and the sequence-specific NF-YA subunit. In plants, the genes encoding each of the three subunits have expanded in number, giving rise to hundreds of potential trimers. While in mammals NF-Y binds a well-characterized motif, with a defined matrix centered on the CCAAT box, the specificity of the plant trimers has yet to be determined. Here we report that Arabidopsis thaliana NF-Y trimeric complexes, containing two different NF-YA subunits, bind DNA in vitro with similar affinities. We assayed precisely sequence-specificity by saturation mutagenesis, and analyzed genomic DNA sites bound in vivo by selected HFDs. The plant NF-Y CCAAT matrix is different in nucleotides flanking CCAAT with respect to the mammalian matrix, in vitro and in vivo. Our data point to flexible DNA-binding rules by plant NF-Ys, serving the scope of adapting to a diverse audience of genomic motifs.

19.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 430-440, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30296497

RESUMO

Cell Penetrating Peptides -CPPs- are short aminoacidic stretches present in proteins that have the ability to translocate the plasma membrane and facilitate delivery of various molecules. They are usually rich in basic residues, and organized as alpha helices. NF-Y is a transcription factor heterotrimer formed by two Histone Fold Domain -HFD- subunits and the sequence-specific NF-YA. NF-YA possesses two α-helices rich in basic residues. We show that it efficiently enters cells at nanomolar concentrations in the absence of carrier peptides. Mutagenesis identified at least two separate CPPs in the A1 and A2, which overlap with previously identified nuclear localization signals (NLS). The half-life of the transduced protein is short in human cancer cells, longer in mouse C2C12 myoblasts. The internalized NF-YA is capable of trimerization with the HFD subunits and binding to the target CCAAT box. Functionality is further suggested by protein transfection in C2C12 cells, leading to inhibition of differentiation to myotubes. In conclusion, NF-YA contains CPPs, hinting at novel -and unexpected- properties of this subunit.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Peptídeos Penetradores de Células/metabolismo , Sequência de Aminoácidos , Animais , Fator de Ligação a CCAAT/genética , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Células HCT116 , Células HeLa , Humanos , Camundongos , Mioblastos/metabolismo , Sinais de Localização Nuclear/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transfecção
20.
Trends Plant Sci ; 23(4): 293-301, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29331540

RESUMO

CONSTANS (CO) is a master regulator of flowering time, although the mechanisms underlying its role as a transcriptional regulator are not well understood. The DNA-binding domain of CO shares homology with that of NUCLEAR FACTOR YA (NF-YA), a subunit of the CCAAT-binding trimer NF-Y. Recent publications indicate that CO and its rice homolog HEADING DATE 1 (Hd1) form heterotrimers with the histone-fold subunits of NF-Y to efficiently bind promoter elements in the florigen genes. Differences in the DNA-binding specificities of NF-Y and NF-CO can be conceptualized based on our knowledge of the 3D structure of the NF-Y/CCAAT complex. Here we discuss the modes of assembly of NF-Y-like heterotrimers and possible models for their activity as flexible sequence-specific transcriptional regulators.


Assuntos
DNA de Plantas/fisiologia , Flores/crescimento & desenvolvimento , Histonas/metabolismo , DNA de Plantas/genética , Flores/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Histonas/genética , Histonas/fisiologia , Fotoperíodo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
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