RESUMO
CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.
Assuntos
Proteínas Serina-Treonina Quinases , Caseína Quinase Idelta , Humanos , Fosforilação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transdução de Sinais , Especificidade por Substrato , TreoninaRESUMO
Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA-binding protein TAR DNA-binding protein (TDP-43), is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1δ-mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic. Multi-scale molecular dynamics simulations reveal reduced homotypic interactions of TDP-43 low-complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We speculate that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Agregados Proteicos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The p53 homolog TAp63α is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By measuring activation kinetics in ovaries and single-site phosphorylation kinetics in vitro with peptides and full-length protein, we show that TAp63α phosphorylation follows a biphasic behavior. Although the first two CK1 phosphorylation events are fast, the third one, which constitutes the decisive step to form the active conformation, is slow. Structure determination of CK1 in complex with differently phosphorylated peptides reveals the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63α substrate interaction in which the product of one phosphorylation step acts as an inhibitor for the following one.
Assuntos
Apoptose/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Domínio Catalítico , Dano ao DNA , Feminino , Humanos , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Oócitos , Fosforilação , Conformação Proteica , Fatores de Tempo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The production of haploid gametes through meiosis is central to the principle of sexual reproduction. The genetic diversity is further enhanced by exchange of genetic material between homologous chromosomes by the crossover mechanism. This mechanism not only requires correct pairing of homologous chromosomes but also efficient repair of the induced DNA double-strand breaks. Oocytes have evolved a unique quality control system that eliminates cells if chromosomes do not correctly align or if DNA repair is not possible. Central to this monitoring system that is conserved from nematodes and fruit fly to humans is the p53 protein family, and in vertebrates in particular p63. In mammals, oocytes are stored for a long time in the prophase of meiosis I which, in humans, can last more than 50 years. During the entire time of this arrest phase, the DNA damage checkpoint remains active. The treatment of female cancer patients with DNA damaging irradiation or chemotherapeutics activates this checkpoint and results in elimination of the oocyte pool causing premature menopause and infertility. Here, we review the molecular mechanisms of this quality control system and discuss potential therapeutic intervention for the preservation of the oocyte pool during chemotherapy.
Assuntos
Morte Celular/genética , Dano ao DNA/genética , Oócitos/fisiologia , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , HumanosRESUMO
Germ cells are totipotent and, in principle, immortal as they are the source for new germ cells in each generation. This very special role requires tight quality control systems. The p53 protein family constitutes one of the most important quality surveillance systems in cells. Whereas p53 has become famous for its role as the guardian of the genome in its function as the most important somatic tumor suppressor, p63 has been nicknamed 'guardian of the female germ line'. p63 is strongly expressed in resting oocytes and responsible for eliminating those that carry DNA double-strand breaks. The third family member, p73, acts later during oocyte and embryo development by ensuring correct assembly of the spindle assembly checkpoint. In addition to its role in the female germ line, p73 regulates cell-cell contacts between developing sperm cells and supporting somatic cells in the male germ line. Here, we review the involvement of the p53 protein family in the development of germ cells with a focus on quality control in the female germ line and discuss medical implications for cancer patients.
RESUMO
Sulfonamides are one of the most important pharmacophores in medicinal chemistry, and sulfonamide analogues have gained substantial interest in recent years. However, the protein interactions of sulfonamides and especially of their analogues are underexplored. Using FKBP12 as a model system, we describe the synthesis of optically pure sulfenamide, sulfinamide, and sulfonimidamide analogues of a well characterized sulfonamide ligand. This allowed us to precisely determine the binding contributions of each sulfonamide oxygen atom and the consequences of nitrogen replacements. We also present high-resolution cocrystal structures of sulfonamide analogues buried in the pocket of a protein target. This revealed intimate contacts with the protein including an unprecedented hydrogen bond acceptor of sulfonimidamides. The use of sulfonamide analogues enabled new exit vectors that allowed remodeling of a subpocket in FKBP12. Our results illuminate the protein interaction potential of sulfonamides/sulfonamide analogues and will aid in their rational design.
RESUMO
The p53 family member TAp63α plays an important role in maintaining the genetic integrity in oocytes. DNA damage, in particular DNA double strand breaks, lead to the transformation of the inhibited, only dimeric conformation into the active tetrameric one that results in the initiation of an apoptotic program. Activation requires phosphorylation by the kinase CK1 which phosphorylates TAp63α at four positions. The third phosphorylation event is the decisive step that transforms TAp63α into the active state. This third phosphorylation, however, is ~ 20 times slower than the first two phosphorylation events. This difference in the phosphorylation kinetics constitutes a safety mechanism that allows oocytes with a low degree of DNA damage to survive. So far these kinetic investigations of the phosphorylation steps have been performed with the isolated CK1 kinase domain. However, all CK1 enzymes contain C-terminal extensions that become auto-phosphorylated and inhibit the activity of the kinase. Here we have investigated the effect of auto-phosphorylation of the C-terminus in the kinase CK1δ and show that it slows down phosphorylation of the first two sites in TAp63α but basically inhibits the phosphorylation of the third site. We have identified up to ten auto-phosphorylation sites in the CK1δ C-terminal domain and show that all of them interact with the kinase domain in a "fuzzy" way in which not a single site is particularly important. Through mutation analysis we further show that hydrophobic amino acids following the phosphorylation site are important for a substrate to be able to successfully compete with the auto-inhibitory effect of the C-terminal domain. This auto-phosphorylation adds a new layer to the regulation of apoptosis in oocytes.
Assuntos
Apoptose , Dano ao DNA , Fosforilação , Oócitos/metabolismo , Quebras de DNA de Cadeia DuplaRESUMO
The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.
Assuntos
Carcinoma de Células Escamosas , Fatores de Transcrição , Animais , Humanos , Camundongos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Proteínas de Repetição de Anquirina Projetadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.
Assuntos
Proteínas de Repetição de Anquirina Projetadas , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Proteína Tumoral p73/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , DNA/químicaRESUMO
Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.
Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Di-Hidropiridinas/farmacologia , Desenho de Fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Células Cultivadas , Di-Hidropiridinas/síntese química , Di-Hidropiridinas/química , Relação Dose-Resposta a Droga , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Masculino , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery.
Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas , NF-kappa B/antagonistas & inibidores , Engenharia de Proteínas , Ubiquitina/antagonistas & inibidores , Produtos Biológicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , NF-kappa B/química , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
Combinatorial selective isotope labeling is a valuable tool to facilitate polypeptide backbone resonance assignment in cases of low sensitivity or extensive chemical shift degeneracy. It involves recording of 15N-HSQC and 2D HN-projections of triple-resonance spectra on a limited set of samples containing different combinations of labeled and unlabeled amino acid types. Using labeling schemes in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes - individually as well as simultaneously - usually yields abundant amino-acid type information of consecutive residues i and iâ¯-â¯1. Although this results in a large number of anchor points that can be used in the sequential assignment process, for most amide signals the exact positioning of the corresponding residue the polypeptide sequence still relies on matching intra- and interresidual 13C chemical shifts obtained from 3D spectra. An obvious way to obtain more sequence-specific assignments directly with combinatorial labeling would be to increase the number of samples. This is, however, undesirable because of increased sample preparation efforts and costs. Irrespective of the number of samples, unambiguous assignments cannot be accomplished for iâ¯-â¯1/i pairs that are not unique in the sequence. Here we show that the ambiguity for non-unique pairs can be resolved by including information about the labeling state of residues iâ¯+â¯1 and iâ¯-â¯2. Application to a 35-residue peptide resulted in complete assignments of all detectable signals in the 15N HSQC which, due to its repetitive sequence and 13C chemical shift degeneracies, was difficult to achieve by other means. For a medium-sized protein (165 residues, rotational correlation time 8.2â¯ns) the improved protocol allowed the extent of backbone amide assignment to be expanded to 88% solely using a suite of 2D 1H-15N correlated spectra.
Assuntos
Técnicas de Química Combinatória , Nitrogênio/química , Peptídeos/química , Amidas/química , Aminoácidos/química , Isótopos de Carbono , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Dobramento de Proteína , Espécies Reativas de NitrogênioRESUMO
The p53 homolog p63 plays important roles in development of epithelial tissues and quality control in germ cells. These two functions are executed by two distinct isoforms of p63. They are created by different promotors resulting in isoforms having either an N-terminal transactivation domain (TAp63) or a truncated form (ΔNp63). In addition to these two N-terminal isoforms a third one with an even longer N-terminus, named TA*p63, has been found. A fourth N-terminal isoform, GTAp63, that closely resembles TA*p63 was discovered in male germ cells where it is involved in genetic quality control. Here, we characterize TA*p63α and GTAp63α and show that their N-terminal extensions stabilize the closed and only dimeric conformation adopted by the shorter TAp63α protein. Both proteins can be activated by the two kinases Chk2 and CK1 resulting in the open tetrameric state. In this conformation, the N-terminal extension acts as an additional transactivation domain enhancing transcriptional activity. Through this mechanism, the difference in transcriptional activity between the repressed and the active state of the protein gets enhanced relative to TAp63α. Finally, we show by mass spectrometry that TA*p63α is expressed in the breast cancer cell line Sum159 at the protein level together with mutant p53. Upon doxorubicin treatment, TA*p63α gets activated, providing a potential new tool to fight cancer.
Assuntos
Fatores de Transcrição/genética , Ativação Transcricional , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Domínios Proteicos , Isoformas de Proteínas , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
We aimed to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy number variants were assessed in three OFC families using microarray analysis. Subsequently, we analyzed TP63 in a cohort of 1072 individuals affected with OFC and 706 population-based controls using molecular inversion probes (MIPs). We identified partial deletions of TP63 in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant c.569_576del (p.(Ala190Aspfs*5)) and a nonsense variant c.997C>T (p.(Gln333*)) that introduces a premature stop codon in the DNA-binding domain. In addition, we identified the first missense variants in the oligomerization domain c.1213G>A (p.(Val405Met)), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant-negative. All of these variants were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Our data indicate that loss-of-function alleles in TP63 can also give rise to OFC as the main phenotype. We have uncovered the dosage-dependent functions of p63, which were previously rejected.
Assuntos
Alelos , Sequência de Bases , Fenda Labial/genética , Fissura Palatina/genética , Mutação com Perda de Função , Deleção de Sequência , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Adulto , Substituição de Aminoácidos , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido IncorretoRESUMO
The p53 family of transcription factors (p53, p63 and p73) covers a wide range of functions critical for development, homeostasis and health of mammals across their lifespan. Beside the well-established tumor suppressor role, recent evidence has highlighted novel non-oncogenic functions exerted by p73. In particular, p73 is required for multiciliated cell (MCC) differentiation; MCCs have critical roles in brain and airways to move fluids across epithelial surfaces and to transport germ cells in the reproductive tract. This novel function of p73 provides a unifying cellular mechanism for the disparate inflammatory and immunological phenotypes of p73-deficient mice. Indeed, mice with Trp73 deficiency suffer from hydrocephalus, sterility and chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance since MCCs are essential for cleaning airways from inhaled pollutants, pathogens and allergens. Cross-species genomic analyses and functional rescue experiments identify TAp73 as the master transcriptional integrator of ciliogenesis, upstream of previously known central nodes. In addition, TAp73 shows a significant ability to regulate cellular metabolism and energy production through direct transcriptional regulation of several metabolic enzymes, such as glutaminase-2 and glucose-6 phosphate dehydrogenase. This recently uncovered role of TAp73 in the regulation of cellular metabolism strongly affects oxidative balance, thus potentially influencing all the biological aspects associated with p73 function, including development, homeostasis and cancer. Although through different mechanisms, p63 isoforms also contribute to regulation of cellular metabolism, thus indicating a common route used by all family members to control cell fate. At the structural level, the complexity of p73's function is further enhanced by its ability to form heterotetramers with some p63 isoforms, thus indicating the existence of an intrafamily crosstalk that determines the global outcome of p53 family function. In this review, we have tried to summarize all the recent evidence that have emerged on the novel non-oncogenic roles of p73, in an attempt to provide a unified view of the complex function of this gene within its family.
Assuntos
Cílios/fisiologia , Proteína Tumoral p73/fisiologia , Aminoácidos/fisiologia , Animais , Axonema/fisiologia , Cílios/ultraestrutura , Epiderme/crescimento & desenvolvimento , Humanos , Metabolismo , Camundongos , Estresse Oxidativo , Sistema Respiratório/ultraestrutura , Fatores de Transcrição/fisiologia , Transcrição Gênica , Proteína Tumoral p73/química , Proteína Tumoral p73/genéticaRESUMO
Despite high sequence homology among the p53 family members, the regulation of their transactivation potential is based on strikingly different mechanisms. Previous studies revealed that the activity of TAp63α is regulated via an autoinhibitory mechanism that keeps inactive TAp63α in a dimeric conformation. While all p73 isoforms are constitutive tetramers, their basal activity is much lower compared with tetrameric TAp63. We show that the dimeric state of TAp63α not only reduces DNA binding affinity, but also suppresses interaction with the acetyltransferase p300. Exchange of the transactivation domains is sufficient to transfer the regulatory characteristics between p63 and p73. Structure determination of the transactivation domains of p63 and p73 in complex with the p300 Taz2 domain further revealed that, in contrast to p53 and p73, p63 has a single transactivation domain. Sequences essential for stabilizing the closed dimer of TAp63α have evolved into a second transactivation domain in p73 and p53.
Assuntos
DNA/química , Proteína p300 Associada a E1A/química , Fatores de Transcrição/química , Ativação Transcricional , Proteína Tumoral p73/química , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de Tumor/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Clonagem Molecular , DNA/genética , DNA/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Neurônios , Osteoblastos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.
Assuntos
Dano ao DNA , Oócitos/fisiologia , Fosfoproteínas/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Transativadores/metabolismo , Animais , Apoptose , Feminino , Camundongos , Fosforilação , Controle de QualidadeRESUMO
Members of the p53 tumor-suppressor family are expressed as multiple isoforms. Isoforms with an N-terminal transactivation domain are transcriptionally active, while those ones lacking this domain often inhibit the transcriptional activity of other family members. In squamous cell carcinomas, the high expression level of ΔNp63α inhibits the tumor-suppressor function of TAp73ß. This can in principle be due to blocking of the promoter or by direct interaction between both proteins. p63 and p73 can hetero-oligomerize through their tetramerization domains and a hetero-tetramer consisting of two p63 and two p73 molecules is thermodynamically more stable than both homo-tetramers. Here we show that cells expressing both p63 and p73 exist in mouse epidermis and hair follicle and that hetero-tetramer complexes can be detected by immunoprecipitation in differentiating keratinocytes. Through structure determination of the hetero-tetramer, we reveal why this hetero-tetramer is the thermodynamically preferred species. We have created mutants that exclusively form either hetero-tetramers or homo-tetramers, allowing to investigate the function of these p63/p73 hetero-tetramers. Using these tools, we show that inhibition of TAp73ß in squamous cell carcinomas is due to promoter squelching and not direct interaction.