RESUMO
Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.
Assuntos
Proteína de Ligação a CREB , Proteínas de Choque Térmico , Transtornos do Neurodesenvolvimento , Síndrome de Rubinstein-Taybi , Fatores de Transcrição , Humanos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histonas/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Síndrome de Rubinstein-Taybi/genética , Síndrome de Rubinstein-Taybi/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismoRESUMO
The oncogenic G-protein-coupled receptor (GPCR) Smoothened (SMO) is a key transducer of the hedgehog (HH) morphogen, which plays an essential role in the patterning of epithelial structures. Here, we examine how HH controls SMO subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. We provide evidence that HH promotes the stabilization of SMO by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of protein kinase A, casein kinase I, and the Fused (FU) kinase. Moreover, in the presence of very high levels of HH, the second effect of FU leads to the local enrichment of SMO in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of HH to the apico-basal distribution of SMO and provide a novel mechanism for the regulation of a GPCR.
Assuntos
Proteínas de Drosophila , Proteínas Hedgehog , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Hedgehog/metabolismo , Fosforilação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMO
Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.
Assuntos
Proteínas Hedgehog , Proteínas Repressoras , Animais , Drosophila/genética , Proteínas Hedgehog/genética , Proteínas Repressoras/química , Transdução de Sinais/genética , Peixe-ZebraRESUMO
From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Ligação a RNA/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Repressoras/metabolismo , Receptor Smoothened/genéticaRESUMO
Suppressor of Fused (SUFU) is a highly conserved protein that acts as a negative regulator of the Hedgehog (HH) signalling pathway, a major determinant of cell differentiation and proliferation. Therefore, SUFU deletion in mammals has devastating effects on embryo development. SUFU is part of a multi-protein cytoplasmic signal-transducing complex. Its partners include the Gli family of transcription factors that function either as repressors, or as transcription activators according to the HH activation state. The crystal structure of SUFU revealed a two-domain arrangement, which undergoes a closing movement upon binding a peptide from Gli1. There remains however, much to be discovered about SUFU's behaviour. To this end, we expressed recombinant, full-length SUFU from Drosophila, Zebrafish and Human. Guided by a sequence analysis that revealed a conserved potential metal binding site, we discovered that SUFU binds zinc. This binding was found to occur with a nanomolar affinity to SUFU from all three species. Mutation of one histidine from the conserved motif induces a moderate decrease in affinity for zinc, while circular dichroism indicates that the mutant remains structured. Our results reveal new metal binding affinity characteristics about SUFU that could be of importance for its regulatory function in HH.
RESUMO
Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.
Assuntos
Caseína Quinase I/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas Hedgehog/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Receptor Smoothened/metabolismo , Animais , Animais Geneticamente Modificados , Caseína Quinase I/genética , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Hedgehog/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Receptor Smoothened/genética , Asas de Animais/embriologia , Asas de Animais/metabolismoRESUMO
The conserved Hedgehog (HH) signals control animal development, adult stem cell maintenance and oncogenesis. In Drosophila, the HH co-receptor Patched (PTC) controls both HH gradient formation and signalling. PTC is post-translationally downregulated by HH, which promotes its endocytosis and destabilization, but the mechanisms of PTC trafficking and its importance in the control of PTC remain to be understood. PTC interacts with E3 Ubiquitin (UB)-ligases of the C2-WW-HECT family; two of them-SMURF and NEDD4-are known to regulate its levels. We demonstrate that mutation of the PTC PY motif, which mediates binding of C2-WW-HECT family members, inhibits its internalization but not its autonomous and non-autonomous signalling activities. In addition, we show that the two related UB-C2-WW-HECT ligases NEDD4 and SU(DX) regulate PTC trafficking and finely tune its accumulation through partially redundant but distinct functions. While both NEDD4 and SU(DX) promote PTC endocytosis, only SU(DX) is able to induce its lysosomal targeting and degradation. In conclusion, PTC trafficking and homeostasis are tightly regulated by a family of UB-ligases.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Homeostase , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , UbiquitinaçãoRESUMO
The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/ß-TrCP protein, as in mammals, both SLIMB/ßTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.
Assuntos
Apoptose , Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , MAP Quinase Quinase 4/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Animais Geneticamente Modificados , Caspases/metabolismo , Drosophila melanogaster , Ativação Enzimática , Fenótipo , Transdução de Sinais , Transgenes , Asas de Animais/metabolismoRESUMO
Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, we investigated the role of the Drosophila MLF homolog during blood cell development. Here we report that mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, our data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO-induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, we show that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO-dependent proliferation. Thus, we propose that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development.
Assuntos
Sequência Conservada/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hematopoese , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Humanos , Larva/citologia , Larva/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fenótipo , Estabilidade Proteica , Proteólise , Ativação Transcricional/genéticaRESUMO
The Hedgehog (Hh) signaling pathway initiates an evolutionarily conserved developmental program required for the proper patterning of many tissues [1]. Although Costal2 (Cos2) is a requisite component of the Hh pathway, its mechanistic role is not well understood. Because of its primary sequence, Cos2 was initially predicted to function as a kinesin-like protein [2]. However, evidence showing that Cos2 function might require kinesin-like properties has been lacking [2-6]. Thus, the prevailing dogma in the field is that Cos2 functions solely as a scaffolding protein [7, 8]. Here, we show that Cos2 motility is required for its biological function and that this motility may be Hh regulated. We show that Cos2 motility requires an active motor domain, ATP, and microtubules. Additionally, Cos2 recruits and transports other components of the Hh signaling pathway, including the transcription factor Cubitus interruptus (Ci). Drosophila expressing cos2 mutations that encode proteins that lack motility are attenuated in their ability to regulate Ci activity and exhibit phenotypes consistent with attenuated Cos2 function [9]. Combined, these results demonstrate that Cos2 motility plays an important role in its function, regulating the amounts and activity of Ci that ultimately interpret the level of Hh to which cells are exposed.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Cinesinas/metabolismo , Proteínas Motores Moleculares/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , DrosophilaRESUMO
Hedgehog (HH) is a major secreted morphogen involved in development, stem cell maintenance and oncogenesis [1, 2]. In Drosophila wing imaginal discs, HH produced in the posterior compartment diffuses into the anterior compartment to control target gene transcription via the transcription factor Cubitus interruptus (CI). The first steps in the reception and transduction of the HH signal are mediated by its receptor Patched (PTC) [3] and the seven-transmembrane-domain protein Smoothened (SMO) [4, 5]. PTC and HH control SMO by regulating its stability, trafficking, and phosphorylation (for review, see [6]). SMO interacts directly with the Ser-Thr protein kinase Fused (FU) and the kinesin-related protein Costal2 (COS2), which interact with each other and with CI in an intracellular Hedgehog transducing complex [7-9]. We show here that HH induces FU targeting to the plasma membrane in a SMO-dependent fashion and that, reciprocally, FU controls SMO stability and phosphorylation. FU anchorage to the membrane is sufficient to make it a potent SMO-dependent, PTC-resistant activator of the pathway. These findings reveal a novel positive-feedback loop in HH transduction and are consistent with a model in which FU and SMO, by mutually enhancing each other's activities, sustain high levels of signaling and render the pathway robust to PTC level fluctuations.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Membrana/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptor SmoothenedRESUMO
The Hedgehog (HH) signaling pathway is crucial for the development of many organisms and its inappropriate activation is involved in numerous cancers. HH signal controls the traffic and activity of the seven-pass transmembrane protein Smoothened (SMO), leading to the transcriptional regulation of HH-responsive genes. In Drosophila, the intracellular transduction events following SMO activation depend on cytoplasmic multimeric complexes that include the Fused (FU) protein kinase. Here we show that the regulatory domain of FU physically interacts with the last 52 amino acids of SMO and that the two proteins colocalize in vivo to vesicles. The deletion of this region of SMO leads to a constitutive activation of SMO, promoting the ectopic transcription of HH target genes. This activation is partially dependent of FU activity. Thus, we identify a novel link between SMO and the cytoplasmic complex(es) and reveal a negative role of the SMO C-terminal region that interacts with FU. We propose that FU could act as a switch, activator in presence of HH signal or inhibitor in absence of HH.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica/genética , Proteínas Hedgehog/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Primers do DNA , Proteínas de Drosophila/genética , Microscopia de Fluorescência , Modelos Biológicos , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Técnicas do Sistema de Duplo-Híbrido , Asas de Animais/embriologiaRESUMO
In human, the myeloid leukemia factor 1 (hMLF1) has been shown to be involved in acute leukemia, and mlf related genes are present in many animals. Despite their extensive representation and their good conservation, very little is understood about their function. In Drosophila, dMLF physically interacts with both the transcription regulatory factor DREF and an antagonist of the Hedgehog pathway, Suppressor of Fused, whose over-expression in the fly suppresses the toxicity induced by polyglutamine. No connection between these data has, however, been established. Here, we show that dmlf is widely and dynamically expressed during fly development. We isolated and analyzed the first dmlf mutants: embryos lacking maternal dmlf product have a low viability with no specific defect, and dmlf(-)- adults display weak phenotypes. We monitored dMLF subcellular localization in the fly and cultured cells. We were able to show that, although generally nuclear, dMLF can also be cytoplasmic, depending on the developmental context. Furthermore, two differently spliced variants of dMLF display differential subcellular localization, allowing the identification of regions of dMLF potentially important for its localization. Finally, we demonstrate that dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Núcleo Celular/metabolismo , Sequência Conservada , Citoplasma/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/química , Embrião não Mamífero , Olho/metabolismo , Olho/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: In Drosophila and vertebrates, suppressor of fused (Su(fu)) proteins act as negative regulators of the Gli/Ci transcription factors, which mediate the transcriptional effects of Hh signalling. RESULTS: We sought for novel partners of Su(fu) in fly using the two-hybrid method. Most of the Su(fu) interactors thus identified are (or are likely to be) able to enter the nucleus. We focused on one of these putative partners, dMLF, which resembles vertebrate myelodysplasia/myeloid leukaemia factors 1 and 2. We demonstrate that dMLF binds specifically to Su(fu) in vitro and in vivo. Using a novel anti-dMLF antibody, we showed, that dMLF is a nuclear, chromosome-associated protein. We over-expressed a dMLF transgene in fly using an inducible expression system and showed that dMLF over-expression disrupts normal development, leading to either a lethal phenotype or adult structural defects associated with apoptosis and increased DNA synthesis. Furthermore, the dMLF-induced eye phenotype is enhanced by the loss of Su(fu) function, suggesting a genetic interaction between Su(fu) and dMLF. CONCLUSION: We propose that dSu(fu) and dMLF act together at the transcriptional level to coordinate patterning and proliferation during development.
Assuntos
Apoptose , Núcleo Celular/metabolismo , DNA/biossíntese , Proteínas de Drosophila , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Animais Geneticamente Modificados , Bromodesoxiuridina , Cromossomos/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Olho/citologia , Olho/metabolismo , Morfogênese , Fenótipo , Proteínas/genética , Proteínas Repressoras/genética , Fase S , Transcrição Gênica , Transgenes , Técnicas do Sistema de Duplo-Híbrido , Asas de Animais/citologia , Asas de Animais/metabolismoRESUMO
BACKGROUND: Hedgehog signaling proteins play important roles in development by controlling growth and patterning in various animals including Drosophila and mammals. Hedgehog signaling triggers changes in responsive cells through a novel transduction mechanism that ultimately controls the transcription of specific target genes via the activity of zinc finger transcription factors of the Cubitus interruptus/GLI family. In flies, key Hedgehog signal transduction components have been identified including the kinesin-related protein Costal2, the serinethreonine kinase Fused, and the PEST-containing protein Suppressor of Fused. These proteins control Cubitus interruptus cleavage, nucleo-cytoplasmic localization and activation. In fly embryos, Costal2, Fused, Suppressor of Fused and Cubitus interruptus are associated in at least one cytoplasmic complex, which interacts with the microtubules in a Hedgehog-dependent manner. RESULTS: Here we identified and mapped direct interactions between Cos2, Fu, and Ci using an in vitro affinity assay and the yeast two-hybrid system. CONCLUSIONS: Our results provide new insights into the possible mechanism of the cytosolic steps of Hedgehog transduction.