RESUMEN
The MYC oncogene was originally identified as a transduced allele (v-myc) in the genome of the highly oncogenic avian retrovirus MC29. The protein product (MYC) of the cellular MYC (c-myc) protooncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. MYC regulates fundamental cellular processes like growth control, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of MYC, leading to increased levels of the MYC protein, is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to quantify the oncogenic potential of MYC alleles, to test MYC inhibitors, and to monitor MYC-specific protein-protein interactions that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of MYC and to analyze structure, function, and regulation of the ancestral MYC proto-oncogenes.
Asunto(s)
Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/genética , Alelos , Secuencia de Aminoácidos , Animales , Carcinogénesis , Transformación Celular Neoplásica/genética , Evolución Molecular , Genes myc/genética , Genes myc/fisiología , Humanos , Mutación , Neoplasias/patología , Oncogenes , Mapeo de Interacción de Proteínas/métodos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/genéticaRESUMEN
The MYC protein is a transcription factor with oncogenic potential controlling fundamental cellular processes such as cell proliferation, metabolism, differentiation, and apoptosis. The MYC gene is a major cancer driver, and elevated MYC protein levels are a hallmark of most human cancers. We have previously shown that the brain acid-soluble protein 1 gene (BASP1) is specifically downregulated by the v-myc oncogene and that ectopic BASP1 expression inhibits v-myc-induced cell transformation. The 11-amino acid effector domain of the BASP1 protein interacts with the calcium sensor calmodulin (CaM) and is mainly responsible for this inhibitory function. We also reported recently that CaM interacts with all MYC variant proteins and that ectopic CaM increases the transactivation and transformation potential of the v-Myc protein. Here, we show that the presence of excess BASP1 or of a synthetic BASP1 effector domain peptide leads to displacement of v-Myc from CaM. The protein stability of v-Myc is decreased in cells co-expressing v-Myc and BASP1, which may account for the inhibition of v-Myc. Furthermore, suppression of v-Myc-triggered transcriptional activation and cell transformation is compensated by ectopic CaM, suggesting that BASP1-mediated withdrawal of CaM from v-Myc is a crucial event in the inhibition. In view of the tumor-suppressive role of BASP1 which was recently also reported for human cancer, small compounds or peptides based on the BASP1 effector domain could be used in drug development strategies aimed at tumors with high MYC expression.
Asunto(s)
Calmodulina/metabolismo , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Calmodulina/antagonistas & inhibidores , Calmodulina/genética , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Codorniz , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The c-Myc protein is a transcription factor with oncogenic potential controlling fundamental cellular processes. Homologs of the human c-myc protooncogene have been identified in the early diploblastic cnidarian Hydra (myc1, myc2). The ancestral Myc1 and Myc2 proteins display the principal design and biochemical properties of their vertebrate derivatives, suggesting that important Myc functions arose very early in metazoan evolution. c-Myc is part of a transcription factor network regulated by several upstream pathways implicated in oncogenesis and development. One of these signaling cascades is the Wnt/ß-Catenin pathway driving cell differentiation and developmental patterning, but also tumorigenic processes including aberrant transcriptional activation of c-myc in several human cancers. Here, we show that genetic or pharmacological stimulation of Wnt/ß-Catenin signaling in Hydra is accompanied by specific downregulation of myc1 at mRNA and protein levels. The myc1 and myc2 promoter regions contain consensus binding sites for the transcription factor Tcf, and Hydra Tcf binds to the regulatory regions of both promoters. The myc1 promoter is also specifically repressed in the presence of ectopic Hydra ß-Catenin/Tcf in avian cell culture. We propose that Hydra myc1 is a negative Wnt signaling target, in contrast to vertebrate c-myc, which is one of the best studied genes activated by this pathway. On the contrary, myc2 is not suppressed by ectopic ß-Catenin in Hydra and presumably represents the structural and functional c-myc ortholog. Our data implicate that the connection between ß-Catenin-mediated signaling and myc1 and myc2 gene regulation is an ancestral metazoan feature. Its impact on decision making in Hydra interstitial stem cells is discussed.
Asunto(s)
Hydra/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Animales , Tipificación del Cuerpo/genética , Regulación de la Expresión Génica/genética , Hydra/crecimiento & desarrollo , Vía de Señalización Wnt/genética , beta Catenina/genéticaRESUMEN
The architectures of central signaling hubs are precisely organized by static and dynamic protein-protein interactions (PPIs). Upon deregulation, these PPI platforms are capable to propagate or initiate pathophysiological signaling events. This causes the acquisition of molecular features contributing to the etiology or progression of many diseases, including cancer, where deregulated molecular interactions of signaling proteins have been best studied. The reasons for PPI-dependent reprogramming of cancer-initiating cells are manifold; in many cases, mutations perturb PPIs, enzyme activities, protein abundance, or protein localization. Consequently, the pharmaceutical targeting of PPIs promises to be of remarkable therapeutic value. For this review we have selected three key players of oncogenic signaling which are differently affected by PPI deregulation: two (the small G proteins of the RAS family and the transcription factor MYC) are considered "undruggable" using classical drug discovery approaches and in the case of the third protein discussed here, PKA, standard kinase inhibitors, may be unsuitable in the clinic. These circumstances require alternative strategies, which may lie in pharmaceutical drug interference of critical PPIs accountable for oncogenic signaling.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Neoplasias/química , Neoplasias/tratamiento farmacológico , Mapas de Interacción de ProteínasRESUMEN
The prototypes of the human MYC and RAF gene families are orthologs of animal proto-oncogenes that were originally identified as transduced alleles in the genomes of highly oncogenic retroviruses. MYC and RAF genes are now established as key regulatory elements in normal cellular physiology, but also as major cancer driver genes. Although the predominantly nuclear MYC proteins and the cytoplasmic RAF proteins have different biochemical functions, they are functionally linked in pivotal signaling cascades and circuits. The MYC protein is a transcription factor and together with its dimerization partner MAX holds a central position in a regulatory network of bHLH-LZ proteins. MYC regulates transcription conducted by all RNA polymerases and controls virtually the entire transcriptome. Fundamental cellular processes including distinct catabolic and anabolic branches of metabolism, cell cycle regulation, cell growth and proliferation, differentiation, stem cell regulation, and apoptosis are under MYC control. Deregulation of MYC expression by rearrangement or amplification of the MYC locus or by defects in kinase-mediated upstream signaling, accompanied by loss of apoptotic checkpoints, leads to tumorigenesis and is a hallmark of most human cancers. The critically controlled serine/threonine RAF kinases are central nodes of the cytoplasmic MAPK signaling cascade transducing converted extracellular signals to the nucleus for reshaping transcription factor controlled gene expression profiles. Specific mutations of RAF kinases, such as the prevalent BRAF(V600E) mutation in melanoma, or defects in upstream signaling or feedback loops cause decoupled kinase activities which lead to tumorigenesis. Different strategies for pharmacological interference with MYC- or RAF-induced tumorigenesis are being developed and several RAF kinase inhibitors are already in clinical use.
Asunto(s)
Carcinogénesis , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Animales , Humanos , Neoplasias/genética , Neoplasias/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencia de Aminoácidos , Animales , Calmodulina/química , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Recombinantes de Fusión/metabolismo , Activación TranscripcionalAsunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Proliferación Celular/genética , Dimerización , Humanos , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
The 2'-trifluoromethylthio (2'-SCF3 ) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2'-SCF3 adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc-protecting group strategy, are reported. RNA solid-phase synthesis provided site-specifically 2'-SCF3 -modified oligoribonucleotides that were investigated intensively. Their excellent behavior in (19) Fâ NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule-RNA interaction. Moreover, comparably to the 2'-SCF3 pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off-target effects and their performance in silencing of the BASP1 gene was demonstrated.
Asunto(s)
Flúor/química , Oligonucleótidos/química , Compuestos Organofosforados/química , Purinas/química , ARN Interferente Pequeño/química , Cristalografía por Rayos X , Ligandos , Espectroscopía de Resonancia Magnética , Oligonucleótidos/metabolismo , ARN Interferente Pequeño/metabolismo , Técnicas de Síntesis en Fase Sólida , TermodinámicaRESUMEN
Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Mapeo de Interacción de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Animales , Técnicas Biosensibles , Línea Celular Tumoral , Embrión no Mamífero/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Ratones , Osteosarcoma/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
The oncogenic bHLH-LZ transcription factor Myc forms binary complexes with its binding partner Max. These and other bHLH-LZ-based protein-protein interactions (PPI) in the Myc-Max network are essential for the physiological and oncogenic activities of Myc. We have generated a genetically determined and highly specific protein-fragment complementation assay based on Renilla luciferase to analyze the dynamic interplay of bHLH-LZ transcription factors Myc, Max, and Mxd1 in vivo. We also applied this PPI reporter to quantify alterations of nuclear Myc-Max complexes in response to mutational events, competitive binding by the transcriptional repressor Mxd1, or perturbations by small-molecule Myc inhibitors, including recently identified potent PPI inhibitors from a Kröhnke pyridine library. We show that the specificity of Myc-Max PPI reduction by the pyridine inhibitors directly correlates with their efficient and highly specific potential to interfere with the proliferation of human and avian tumor cells displaying deregulated Myc expression. In a direct comparison with known Myc inhibitors using human and avian cell systems, the pyridine compounds reveal a unique inhibitory potential even at sub-micromolar concentrations combined with remarkable specificity for the inhibition of Myc-driven tumor cell proliferation. Furthermore, we show in direct comparisons using defined avian cell systems that different Max PPI profiles for the variant members of the Myc protein family (c-Myc, v-Myc, N-Myc, L-Myc) correlate with their diverse oncogenic potential and their variable sensitivity to the novel pyridine inhibitors.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HEK293 , Humanos , Luciferasas de Renilla , Neoplasias/metabolismo , Oxadiazoles/farmacología , Unión Proteica , Multimerización de Proteína , Piridinas/farmacología , Tiazoles/farmacologíaRESUMEN
In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Piridinas/farmacología , Pirimidinas/farmacología , Animales , Antineoplásicos/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/antagonistas & inhibidores , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Células Cultivadas , Embrión de Pollo , Evaluación Preclínica de Medicamentos , Femenino , Polarización de Fluorescencia , Genes myc , Humanos , Interferometría , Ratones , Ratones Desnudos , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/química , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/química , Piridinas/química , Pirimidinas/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The myc protooncogene encodes the Myc transcription factor which is the essential part of the Myc-Max network controlling fundamental cellular processes. Deregulation of myc leads to tumorigenesis and is a hallmark of many human cancers. We have recently identified homologs of myc (myc1, myc2) and max in the early diploblastic cnidarian Hydra and have characterized myc1 in detail. Here we show that myc2 is transcriptionally activated in the interstitial stem cell system. Furthermore, in contrast to myc1, myc2 expression is also detectable in proliferating epithelial stem cells throughout the gastric region. myc2 but not myc1 is activated in cycling precursor cells during early oogenesis and spermatogenesis, suggesting that the Hydra Myc2 protein has a possible non-redundant function in cell cycle progression. The Myc2 protein displays the principal design and properties of vertebrate Myc proteins. In complex with Max, Myc2 binds to DNA with similar affinity as Myc1-Max heterodimers. Immunoprecipitation of Hydra chromatin revealed that both Myc1 and Myc2 bind to the enhancer region of CAD, a classical Myc target gene in mammals. Luciferase reporter gene assays showed that Myc1 but not Myc2 transcriptionally activates the CAD promoter. Myc2 has oncogenic potential when tested in primary avian fibroblasts but to a lower degree as compared to Myc1. The identification of an additional myc gene in Cnidaria, a phylum that diverged prior to bilaterians, with characteristic expression patterns in tissue homeostasis and developmental processes suggests that principle functions of myc genes have arisen very early in metazoan evolution.
RESUMEN
Labeled RNA becomes increasingly important for molecular diagnostics and biophysical studies on RNA with its diverse interaction partners, which range from small metabolites to large macromolecular assemblies, such as the ribosome. Here, we introduce a fast synthesis path to 3'-terminal 2'-O-(2-azidoethyl) modified oligoribonucleotides for subsequent bioconjugation, as exemplified by fluorescent labeling via Click chemistry for an siRNA targeting the brain acid-soluble protein 1 gene (BASP1). Importantly, the functional group pattern is inverse to commonly encountered alkyne-functionalized "click"-able RNA and offers increased flexibility with respect to multiple and stepwise labeling of the same RNA molecule. Additionally, our route opens up a minimal step synthesis of 2'-O-(2-aminoethyl) modified pyrimidine nucleoside phosphoramidites which are of widespread use to generate amino-modified RNA for N-hydroxysuccinimide (NHS) ester-based conjugations.
Asunto(s)
Azidas/química , Proteínas del Citoesqueleto/química , Oligorribonucleótidos/química , ARN Interferente Pequeño/química , Animales , Línea Celular , Pollos , Química Clic , Proteínas del Citoesqueleto/genética , Fluorescencia , Microscopía Fluorescente , Estructura Molecular , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
The c-myc protooncogene encodes the Myc transcription factor, a global regulator of fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis, and c-myc is an important driver in human cancer. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins involved in transcriptional regulation of target genes. Non-transcriptional functions have also been attributed to the Myc protein, notably direct interaction with the pre-replicative complex (pre-RC) controlling the initiation of DNA replication. A key component of the pre-RC is the Cdt1 protein, an essential factor in origin licensing. Here we present data suggesting that the CDT1 gene is a transcriptional target of the Myc-Max complex. Expression of the CDT1 gene in v-myc-transformed cells directly correlates with myc expression. Also, human tumor cells with elevated c-myc expression display increased CDT1 expression. Occupation of the CDT1 promoter by Myc-Max is demonstrated by chromatin immunoprecipitation, and transactivation by Myc-Max is shown in reporter assays. Ectopic expression of CDT1 leads to cell transformation. Our results provide a possible direct mechanistic link of Myc's canonical function as a transcription factor to DNA replication. Furthermore, we suggest that aberrant transcriptional activation of CDT1 by deregulated myc alleles contributes to the genomic instabilities observed in tumor cells.
Asunto(s)
Replicación del ADN , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Pollos , Orden Génico , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Activación TranscripcionalRESUMEN
Cellular membrane receptors sense environmental changes and relay the reshaped signal through spatially and temporally organized protein-protein interactions (PPI). Many of such PPI are transient and occur in a certain cell-dependent context. Molecular switches such as kinases and GTPases are engaged in versatile PPI. Recently, we have identified dynamic interaction and reciprocal regulation of cAMP-dependent protein kinase A (PKA) and Rho-GTPase Rac signaling. We demonstrated that GTP-activated Rac acts as a dual kinase-tuning scaffold for p21-activated kinase (PAK) and PKA activities. We showed that receptor-triggered PKA trans-phosphorylation of GTP-Rac-organized PAK contributes to elevations of nuclear Erk1/2 signaling and proliferation. We discuss these recent observations and we provide additional insights how the cAMP-PKA axis might also participate in the regulation of Rac localization.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Complejos Multienzimáticos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Femenino , HumanosRESUMEN
The myc oncogene was originally identified as a transduced allele (v-myc) in the genome of a highly oncogenic avian retrovirus. The protein product (Myc) of the cellular c-myc proto-oncogene represents the key component of a transcription factor network controlling the expression of a large fraction of all human genes. Myc regulates fundamental cellular processes like growth, metabolism, proliferation, differentiation, and apoptosis. Mutational deregulation of c-myc leading to increased levels of the Myc protein is a frequent event in the etiology of human cancers. In this chapter, we describe cell systems and experimental strategies to monitor and quantify the oncogenic potential of myc alleles and to isolate and characterize transcriptional targets of Myc that are relevant for the cell transformation process. We also describe experimental procedures to study the evolutionary origin of myc and to analyze structure and function of the ancestral myc proto-oncogenes.
Asunto(s)
Transformación Celular Neoplásica/genética , Evolución Molecular , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética/métodos , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Vectores Genéticos/genética , Humanos , Hibridación in Situ/métodos , Cultivo Primario de Células/métodos , Unión Proteica , Proto-Oncogenes Mas , Codorniz , Retroviridae/genética , Activación Transcripcional , Transducción Genética , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
The oncogenic transcription factor Myc is one of the most interesting members of the basic-helix-loop-helix-zipper (bHLHZip) protein family. Deregulation of Myc via gene amplification, chromosomal translocation or other mechanisms lead to tumorigenesis including Burkitt lymphoma, multiple myeloma, and many other malignancies. The oncogene myc is a highly potent transforming gene and capable to transform various cell types in vivo and in vitro. Its oncogenic activity initialized by deregulated expression leads to a shift of the equilibrium in the Myc/Max/Mad network towards Myc/Max complexes. The Myc/Max heterodimerization is a prerequisite for transcriptional functionality of Myc. Primarily, we are focusing on the apo-state of the C-terminal domain of v-Myc, the retroviral homolog of human c-Myc. Based on multi-dimensional NMR measurements v-Myc appears to be neither a fully structured nor a completely unstructured protein. The bHLHZip domain of v-Myc does not exist as a random coil but exhibits partially pre-formed α-helical regions in its apo-state. In order to elucidate the structural propensities of Myc in more detail, the backbone and side-chain assignments obtained here for apo-Myc are a crucial prerequisite for further NMR measurements.
Asunto(s)
ADN/metabolismo , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/metabolismo , Protones , Secuencia de Aminoácidos , Isótopos de Carbono , Humanos , Isótopos de Nitrógeno , Estructura Terciaria de ProteínaRESUMEN
Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2'-azido cytidine and 2'-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2'-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2'-azido uridine and 2'-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2'-azido group supports the C3'-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2'-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2'-azido modifications are compatible with 2'-fluoro and/or 2'-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2'-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2'-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.
Asunto(s)
Azidas/química , Azidas/síntesis química , Nucleótidos/química , ARN Interferente Pequeño/química , Coloración y Etiquetado , Química Clic , Cristalografía por Rayos X , Modelos Moleculares , Conformación Molecular , Nucleótidos/síntesis químicaRESUMEN
Siderocalins are particular lipocalins that participate in the innate immune response by interfering with bacterial siderophore-mediated iron uptake. Additionally, siderocalins are involved in several physiological and pathological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. Here we show that siderocalin Q83 displays an unexpected dual ligand binding mode as it can bind enterobactin and unsaturated fatty acids simultaneously. The solution structure of the siderocalin Q83 in complex with arachidonic acid and enterobactin reveals molecular details of this novel dual binding mode and the determinants of fatty acid binding specificity. Our results suggest that Q83 is a metabolic hub linking iron and fatty acid pathways. This unexpected coupling might contribute to the pleiotropic functions of siderocalins.