RESUMEN
We previously showed that MYC promoted Burkitt lymphoma (BL) growth by inhibiting the tumor suppressor miR-150, resulting in release of miR-150 targets MYB and ZDHHC11. The ZDHHC11 gene encodes three different transcripts including a mRNA (pcZDHHC11), a linear long non-coding RNA (lncZDHHC11) and a circular RNA (circZDHHC11). All transcripts contain the same region with 18 miR-150 binding sites. Here we studied the relevance of circZDHHC11, including this miR-150 binding site region, for growth of BL cells. CircZDHHC11 was mainly present in the cytoplasmic fraction in BL cells and its localization was not altered upon miR-150 overexpression. Knockdown of circZDHHC11 caused a strong inhibition of BL growth without affecting the expression levels of MYC, MYB, miR-150 and other genes. Overexpression of circZDHHC11 neither affected cell growth, nor rescued the phenotype induced by miR-150 overexpression. Genomic deletion of the miR-150 binding site region did not affect growth, nor did it change the effect of circZDHHC11 knockdown. This indicated that the miR-150 binding site region is dispensable for the growth promoting role of circZDHHC11. To conclude, our results show that circZDHHC11 is a crucial factor supporting BL cell growth independent of its ability to sponge miR-150.
Asunto(s)
Linfoma de Burkitt , MicroARNs , Humanos , Linfoma de Burkitt/patología , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , ARN Mensajero/genética , ARN CircularRESUMEN
The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. The central role of vitamin transport in the metabolism of bacteria and absence from humans make the ECF transporters an attractive target for inhibition with selective chemical probes. Here, we report on the identification of a promising class of inhibitors of the ECF transporters. We used coarse-grained molecular dynamics simulations on Lactobacillus delbrueckii ECF-FolT2 and ECF-PanT to profile the binding mode and mechanism of inhibition of this novel chemotype. The results corroborate the postulated mechanism of transport and pave the way for further drug-discovery efforts.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas , Humanos , Proteínas Bacterianas/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Bacterias/metabolismo , Vitaminas/metabolismo , Simulación de Dinámica MolecularRESUMEN
We previously described involvement of the MYC/miR-150/MYB/ZDHHC11 network in the growth of Burkitt lymphoma (BL) cells. Here we studied the relevance of this network in the two other B-cell lymphomas: Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL). Expression levels of the network components were assessed at the RNA and protein level. The effect of modulating levels of the network components on cell growth was determined through GFP competition assay. AGO2-RNA immunoprecipitation was performed to validate targeting by miR-150. Expression levels of MYC, MYB and ZDHHC11 were increased, while miR-150 levels were decreased similar to the pattern observed in BL. The knockdown of MYC, MYB and ZDHHC11 decreased the growth of HL and DLBCL cells. In contrast, overexpression of miR-150 did not induce clear phenotypes in HL, and limited the effects in DLBCL. This could not be explained by the differences in overexpression levels. Furthermore, we showed that in HL, ZDHHC11 and MYB are efficiently targeted by miR-150. To conclude, MYC, MYB and ZDHHC11 are critical for the growth of HL and DLBCL cells consistent with the role observed in BL cells, while low endogenous miR-150 levels appeared to be less critical for the growth of HL and DLBCL cells despite the effective targeting of ZDHHC11 and MYB.
Asunto(s)
Linfoma de Burkitt , Enfermedad de Hodgkin , Linfoma de Células B Grandes Difuso , MicroARNs , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Proliferación Celular , Enfermedad de Hodgkin/genética , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , MicroARNs/genéticaRESUMEN
Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico Activo/fisiología , Técnicas In Vitro , Lactobacillus delbrueckii , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Myelocytomatosis viral oncogene homolog (MYC) plays an important role in the regulation of many cellular processes, and its expression is tightly regulated at the level of transcription, translation, protein stability, and activity. Despite this tight regulation, MYC is overexpressed in many cancers and contributes to multiple hallmarks of cancer. In recent years, it has become clear that noncoding RNAs add a crucial additional layer to the regulation of MYC and its downstream effects. So far, twenty-five microRNAs and eighteen long noncoding RNAs that regulate MYC have been identified. Thirty-three miRNAs and nineteen lncRNAs are downstream effectors of MYC that contribute to the broad oncogenic role of MYC, including its effects on diverse hallmarks of cancer. In this review, we give an overview of this extensive, multilayered noncoding RNA network that exists around MYC. Current data clearly show explicit roles of crosstalk between MYC and ncRNAs to allow tumorigenesis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Largo no Codificante/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Humanos , MicroARNs/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genéticaRESUMEN
We applied dynamic combinatorial chemistry (DCC) to identify ligands of ThiT, the S-component of the energy-coupling factor (ECF) transporter for thiamine in Lactococcus lactis. We used a pre-equilibrated dynamic combinatorial library (DCL) and saturation-transfer difference (STD) NMR spectroscopy to identify ligands of ThiT. This is the first report in which DCC is used for fragment growing to an ill-defined pocket, and one of the first reports for its application with an integral membrane protein as target.
Asunto(s)
Tiamina/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Proteínas Portadoras , Técnicas Químicas Combinatorias , Diseño de Fármacos , Lactococcus lactis , Modelos Moleculares , Estructura Molecular , Subunidades de Proteína , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
In chemical biology, azides are used to chemically manipulate target structures in a bioorthogonal manner for a plethora of applications ranging from target identification to the synthesis of homogeneously modified protein conjugates. While a variety of methods have been established to introduce the azido group into recombinant proteins, a method that directly converts specific amino groups in endogenous proteins is lacking. Here, we report the first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies the model proteins streptavidin and avidin and the membrane protein BioY on cell surface. The reagent converts amines in the proximity of the binding pocket to azides and leaves the remaining amino groups in streptavidin untouched. Reagents of this novel class will find use in target identification as well as the selective functionalization and bioorthogonal protection of proteins.
Asunto(s)
Avidina/química , Azidas/química , Proteínas Bacterianas/química , Biotina/química , Escherichia coli/química , Lactococcus lactis/química , Estreptavidina/química , Alquinos/química , Compuestos de Boro/química , Modelos MolecularesRESUMEN
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
Asunto(s)
Carbono/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Serina/biosíntesis , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Carbono/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Femenino , Glucólisis/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/metabolismo , Purinas/biosíntesis , Serina/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Timidina/biosíntesis , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Energy-coupling factor (ECF) transporters mediate uptake of micronutrients in prokaryotes. The transporters consist of an S-component that binds the transported substrate and an ECF module (EcfAA'T) that binds and hydrolyses ATP. The mechanism of transport is poorly understood but presumably involves an unusual step in which the membrane-embedded S-component topples over to carry the substrate across the membrane. In many ECF transporters, the S-component dissociates from the ECF module after transport. Subsequently, substrate-bound S-components out-compete the empty proteins for re-binding to the ECF module in a new round of transport. Here we present crystal structures of the folate-specific transporter ECF-FolT from Lactobacillus delbrueckii. Interaction of the ECF module with FolT stabilizes the toppled state, and simultaneously destroys the high-affinity folate-binding site, allowing substrate release into the cytosol. We hypothesize that differences in the kinetics of toppling can explain how substrate-loaded FolT out-competes apo-FolT for association with the ECF module.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenilil Imidodifosfato/metabolismo , Cristalografía por Rayos X , Ácido Fólico/metabolismo , Lactobacillus/metabolismo , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Especificidad por SustratoRESUMEN
Energy-coupling factor (ECF) transporters are membrane-protein complexes that mediate vitamin uptake in prokaryotes. They bind the substrate through the action of a specific integral membrane subunit (S-component) and power transport by hydrolysis of ATP in the three-subunit ECF module. Here, we have studied the binding of thiamine derivatives to ThiT, a thiamine-specific S-component. We designed and synthesized derivatives of thiamine that bind to ThiT with high affinity; this allowed us to evaluate the contribution of the functional groups to the binding affinity. We determined six crystal structures of ThiT in complex with our derivatives. The structure of the substrate-binding site in ThiT remains almost unchanged despite substantial differences in affinity. This work indicates that the structural organization of the binding site is robust and suggests that substrate release, which is required for transport, requires additional changes in conformation in ThiT that might be imposed by the ECF module.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Diseño de Fármacos , Bibliotecas de Moléculas Pequeñas/metabolismo , Tiamina/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Transporte Biológico , Lactococcus lactis/química , Modelos Moleculares , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Tiamina/síntesis química , Tiamina/químicaRESUMEN
Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects.