RESUMEN
Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.
Asunto(s)
Empalme Alternativo , MicroARNs , Ribonucleasa III , MicroARNs/genética , Humanos , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN/genética , Mutación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Intrones/genéticaRESUMEN
Although new genes can arrive from modes other than duplication, few examples are well characterized. Given high expression in some human brain subregions and a putative link to psychological disorders [e.g., schizophrenia (SCZ)], suggestive of brain functionality, here we characterize piggyBac transposable element-derived 1 (PGBD1). PGBD1 is nonmonotreme mammal-specific and under purifying selection, consistent with functionality. The gene body of human PGBD1 retains much of the original DNA transposon but has additionally captured SCAN and KRAB domains. Despite gene body retention, PGBD1 has lost transposition abilities, thus transposase functionality is absent. PGBD1 no longer recognizes piggyBac transposon-like inverted repeats, nonetheless PGBD1 has DNA binding activity. Genome scale analysis identifies enrichment of binding sites in and around genes involved in neuronal development, with association with both histone activating and repressing marks. We focus on one of the repressed genes, the long noncoding RNA NEAT1, also dysregulated in SCZ, the core structural RNA of paraspeckles. DNA binding assays confirm specific binding of PGBD1 both in the NEAT1 promoter and in the gene body. Depletion of PGBD1 in neuronal progenitor cells (NPCs) results in increased NEAT1/paraspeckles and differentiation. We conclude that PGBD1 has evolved core regulatory functionality for the maintenance of NPCs. As paraspeckles are a mammal-specific structure, the results presented here show a rare example of the evolution of a novel gene coupled to the evolution of a contemporaneous new structure.
Asunto(s)
Elementos Transponibles de ADN , ARN Largo no Codificante , Animales , Núcleo Celular/genética , Histonas/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Proteínas del Tejido Nervioso , Paraspeckles , ARN Largo no Codificante/metabolismo , Transposasas/genética , Transposasas/metabolismoRESUMEN
The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transposase are well described. There is an RNase H-like fold containing the DDD motif responsible for the catalytic DNA cleavage and joining reactions and a C-terminal cysteine-rich domain important for interaction with the transposon DNA. However, the protein also contains a ~100 amino acid long N-terminal disordered region (NTDR) whose function is currently unknown. Here we show that deletion of the NTDR significantly impairs piggyBac transposition, although the extent of decrease is strongly cell-type specific. Moreover, replacing the NTDR with scrambled but similarly disordered sequences did not rescue transposase activity, indicating the importance of sequence conservation. Cell-based transposon excision and integration assays reveal that the excision step is more severely affected by NTDR deletion. Finally, bioinformatic analyses indicated that the NTDR is specific for the piggyBac superfamily and is also present in domesticated, transposase-derived proteins incapable of catalyzing transposition. Our results indicate an essential role of the NTDR in the "fine-tuning" of transposition and its significance in the functions of piggyBac-originated co-opted genes.
Asunto(s)
ADN Catalítico , Transposasas , Cisteína/genética , Elementos Transponibles de ADN/genética , ADN Catalítico/metabolismo , Ribonucleasa H/metabolismo , Transposasas/metabolismoRESUMEN
One of the longest human microRNA (miRNA) clusters is located on chromosome 19 (C19MC), containing 46 miRNA genes, which were considered to be expressed simultaneously and at similar levels from a common long noncoding transcript. Investigating the two tissue types where C19MC is exclusively expressed, we could show that there is a tissue-specific and chromosomal position-dependent decrease in mature miRNA levels towards the 3' end of the cluster in embryonic stem cells but not in placenta. Although C19MC transcription level is significantly lower in stem cells, this gradual decrease is not present at the primary miRNA levels, indicating that a difference in posttranscriptional processing could explain this observation. By depleting Drosha, the nuclease component of the Microprocessor complex, we could further enhance the positional decrease in stem cells, demonstrating that a tissue-specific, local availability of the Microprocessor complex could lie behind the phenomenon. Moreover, we could describe a tissue-specific promoter being exclusively active in placenta, and the epigenetic mark analysis suggested the presence of several putative enhancer sequences in this region. Performing specific chromatin immunoprecipitation followed by quantitative real-time PCR experiments we could show a strong association of Drosha with selected enhancer regions in placenta, but not in embryonic stem cells. These enhancers could provide explanation for a more efficient co-transcriptional recruitment of the Microprocessor, and therefore a more efficient processing of pri-miRNAs throughout the cluster in placenta. Our results point towards a new model where tissue-specific, posttranscriptional 'fine-tuning' can differentiate among miRNAs that are expressed simultaneously from a common precursor.
Asunto(s)
Cromosomas Humanos Par 19/química , Células Madre Embrionarias Humanas/metabolismo , MicroARNs/genética , Placenta/metabolismo , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , Ribonucleasa III/genética , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Epigénesis Genética , Femenino , Células Madre Embrionarias Humanas/citología , Humanos , MicroARNs/metabolismo , Familia de Multigenes , Especificidad de Órganos , Placenta/citología , Embarazo , Precursores del ARN/metabolismo , Ribonucleasa III/deficiencia , Transcripción GenéticaRESUMEN
The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Resistencia a Antineoplásicos/genética , Variación Genética/genética , Proteínas de Neoplasias/genética , Adenosina Trifosfatasas/genética , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Transporte de Proteínas/genéticaRESUMEN
Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Dinaminas/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Fagocitosis/fisiología , Actinas/metabolismo , Animales , Apoptosis/fisiología , Caenorhabditis elegans/metabolismo , Células Cultivadas , Humanos , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fagosomas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5' cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , MicroARNs/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/fisiología , Polirribosomas/metabolismo , División del ARN , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.
Asunto(s)
Calcio/metabolismo , Giro Dentado/citología , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Células Madre Pluripotentes/citología , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Hipocampo/citología , Humanos , Células-Madre Neurales/metabolismo , Neurogénesis/fisiologíaRESUMEN
ABCG2 is a multidrug transporter with wide substrate specificity, and is believed to protect several cell types from various xenobiotics and endobiotics. This "guardian" function is important in numerous cell types and tissue barriers but becomes disadvantageous by being responsible for the multidrug resistance phenotype in certain tumor cells. ABCG2 regulation at the protein level has already been extensively studied, however, regulation at the mRNA level, especially the functional role of the various 5' untranslated exon variants (5' UTRs) has been elusive. In the present work, we describe a comprehensive characterization of four ABCG2 mRNA variants with different exon 1 sequences, investigate drug inducibility, stem cell specificity, mRNA stability, and translation efficiency. Although certain variants (E1B and E1C) are considered as "constitutive" mRNA isoforms, we show that chemotoxic drugs significantly alter the expression pattern of distinct ABCG2 mRNA isoforms. When examining human embryonic stem cell lines, we provide evidence that variant E1A has an expression pattern coupled to undifferentiated stem cell stage, as its transcript level is regulated parallel to mRNAs of Oct4 and Nanog pluripotency marker genes. When characterizing the four exon 1 variants we found no significant differences in terms of mRNA stabilities and half-lives of the isoforms. In contrast, variant E1U showed markedly lower translation efficiency both at the total protein level or regarding the functional presence in the plasma membrane. Taken together, these results indicate that the different 5' UTR variants play an important role in cell type specific regulation and fine tuning of ABCG2 expression.
Asunto(s)
Regiones no Traducidas 5' , Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genética , Polimorfismo Genético , Células Madre/fisiología , Regiones no Traducidas 5'/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Células Cultivadas , Exones/genética , Células HEK293 , Humanos , Células MCF-7 , Ratones , Especificidad de Órganos/genéticaRESUMEN
Supply of major metabolites such as γ-aminobutyric acid (GABA), ß-alanine and taurine is an essential instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs. This background motivates the synthesis of novel classes of compounds regulating their selective transport through various fluid-organ barriers via the low-affinity γ-aminobutyric acid (GABA) transporter subtype 2 (GAT2). Natural and synthetic spirocyclic compounds or therapeutics with a range of structures and biological activity are increasingly recognised in this regard. Based on pre-validated GABA transport activity, straightforward and efficient synthesis method was developed to provide an azaspiro[4.5]decane scaffold, holding a variety of charge, substituent and 3D constrain of spirocyclic amine. Investigation of the azaspiro[4.5]decane scaffold in cell lines expressing the four GABA transporter subtypes led to the discovery of a subclass of a GAT2-selective compounds with acyl-substituted azaspiro[4.5]decane core.
Asunto(s)
Alcanos/química , Alcanos/farmacología , Compuestos Aza/química , Compuestos Aza/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Acilación , Alcanos/síntesis química , Animales , Compuestos Aza/síntesis química , Humanos , Compuestos de Espiro/síntesis química , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations.
Asunto(s)
Calcio/metabolismo , Túbulos Renales Proximales/metabolismo , Microscopía Confocal , Transgenes , Animales , Animales Modificados Genéticamente , Citoplasma/metabolismo , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Homocigoto , Hipoxia/patología , Isquemia/patología , Riñón/metabolismo , Riñón/patología , Corteza Renal/metabolismo , Enfermedades Renales/patología , Túbulos Renales/metabolismo , Túbulos Renales Proximales/patología , Ligandos , Ratas , Daño por Reperfusión , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Introduction: Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. Methods: We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Results: Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Discussion: Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.
Asunto(s)
MicroARNs , Preeclampsia , Embarazo , Recién Nacido , Humanos , Femenino , Primer Trimestre del Embarazo , Preeclampsia/diagnóstico , Preeclampsia/genética , MicroARNs/genética , Biomarcadores , Retardo del Crecimiento Fetal/genética , Retardo del Crecimiento Fetal/metabolismoRESUMEN
ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estrés Oxidativo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Línea Celular , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/efectos de la radiación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Xenobióticos/farmacologíaRESUMEN
With the development of modern molecular genetics, the original "one gene-one enzyme" hypothesis has been outdated. For protein coding genes, the discovery of alternative splicing and RNA editing provided the biochemical background for the RNA repertoire of a single locus, which also serves as an important pillar for the enormous protein variability of the genomes. Non-protein coding RNA genes were also revealed to produce several RNA species with distinct functions. The loci of microRNAs (miRNAs), encoding for small endogenous regulatory RNAs, were also found to produce a population of small RNAs, rather than a single defined product. This review aims to present the mechanisms contributing to the astonishing variability of miRNAs revealed by the new sequencing technologies. One important source is the careful balance of arm selection, producing sequentially different 5p- or 3p-miRNAs from the same pre-miRNA, thereby broadening the number of regulated target RNAs and the phenotypic response. In addition, the formation of 5', 3' and polymorphic isomiRs, with variable end and internal sequences also leads to a higher number of targeted sequences, and increases the regulatory output. These miRNA maturation processes, together with other known mechanisms such as RNA editing, further increase the potential outcome of this small RNA pathway. By discussing the subtle mechanisms behind the sequence diversity of miRNAs, this review intends to reveal this engaging aspect of the inherited "RNA world", how it contributes to the almost infinite molecular variability among living organisms, and how this variability can be exploited to treat human diseases.
Asunto(s)
MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.
Asunto(s)
Adenosina Trifosfato , Adenosina Trifosfato/metabolismo , Conformación ProteicaRESUMEN
ABC multidrug transporter proteins expel a wide variety of structurally unrelated, mostly hydrophobic compounds from cells. The special role of these transporters both at the physiological barriers and in cancer cells is based on their extremely broad substrate recognition. Since hydrophobic compounds are known to partition into the lipid bilayer and accumulate in membranes, the "classical pump" model for the mechanism of multidrug transporter proteins has been challenged, and alternative models suggesting substrate recognition within the lipid bilayer have been proposed. Although much effort has been made to validate this concept, unambiguous evidence for direct drug extrusion from the plasma membrane has not been provided yet. Here we show a detailed on-line microscopic analysis of cellular extrusion of fluorescent anti-cancer drugs, mitoxantrone and pheophorbide A, by a key human multidrug transporter, ABCG2. Using the fully active GFP-tagged ABCG2 and exploiting the special character of mitoxantrone that gains fluorescence in the lipid environment, we were able to determine transporter-modulated drug concentrations separately in the plasma membrane and the intracellular compartments. Different kinetic models describing the various transport mechanisms were generated and the experimental data were analyzed using these models. On the basis of the kinetic analysis, drug extrusion from the cytoplasm can be excluded, thus, our results indicate that ABCG2 extrudes mitoxantrone directly from the plasma membrane.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Mitoxantrona/química , Proteínas de Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Antineoplásicos/farmacología , Transporte Biológico , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/química , Humanos , Cinética , Lípidos/química , Mitoxantrona/farmacología , Modelos Estadísticos , Neoplasias/tratamiento farmacológico , Factores de Tiempo , TransfecciónRESUMEN
The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Androstadienos/farmacología , Línea Celular , Cromonas/farmacología , Humanos , Morfolinas/farmacología , Sirolimus/farmacología , WortmaninaRESUMEN
The ATP-binding cassette G subfamily member ABCG2 protein is involved in drug resistance of various types of cancer including hepatocellular carcinoma (HCC). The transcriptional regulation of the ABCG2 gene was shown to depend on various transcription factors, and three alternative promoters were described. Here we aimed to decipher the role of hepatocyte growth factor (HGF) and the related kinase cascades on the expression of ABCG2 and the role of the different promoters in this process in the HepG2 human HCC cell line. We observed that HGF treatment increased the amount of ABCG2 on the cell surface in parallel with an increased ABCG2 transcription. ABCG2 mRNA expression was also increased by EGF, oxidative stress or activation of the aryl hydrocarbon receptor, while decreased by TGFb. Treatment with U0126, a specific inhibitor of the ERK1/2 cascade, prevented the HGF and the oxidative stress induced ABCG2 upregulation. We also show that the regulation of ABCG2 by various modulators involve specific alternative promoters. In conclusion, we demonstrate a unique role of the ERK1/2 cascade on ABCG2 modulation in HepG2, and the differential use of the alternative ABCG2 promoters in this cell line. This study reveals the molecular participants of ABCG2 overexpression as new potential treatment targets in HCC.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Carcinoma Hepatocelular/metabolismo , Resistencia a Antineoplásicos , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Hepáticas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Proteínas de Neoplasias/genética , Estrés Oxidativo , Ésteres del Forbol/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Dibenzodioxinas Policloradas/farmacología , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/agonistas , Transcripción GenéticaRESUMEN
Mirtrons are short intronic microRNA (miRNA) precursors representing an alternative, Drosha/DGCR8-independent miRNA biogenesis pathway. In this study we characterized three predicted human mirtrons. Their expression was proven to be context-independent, since functional mirtrons could be derived either from their endogenous or from a heterologous coding environment. Systematic testing revealed that both 5'- and 3'-arms of mir-877 are capable of producing functional miRNA simultaneously in the various cell types examined. On the other hand, experimental validations revealed that the predicted mir-1233 is not a bona fide mirtron. For functional mirtrons, we were able to detect mature mirtron-derived miRNAs for the first time by qRT-PCR or northern blot analysis, when silencing activity was proven by functional assays. Our results emphasize the need for functional testing of both arms of miRNAs and the importance of experimentally validating human mirtrons since, in spite of being localized in a short intron, predicted species could mature via other miRNA processing pathways.
Asunto(s)
Intrones/fisiología , MicroARNs/biosíntesis , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Ratones , MicroARNs/genética , Proteínas/genética , Proteínas/metabolismo , Precursores del ARN/genética , Proteínas de Unión al ARN , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Maturation of microRNAs (miRNAs) begins by the "Microprocessor" complex, containing the Drosha endonuclease and its partner protein, "DiGeorge Syndrome Critical Region 8" (DGCR8). Although the main function of the two proteins is to coordinate the first step of precursor miRNAs formation, several studies revealed their miRNA-independent functions in other RNA-related pathways (e.g., in snoRNA decay) or, for the DGCR8, the role in tissue development. To investigate the specific roles of DGCR8 in various cellular pathways, we previously established a human embryonic stem-cell (hESC) line carrying a monoallelic DGCR8 mutation by using the CRISPR-Cas9 system. In this study, we genetically characterized single-cell originated progenies of the cell line and showed that DGCR8 heterozygous mutation results in only a modest effect on the mRNA level but a significant decrease at the protein level. Self-renewal and trilineage differentiation capacity of these hESCs were not affected by the mutation. However, partial disturbance of the Microprocessor function could be revealed in pri-miRNA processing along the human chromosome 19 miRNA cluster in several clones. With all these studies, we can demonstrate that the mutant hESC line is a good model to study not only miRNA-related but also other "noncanonical" functions of the DGCR8 protein.