RESUMEN
QM protein was previously discovered as a tumor suppressor, and numerous studies have shown that QM protein also played important roles in the immune responses. To investigate the potential roles of the QM protein gene in Eriocheir sinensis, the QM protein gene (designated as EsQM) has been cloned from E. sinensis using the rapid amplification of cDNA ends (RACE) technique. The cDNA of EsQM is 781 bp in length, consisting of a 654 bp open reading frame (ORF), encoding 219 amino acids, a 27 bp 5' untranslated region (UTR) and a 94 bp 3' UTR. The EsQM protein has a calculated molecular weight of 25.4 kDa and a theoretical isoelectric point of 10.10. The deduced protein sequence of EsQM contains a Ribosomal_L16 domain, an SH3-binding motif, an N-acylation site, two putative antibiotic binding sites, two putative protein kinase C phosphorylation sites, and two amidation sites. EsQM is extremely conserved and exhibits more than 85% similarities to previously identified arthropod QM protein genes. By real-time quantitative PCR (qPCR) analysis, we found that EsQM mRNA transcripts were detectable in all the examined tissues, with the highest expression in hemocytes. The mRNA expression of EsQM in hemocytes was significantly upregulated after the stimulation of Aeromonas hydrophila or polybrominated diphenyl ether-47 (BDE-47). Moreover, EsQM mRNA expression in hemocytes responded more quickly and lasted longer when stimulated by A.hydrophila than BDE-47. Thus, EsQM can respond to bacterial infection and environmental pollution, and might be involved in the defense mechanism to both biological and non-biological stimulation of arthropods.
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Braquiuros , Animales , Secuencia de Bases , Alineación de Secuencia , ADN Complementario/genética , Proteína Ribosómica L10/metabolismo , Clonación Molecular , ARN Mensajero/metabolismo , Braquiuros/genética , Braquiuros/metabolismo , FilogeniaRESUMEN
Pancreatic adenocarcinoma (PAAD) is the most malignant cancer with a high mortality rate. Despite the association of ribosomal protein L10 (RPL10) with PAAD and previous reports on RPL26 ufmylation, the relationship between RPL10 ufmylation and PAAD development remains unexplored. Here, we report the dissection of ufmylating process of RPL10 and potential roles of RPL10 ufmylation in PAAD development. The ufmylation of RPL10 was confirmed in both pancreatic patient tissues and cell lines, and specific modification sites were identified and verified. Phenotypically, RPL10 ufmylation significantly increased cell proliferation and stemness, which is principally resulted from higher expression of transcription factor KLF4. Moreover, the mutagenesis of ufmylation sites in RPL10 further demonstrated the connection of RPL10 ufmylation with cell proliferation and stemness. Collectively, this study reveals that PRL10 ufmylation plays an important role to enhance the stemness of pancreatic cancer cells for PAAD development.
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Adenocarcinoma , Neoplasias Pancreáticas , Humanos , Adenocarcinoma/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteína Ribosómica L10/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Neoplasias PancreáticasRESUMEN
To ascertain the role of Zn(II) as an allosteric modulator on P2X4R, QM/MM molecular dynamic simulations were performed on the WT and two P2X4R mutants suggested by previous electrophysiological data to affect Zn(II) binding. The Gibbs free energy for the reduction of the putative P2X4R Zn(II) binding site by glutathione was estimated at -22 kcal/mol. Simulations of the WT P2X4R head domain revealed a flexible coordination sphere dominated by an octahedral geometry encompassing C126, N127, C132, C149, C159 and a water molecule. The C132A mutation disrupted the metal binding site, leading to a coordination sphere with a majority of water ligands, and a displacement of the metal ion towards the solvent. The C132A/C159A mutant exhibited a tendency towards WT-like stability by incorporating the R148 backbone to the coordination sphere. Thus, the computational findings agree with previous experimental data showing Zn(II) modulation for the WT and C132A/C159A variants, but not for the C132A mutant. The results provide molecular insights into the nature of the Zn(II) modulation in P2X4R, and the effect of the C132A and C132A/C159A mutations, accounting for an elusive modulation mechanism possibly occurring in other extracellular or membrane protein.
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Cisteína/metabolismo , Dominios Proteicos/fisiología , Proteína Ribosómica L10/metabolismo , Zinc/metabolismo , Ligandos , Proteínas de la Membrana/metabolismo , Metales/metabolismo , Simulación de Dinámica Molecular , Unión Proteica/fisiología , Receptores Purinérgicos P2X4 , Agua/metabolismoRESUMEN
Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.
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Antibacterianos/farmacología , Regulación de la Expresión Génica , Técnicas Genéticas , Vectores Genéticos/genética , Animales , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Proteína Ribosómica L10/genética , Tetraciclina/farmacología , Transactivadores/genética , TransgenesRESUMEN
The ruthenium-based anticancer agent BOLD-100/KP1339 has shown promising results in several in vitro and in vivo tumour models as well as in early clinical trials. However, its mode of action remains to be fully elucidated. Recent evidence identified stress induction in the endoplasmic reticulum (ER) and concomitant down-modulation of HSPA5 (GRP78) as key drug effects. By exploiting the naturally formed adduct between BOLD-100 and human serum albumin as an immobilization strategy, we were able to perform target-profiling experiments that revealed the ribosomal proteins RPL10, RPL24, and the transcription factor GTF2I as potential interactors of this ruthenium(III) anticancer agent. Integrating these findings with proteomic profiling and transcriptomic experiments supported ribosomal disturbance and concomitant induction of ER stress. The formation of polyribosomes and ER swelling of treated cancer cells revealed by TEM validated this finding. Thus, the direct interaction of BOLD-100 with ribosomal proteins seems to accompany ER stress-induction and modulation of GRP78 in cancer cells.
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Antineoplásicos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Compuestos Organometálicos/farmacología , Proteína Ribosómica L10/metabolismo , Proteínas Ribosómicas/metabolismo , Antineoplásicos/química , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Células HCT116 , Humanos , Compuestos Organometálicos/química , Polirribosomas/metabolismo , Rutenio/química , Factores de Transcripción TFII/metabolismo , TranscriptomaRESUMEN
Eukaryotic cytoplasmic ribosomes are highly structured macromolecular complexes made up of four different ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs), which play a central role in the decoding of genetic code for the synthesis of new proteins. Over the past 25 years, studies on yeast and human models have made it possible to identify RPL10 (ribosomal protein L10 gene), which is a constituent of the large subunit of the ribosome, as an important player in the final stages of ribosome biogenesis and in ribosome function. Here, we reviewed the literature to give an overview of the role of RPL10 in physiologic and pathologic processes, including inherited disease and cancer.
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Enfermedades Raras/genética , Proteína Ribosómica L10/metabolismo , HumanosRESUMEN
Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10-silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA-sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two-hybrid assay. Silencing or mutants of genes encoding these RPL10-interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation-associated genes during nonhost pathogen infection.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Enfermedades de las Plantas/inmunología , Pseudomonas syringae/patogenicidad , Proteína Ribosómica L10/metabolismo , Proteínas Ribosómicas/metabolismo , Solanum lycopersicum/microbiología , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Resistencia a la Enfermedad/genética , Expresión Génica , Mutación , Enfermedades de las Plantas/microbiología , Biosíntesis de Proteínas , Interferencia de ARN , Proteína Ribosómica L10/genética , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Análisis de Secuencia de ARN , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/microbiología , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: The X-chromosome has been suggested to play a role in prostate cancer (PrCa) since epidemiological studies have provided evidence for an X-linked mode of inheritance for PrCa based on the higher relative risk among men who report an affected brother(s) as compared to those reporting an affected father. The aim of this study was to examine the potential association between the forensic STR markers located at four regions Xp22.31, Xq11.2-12, Xq26.2, and Xq28 and the risk of BPH and PrCa to confirm the impact of ChrX in the PrCa incidence. This may be helpful in the incorporation of STRs genetic variation in the early detection of men population at risk of developing PrCa. METHODS: DNA samples from 92 patients and 156 healthy controls collected from two medical centers in Riyadh, Saudi Arabia were analyzed for four regions located at X-chromosome using the Investigator® Argus X-12 QS Kit. RESULTS: The results demonstrated that microvariant alleles of (DXS7132, DXS10146, HPRTB, DXS10134, and DXS10135) are overrepresented in the BPH group (p < 0.00001). Allele 28 of DXS10135 and allele 15 of DXS7423 could have a protective effect, OR 0.229 (95%CI, 0.066-0.79); and OR 0.439 (95%CI, 0.208-0.925). On the other hand, patients carrying allele 23 of DXS10079 and allele 26 of DXS10148 presented an increased risk to PrCa OR 4.714 (95%CI, 3.604-6.166). CONCLUSION: The results are in concordance with the involvement of the X chromosome in PrCa and BPH development. STR allele studies may add further information from the definition of a genetic profile of PrCa resistance or susceptibility. As TBL1, AR, LDOC1, and RPL10 genes are located at regions Xp22.31, Xq11.2-12, Xq26.2, and Xq28, respectively, these genes could play an essential role in PrCa or BPH.
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Cromosomas Humanos X/genética , Repeticiones de Microsatélite , Proteínas Nucleares/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/genética , Proteína Ribosómica L10/genética , Transducina/genética , Proteínas Supresoras de Tumor/genética , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Estudios de Seguimiento , Predisposición Genética a la Enfermedad , Variación Genética , Genética de Población , Humanos , Masculino , Pronóstico , Hiperplasia Prostática/epidemiología , Hiperplasia Prostática/genética , Hiperplasia Prostática/patología , Neoplasias de la Próstata/epidemiología , Neoplasias de la Próstata/genética , Arabia Saudita/epidemiologíaRESUMEN
Neuroscience relies on techniques for imaging the structure and dynamics of neural circuits, but the cell bodies of individual neurons are often obscured by overlapping fluorescence from axons and dendrites in surrounding neuropil. Here, we describe two strategies for using the ribosome to restrict the expression of fluorescent proteins to the neuronal soma. We show first that a ribosome-tethered nanobody can be used to trap GFP in the cell body, thereby enabling direct visualization of previously undetectable GFP fluorescence. We then design a ribosome-tethered GCaMP for imaging calcium dynamics. We show that this reporter faithfully tracks somatic calcium dynamics in the mouse brain while eliminating cross-talk between neurons caused by contaminating neuropil. In worms, this reporter enables whole-brain imaging with faster kinetics and brighter fluorescence than commonly used nuclear GCaMPs. These two approaches provide a general way to enhance the specificity of imaging in neurobiology.
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Encéfalo/diagnóstico por imagen , Calcio/metabolismo , Cuerpo Celular/patología , Neuronas/patología , Imagen Óptica/métodos , Ribosomas/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Caenorhabditis elegans , Proteínas de Unión al Calcio , Cuerpo Celular/metabolismo , Proteínas Fluorescentes Verdes , Ratones , Neuronas/metabolismo , Neurópilo , Proteína Ribosómica L10/metabolismo , Anticuerpos de Dominio ÚnicoRESUMEN
Plastid casein kinase 2 (CK2), which is a major Ser/Thr-specific enzyme in higher organisms, plays an essential role in plant development and diverse abiotic stresses. CKB1 is a regulatory subunit beta of CK2. To expand our understand of functions of the CKB1 gene in Arabidopsis thaliana, protein changes among wild-type (WT) and CKB1 gain- and loss-of-function mutants were compared. Proteins extracted from the CKB1 knockout mutant and overexpressing mutant were compared with Col-0 plants using 2D-PAGE. Proteins regulated by CKB1 were identified with matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF), and its transcript was verified by qRT-PCR. Bioinformatics analysis, including gene ontology and protein-protein interaction analysis, were employed. The results of mass spectra and bioinformatics analysis suggest that CKB1 may have functions in regulation of the ribosomal protein L10 (RPL10) family and is involved in ultraviolet-B (UV-B) response. Furthermore, qRT-PCR verification showed CKB1 expression was up-regulated by UV-B stress. The expression levels of five genes in the RPL10 family were reduced in the ckb1 T-DNA insertion mutants, whereas they increased in the CKB1 overexpressing mutants under both normal conditions and UV-B treatment. In conclusion, CKB1 has important functions in UV-B radiation stress. Our study implies that CKB1 positively regulates UV-B radiation stress signalling, possibly through modulating expression of the RPL10 family.
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Proteínas de Arabidopsis , Arabidopsis , Quinasa de la Caseína II , Regulación de la Expresión Génica de las Plantas , Proteína Ribosómica L10 , Rayos Ultravioleta , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Quinasa de la Caseína II/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Proteína Ribosómica L10/genética , Estrés Fisiológico/efectos de la radiaciónAsunto(s)
Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/patología , ARN Circular/sangre , Proteína Ribosómica L10/sangre , Proteína Ribosómica L10/genética , Proteínas Ribosómicas/sangre , Proteínas Ribosómicas/genética , Anciano , Biomarcadores de Tumor/sangre , Carcinogénesis/genética , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Células HEK293 , Humanos , Leucocitos Mononucleares/citología , Masculino , Persona de Mediana Edad , Mutación , Riesgo , Resultado del TratamientoRESUMEN
Abstract Background Anti-ribosomal P (anti-Rib-P) antibody is a specific serological marker for systemic lupus erythematosus (SLE) and routinely tested by targeting the common epitope of three ribosomal proteins of P0, P1 and P2. This study aimed to investigate if testing antibodies against individual ribosomal protein, but not the common epitope, is required to achieve the best diagnostic benefit in SLE. Methods The study included 82 patients with SLE and 22 healthy donors. Serum antibodies were determined by ELISA and immunoblot. Results The prevalence of each antibody determined by ELISA was 35.4% (anti-Rib-P), 45.1% (anti-Rib-P0), 32.9% (anti-Rib-P1) and 40.2% (anti-Rib-P2) at 99% specificity, respectively. Of 53 patients with negative anti-Rib-P antibody, 21 (39.6%) were positive for anti-Rib-P0, 9 (17.0%) for anti-Rib-P1 and 12 (22.6%) for anti-Rib-P2 antibody. The positive rate of anti-Rib-P antibody detected by ELISA was close to the results by immunoblot (33.4%). Patients with any of these antibodies were featured by higher disease activity and prevalence of skin rashes than those with negative antibodies. Moreover, each antibody was particularly related to some clinical and laboratory disorders. The distribution of subclasses of IgG1-4 was varied with each antibody. Anti-Rib-P0 IgG1 and IgG3 were strongly correlated with disease activity and lower serum complement components 3 and 4. Conclusions Anti-Rib-P antibody is not adequate to predict the existence of antibodies against ribosomal P0, P1 and P2 protein. The examination of antibodies against each ribosomal protein is required to achieve additional diagnostic benefit and to evaluate the association with clinical and serological disorders as well.(AU)
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Humanos , Proteína Ribosómica L10/sangre , Lupus Eritematoso Sistémico/diagnóstico , Anticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática/instrumentación , Immunoblotting/instrumentaciónRESUMEN
In quantitative real-time PCR (qRT-PCR), data are normalized using reference genes, which helps to control for internal differences and reduce error among samples. In this study, the expression profiles of eight candidate housekeeping genes, 18S ribosomal (18S rRNA), elongation factor (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein L17 (RPL17), histone 3 (H3), arginine kinase (AK), amd ß-Actin (ACTB), were evaluated in the parasitic wasp Cotesia chilonis in response to different temperatures. Specifically, the performance and stabilities of these genes were compared in adult wasps maintained in a growth condition at 27°C (normal storage conditions) and in adults obtained from pupae refrigerated at 4°C for five days (cold storage conditions). Data were analyzed using the ΔCt method, BestKeeper, NormFinder, and geNorm. The optimal numbers and stabilities of reference genes varied between the two temperature treatments (27°C and 4°C). In samples stored at normal developmental temperature (27°C), the requirement for normalization in response to low temperature exposures was three genes (18S, H3, AK), whereas normalization in response to high temperature exposures required only two reference genes (GAPDH, ACTB). In samples stored at cold temperature (4°C), for low temperature exposures two reference genes (RPL17, RPL10) were required for standardization, while following high temperature exposures three reference genes (18S, H3, ACTB) were needed. This study strengthens understanding of the selection of reference genes before qRT-PCR analysis in C. chilonis. The reference genes identified here will facilitate further investigations of the biological characteristics of this important parasitoid.
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Perfilación de la Expresión Génica/normas , Genes Esenciales , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Avispas/fisiología , Actinas/genética , Animales , Arginina Quinasa/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Histonas/genética , Calor , Proteínas de Insectos/genética , Factor 1 de Elongación Peptídica/genética , ARN Ribosómico 18S/genética , Estándares de Referencia , Proteína Ribosómica L10/genética , Proteínas Ribosómicas/genética , Estrés Fisiológico , Avispas/genéticaRESUMEN
Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.
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Glicina/metabolismo , Mutación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Serina/metabolismo , Animales , Línea Celular , Perfilación de la Expresión Génica , Ratones , Monoéster Fosfórico Hidrolasas , Polirribosomas/genética , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteína Ribosómica L10 , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Análisis de Secuencia de ARNRESUMEN
Ghost proteins are issued from alternative Open Reading Frames (ORFs) and are missing a genome annotation. Indeed, historical filters applied for the detection of putative translated ORFs led to a wrong classification of transcripts considered as non-coding although translated proteins can be detected by proteomics. This Ghost (also called Alternative) proteome was neglected, and one major issue is to identify the implication of the Ghost proteins in the biological processes. In this context, we aimed to identify the protein-protein interactions (PPIs) of the Ghost proteins. For that, we re-explored a cross-link MS study performed on nuclei of HeLa cells using cross-linking mass spectrometry (XL-MS) associated with the HaltOrf database. Among 1679 cross-link interactions identified, 292 are involving Ghost Proteins. Forty-Four of these Ghost proteins are found to interact with 7 Reference proteins related to ribonucleoproteins, ribosome subunits and zinc finger proteins network. We, thus, have focused our attention on the heterotrimer between the RE/poly(U)-binding/degradation factor 1 (AUF1), the Ribosomal protein 10 (RPL10) and AltATAD2. Using I-Tasser software we performed docking models from which we could suggest the attachment of AUF1 on the external part of RPL10 and the interaction of AltATAD2 on the RPL10 region interacting with 5S ribosomal RNA as a mechanism of regulation of the ribosome. Taken together, these results reveal the importance of Ghost Proteins within known protein interaction networks.
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Núcleo Celular/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , Bases de Datos de Proteínas , Células HeLa , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Humanos , Espectrometría de Masas , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta , Unión Proteica , Proteínas/química , Proteína Ribosómica L10/metabolismo , Ribosomas/metabolismoRESUMEN
The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock-down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far-western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP-1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase-activating system, and it may play an important role in regulation of the innate immunity in insects.
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Bombyx/genética , Catecol Oxidasa/genética , Precursores Enzimáticos/genética , Proteínas de Insectos/genética , Proteína Ribosómica L10/genética , Animales , Bombyx/enzimología , Bombyx/crecimiento & desarrollo , Bombyx/inmunología , Catecol Oxidasa/metabolismo , Precursores Enzimáticos/metabolismo , Perfilación de la Expresión Génica , Inmunidad Innata/genética , Proteínas de Insectos/metabolismo , Larva/enzimología , Larva/genética , Larva/crecimiento & desarrollo , Larva/inmunología , Micrococcus luteus/fisiología , Pupa/enzimología , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/inmunología , Proteína Ribosómica L10/metabolismoRESUMEN
We combined translating ribosome affinity purification (TRAP) with in utero electroporation (IUE), called iTRAP to identify the molecular profile of specific neuronal populations during neonatal development without the need for viral approaches and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14-15 to target layer 2-4 cortical neurons of the somatosensory cortex. At three postnatal (P) ages-P0, P7, and P14-when morphogenesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found that ribosome bound (Ribo)-mRNAs from â¼7300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbr1, Dcx) or increase (e.g., Eno2, Camk2a, Syn1) as neurons mature. This approach led to the identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of their role in selective biological processes. These include upregulation of Lynx1, Nrn1, Cntnap1 over time; downregulation of St8sia2 and Draxin; and bidirectional changes to Fkbp1b. iTRAP is a versatile approach that allows researchers to easily assess the molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings.
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Electroporación/métodos , Biosíntesis de Proteínas , Células Piramidales/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Proteína Doblecortina , Desarrollo Embrionario , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Células Piramidales/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Ribosómica L10 , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Corteza Somatosensorial/citología , Corteza Somatosensorial/metabolismo , Sinapsinas/metabolismoRESUMEN
Ribosomopathies are congenital disorders caused by mutations in ribosomal proteins (RP) or assembly factors and are characterized by cellular hypoproliferation at an early stage. Paradoxically, many of these disorders have an elevated risk to progress to hyperproliferative cancer at a later stage. In addition, somatic RP mutations have recently been identified in various cancer types, for example, the recurrent RPL10-R98S mutation in T-cell acute lymphoblastic leukemia (T-ALL) and RPS15 mutations in chronic lymphocytic leukemia (CLL). We previously showed that RPL10-R98S promotes expression of oncogenes, but also induces a proliferative defect due to elevated oxidative stress. In this study, we demonstrate that this proliferation defect is eventually rescued by RPL10-R98S mouse lymphoid cells that acquire 5-fold more secondary mutations than RPL10-WT cells. The presence of RPL10-R98S and other RP mutations also correlated with a higher mutational load in patients with T-ALL, with an enrichment in NOTCH1-activating lesions. RPL10-R98S-associated cellular oxidative stress promoted DNA damage and impaired cell growth. Expression of NOTCH1 eliminated these phenotypes in RPL10-R98S cells, in part via downregulation of PKC-θ, with no effect on RPL10-WT cells. Patients with RP-mutant CLL also demonstrated a higher mutational burden, enriched for mutations that may diminish oxidative stress. We propose that oxidative stress due to ribosome dysfunction causes hypoproliferation and cellular insufficiency in ribosomopathies and RP-mutant cancer. This drives surviving cells, potentiated by genomic instability, to acquire rescuing mutations, which ultimately promote transition to hyperproliferation. SIGNIFICANCE: Ribosomal lesions cause oxidative stress and increase mutagenesis, promoting acquisition of rescuing mutations that stimulate proliferation.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Ribosomas/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Mutagénesis , Mutación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteína Ribosómica L10 , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Ribosomas/patologíaRESUMEN
The R98S mutation in ribosomal protein L10 (RPL10 R98S) affects 8% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases, and was previously described to impair cellular proliferation. The current study reveals that RPL10 R98S cells accumulate reactive oxygen species which promotes mitochondrial dysfunction and reduced ATP levels, causing the proliferation defect. RPL10 R98S mutant leukemia cells can survive high oxidative stress levels via a specific increase of IRES-mediated translation of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2), mediating BCL-2 protein overexpression. RPL10 R98S selective sensitivity to the clinically available Bcl-2 inhibitor Venetoclax (ABT-199) was supported by suppression of splenomegaly and the absence of human leukemia cells in the blood of T-ALL xenografted mice. These results shed new light on the oncogenic function of ribosomal mutations in cancer, provide a novel mechanism for BCL-2 upregulation in leukemia, and highlight BCL-2 inhibition as a novel therapeutic opportunity in RPL10 R98S defective T-ALL.
Asunto(s)
Sitios Internos de Entrada al Ribosoma , Mutación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Animales , Regulación Leucémica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína Ribosómica L10 , Proteínas Ribosómicas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumorigenesis is commonly known as a complicated process, in which reactive oxygen species (ROS) plays a critical role to involve in signal transduction, metabolism, cell proliferation and differentiation. Previously, ribosomal protein L10 (RPL10) was suggested to possess extra-ribosomal functions in pancreatic cancer cells in addition to being proposed as a tumor suppressor or transcription co-regulator. To better understand the relationship between RPL10 and tumorigenic potential in pancreatic cancer cells, chromatin immunoprecipitation sequencing reveals that RPL10 is unlikely to be a transcription factor without a specific binding motif for gene transcription. Additionally, transcriptome analysis indicates that RPL10 could regulate the expression of proteins related to ROS production. Moreover, RPL10 in mitochondria is closely associated with the regulation of ROS level by affecting Complex I activity and the subsequent events. Together, the present study suggests that the regulation of ROS level by mitochondrial RPL10 is one of the major extra-ribosomal functions in pancreatic cancer cells, which could be used as an indicator for the tumorigenesis of pancreatic cancer.