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1.
J Biol Chem ; 299(9): 105154, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37572851

RESUMEN

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.


Asunto(s)
Anomalías Múltiples , Humanos , Trastorno del Espectro Autista , Células HEK293 , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosforilación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteómica , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología
2.
J Allergy Clin Immunol ; 152(3): 807-813.e7, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37211057

RESUMEN

BACKGROUND: Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T- and B-cell defects. OBJECTIVE: We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. METHODS: The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. RESULTS: Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. CONCLUSIONS: Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.


Asunto(s)
Fosfatidilinositol 3-Quinasas , Inmunodeficiencia Combinada Grave , Masculino , Niño , Humanos , Preescolar , Adulto , Fosfatidilinositol 3-Quinasas/genética , Linfocitos T CD8-positivos , Ligandos , Proteína S6 Ribosómica/genética , Transducción de Señal/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/diagnóstico , Mutación
3.
PLoS Genet ; 19(1): e1010595, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36656901

RESUMEN

Defective ribosome biogenesis (RiBi) underlies a group of clinically diverse human diseases collectively known as the ribosomopathies, core manifestations of which include cytopenias and developmental abnormalities that are believed to stem primarily from an inability to synthesize adequate numbers of ribosomes and concomitant activation of p53. The importance of a correctly functioning RiBi machinery for maintaining tissue homeostasis is illustrated by the observation that, despite having a paucity of certain cell types in early life, ribosomopathy patients have an increased risk for developing cancer later in life. This suggests that hypoproliferative states trigger adaptive responses that can, over time, become maladaptive and inadvertently drive unchecked hyperproliferation and predispose to cancer. Here we describe an experimentally induced ribosomopathy in the mouse and show that a normal level of hepatic ribosomal protein S6 (Rps6) is required for proper bile duct development and preservation of hepatocyte viability and that its insufficiency later promotes overgrowth and predisposes to liver cancer which is accelerated in the absence of the tumor-suppressor PTEN. We also show that the overexpression of c-Myc in the liver ameliorates, while expression of a mutant hyperstable form of p53 partially recapitulates specific aspects of the hepatopathies induced by Rps6 deletion. Surprisingly, co-deletion of p53 in the Rps6-deficient background fails to restore biliary development or significantly improve hepatic function. This study not only reveals a previously unappreciated dependence of the developing liver on adequate levels of Rps6 and exquisitely controlled p53 signaling, but suggests that the increased cancer risk in ribosomopathy patients may, in part, stem from an inability to preserve normal tissue homeostasis in the face of chronic injury and regeneration.


Asunto(s)
Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Animales , Ratones , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Hepatocitos/metabolismo , Fenotipo , Conductos Biliares/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo
4.
Reproduction ; 164(5): 221-230, 2022 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-36111643

RESUMEN

In brief: Several factors affect the reprogramming efficiency of nuclear transfer embryos. This study shows that inhibiting 18S rRNA m6A methyltransferase METTL5 during nuclear transfer can improve the developmental rate of nuclear transfer embryos. Abstract: N6-methyladenosine (m6A) is one of the most important epigenetic modifications in eukaryotic RNAs, which regulates development and diseases. It is identified by several proteins. Methyltransferase-like 5 (METTL5), an enzyme that methylates 18S rRNA m6A, controls the translation of proteins and regulates pluripotency in embryonic stem cells. However, the functions of METTL5 in embryonic development have not been explored. Here, we found that Mettl5 was upregulated in somatic cell nuclear transfer (SCNT) embryos compared with normal fertilized embryos. Therefore, we hypothesized that METTL5 knockdown during the early stage of SCNT would improve the developmental rate of SCNT embryos. Notably, injection of Mettl5 siRNA (si-Mettl5) into enucleated oocytes during nuclear transfer increased the rate of development and the number of cells in blastocysts. Moreover, inhibition of METTL5 reduced the activity of phosphorylated ribosomal protein S6, decreased the levels of the repressive histone modification H3K27me3 and increased the expression of activating histone modifications H3K27ac and H3K4me3 and mRNA levels of some 2-cell-specific genes. These results expand our understanding of the role of METTL5 in early embryonic development and provide a novel idea for improving the efficiency of nuclear transfer cloning.


Asunto(s)
Reprogramación Celular , Histonas , Animales , Blastocisto/metabolismo , Desarrollo Embrionario , Femenino , Histonas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Técnicas de Transferencia Nuclear , Embarazo , ARN Mensajero/metabolismo , ARN Ribosómico 18S/metabolismo , ARN Interferente Pequeño/genética , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo
5.
Mol Cancer Res ; 20(8): 1320-1336, 2022 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-35503453

RESUMEN

Uveal melanoma is a rare form of melanoma that originates in the eye, exerts widespread therapeutic resistance, and displays an inherent propensity for hepatic metastases. Because metastatic disease is characterized by poor survival, there is an unmet clinical need to identify new therapeutic targets in uveal melanoma. Here, we show that the pleiotropic cytokine midkine is expressed in uveal melanoma. Midkine expression in primary uveal melanoma significantly correlates with poor survival and is elevated in patients that develop metastatic disease. Monosomy 3 and histopathologic staging parameters are associated with midkine expression. In addition, we demonstrate that midkine promotes survival, migration across a barrier of hepatic sinusoid endothelial cells and resistance to AKT/mTOR inhibition. Furthermore, midkine is secreted and mediates mTOR activation by maintaining phosphorylation of the mTOR target RPS6 in uveal melanoma cells. Therefore, midkine is identified as a uveal melanoma cell survival factor that drives metastasis and therapeutic resistance, and could be exploited as a biomarker as well as a new therapeutic target. IMPLICATIONS: Midkine is identified as a survival factor that drives liver metastasis and therapeutic resistance in melanoma of the eye.


Asunto(s)
Neoplasias Hepáticas , Melanoma , Midkina , Proteína S6 Ribosómica , Serina-Treonina Quinasas TOR , Neoplasias de la Úvea , Resistencia a Antineoplásicos , Células Endoteliales/metabolismo , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Midkina/genética , Midkina/metabolismo , Metástasis de la Neoplasia/patología , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Úvea/tratamiento farmacológico , Neoplasias de la Úvea/genética
6.
PLoS Genet ; 17(12): e1009980, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34941873

RESUMEN

The liver is a crucial center in the regulation of energy homeostasis under starvation. Although downregulation of mammalian target of rapamycin complex 1 (mTORC1) has been reported to play pivotal roles in the starvation responses, the underpinning mechanisms in particular upstream factors that downregulate mTORC1 remain largely unknown. To identify genetic variants that cause liver energy disorders during starvation, we conduct a zebrafish forward genetic screen. We identify a liver hulk (lvh) mutant with normal liver under feeding, but exhibiting liver hypertrophy under fasting. The hepatomegaly in lvh is caused by enlarged hepatocyte size and leads to liver dysfunction as well as limited tolerance to starvation. Positional cloning reveals that lvh phenotypes are caused by mutation in the ftcd gene, which encodes the formimidoyltransferase cyclodeaminase (FTCD). Further studies show that in response to starvation, the phosphorylated ribosomal S6 protein (p-RS6), a downstream effector of mTORC1, becomes downregulated in the wild-type liver, but remains at high level in lvh. Inhibition of mTORC1 by rapamycin rescues the hepatomegaly and liver dysfunction of lvh. Thus, we characterize the roles of FTCD in starvation response, which acts as an important upstream factor to downregulate mTORC1, thus preventing liver hypertrophy and dysfunction.


Asunto(s)
Amoníaco-Liasas/genética , Glutamato Formimidoiltransferasa/genética , Hepatomegalia/genética , Hígado/metabolismo , Enzimas Multifuncionales/genética , Proteína S6 Ribosómica/genética , Animales , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatomegalia/metabolismo , Hepatomegalia/patología , Humanos , Hígado/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Complejos Multiproteicos/genética , Mutación/genética , Fosforilación , Transducción de Señal/genética , Inanición/genética , Inanición/metabolismo , Inanición/patología , Pez Cebra/genética
7.
Nucleic Acids Res ; 49(22): 13062-13074, 2021 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-34871442

RESUMEN

Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, the molecular consequences of RPS6 phosphorylation on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. We test whether RPS6 phosphorylation differentially affects mRNA translation based on CDS length by genetic removal of RPS6 phosphorylation. We find that RPS6 phosphorylation promotes translation of mRNAs with short CDSs more strongly than mRNAs with long CDSs. Interestingly, RPS6 phosphorylation does not promote translation of mRNAs with 5' TOP motifs despite their short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.


Asunto(s)
Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteína S6 Ribosómica/genética , Animales , Células Cultivadas , Células HeLa , Humanos , Immunoblotting , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Mutación , Fosforilación , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , RNA-Seq/métodos , Proteína S6 Ribosómica/metabolismo , Ribosomas/genética , Ribosomas/metabolismo
8.
Nucleic Acids Res ; 49(12): 6908-6924, 2021 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-34133725

RESUMEN

Reinitiation supporting protein, RISP, interacts with 60S (60S ribosomal subunit) and eIF3 (eukaryotic initiation factor 3) in plants. TOR (target-of-rapamycin) mediates RISP phosphorylation at residue Ser267, favoring its binding to eL24 (60S ribosomal protein L24). In a viral context, RISP, when phosphorylated, binds the CaMV transactivator/ viroplasmin, TAV, to assist in an exceptional mechanism of reinitiation after long ORF translation. Moreover, we show here that RISP interacts with eIF2 via eIF2ß and TOR downstream target 40S ribosomal protein eS6. A RISP phosphorylation knockout, RISP-S267A, binds preferentially eIF2ß, and both form a ternary complex with eIF3a in vitro. Accordingly, transient overexpression in plant protoplasts of RISP-S267A, but not a RISP phosphorylation mimic, RISP-S267D, favors translation initiation. In contrast, RISP-S267D preferentially binds eS6, and, when bound to the C-terminus of eS6, can capture 60S in a highly specific manner in vitro, suggesting that it mediates 60S loading during reinitiation. Indeed, eS6-deficient plants are highly resistant to CaMV due to their reduced reinitiation capacity. Strikingly, an eS6 phosphomimic, when stably expressed in eS6-deficient plants, can fully restore the reinitiation deficiency of these plants in cellular and viral contexts. These results suggest that RISP function in translation (re)initiation is regulated by phosphorylation at Ser267.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Iniciación de la Cadena Peptídica Traduccional , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Caulimovirus , Factor 2B Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Fosforilación , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo
9.
Endocrinology ; 162(1)2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33094326

RESUMEN

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a nonhormonal male contraceptive, since it effectively induces reversible male infertility without perturbing the serum concentrations of follicle stimulating hormone (FSH), testosterone, and inhibin B based on studies in rats and rabbits. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli-spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively inducing spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell-cell AJ called basal ectoplasmic specialization (basal ES), which, together with tight junction constitute the blood-testis barrier (BTB), unless an acute dose of adjudin was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to not only study adjudin-mediated male infertility but also the biology of spermatogenesis.


Asunto(s)
Quinasa 1 de Adhesión Focal/metabolismo , Hidrazinas/farmacología , Indazoles/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína S6 Ribosómica/metabolismo , Animales , Cloruro de Cadmio/toxicidad , Quinasa 1 de Adhesión Focal/genética , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas Asociadas a Microtúbulos/genética , Ratas , Ratas Sprague-Dawley , Proteína S6 Ribosómica/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología
10.
Sci Rep ; 10(1): 19000, 2020 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-33149163

RESUMEN

Alterations of RNA homeostasis can lead to severe pathological conditions. The Survival of Motor Neuron (SMN) protein, which is reduced in Spinal Muscular Atrophy, impacts critical aspects of the RNA life cycle, such as splicing, trafficking, and translation. Increasing evidence points to a potential role of SMN in ribosome biogenesis. Our previous study revealed that SMN promotes membrane-bound ribosomal proteins (RPs), sustaining activity-dependent local translation. Here, we suggest that plasma membrane domains could be a docking site not only for RPs but also for their encoding transcripts. We have shown that SMN knockdown perturbs subcellular localization as well as translation efficiency of RPS6 mRNA. We have also shown that plasma membrane-enriched fractions from human fibroblasts retain RPS6 transcripts in an SMN-dependent manner. Furthermore, we revealed that SMN traffics with RPS6 mRNA promoting its association with caveolin-1, a key component of membrane dynamics. Overall, these findings further support the SMN-mediated crosstalk between plasma membrane dynamics and translation machinery. Importantly, our study points to a potential role of SMN in the ribosome assembly pathway by selective RPs synthesis/localization in both space and time.


Asunto(s)
Compartimento Celular , ARN Mensajero/metabolismo , Proteína S6 Ribosómica/genética , Proteína 1 para la Supervivencia de la Neurona Motora/fisiología , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Humanos , Biosíntesis de Proteínas , Transporte de Proteínas , Ribosomas/metabolismo
11.
J Ovarian Res ; 13(1): 100, 2020 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-32862831

RESUMEN

BACKGROUND: Ovarian cancer typically is diagnosed late because insensitivity and lack of specificity of current biomarkers prior to its clinical detection. Ribosomal protein S6 (RPS6) is a ribosomal protein involved in the ribosomal 40S subunit, but its biological role in epithelial ovarian cancer (EOC) is still unknown. RESULTS: RPS6 was elevated in EOC compared to normal ovarian tissues and adenomas. Higher expression of RPS6 predicted worse prognosis in EOC. The level of RPS6 was correlated with clinical stage, histological type and pathological grade. Knockdown of RPS6 reduced the proliferation of ovarian cancer cell lines SKOV-3 and HO8910, and inhibit the migration and invasion ability. It revealed that cells arrested at G0G1 phase after knockdown of RPS6, and the expressions of CyclinD1, Cyclin E, CDK2, CDK4, CDK6 and pRb were also reduced. CONCLUSIONS: RPS6 is involved in EOC and knockdown of RPS6 could inhibit the proliferation, invasion and migration ability of EOC in vitro by inducing G0/G1 phase arrest. RPS6 is expected to be a novel biomarker and molecular target to the EOC.


Asunto(s)
Carcinoma Epitelial de Ovario/patología , Neoplasias Ováricas/patología , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Regulación hacia Arriba , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Pronóstico , Análisis de Supervivencia
12.
Cancer Sci ; 111(4): 1291-1302, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31997546

RESUMEN

Postoperative distant metastasis dramatically affects rectal cancer patients who have undergone neoadjuvant chemoradiotherapy (NACRT). Here, we clarified the association between NACRT-mediated mammalian target of rapamycin (mTOR) signaling pathway activation and rectal cancer metastatic potential. We performed immunohistochemistry for phosphorylated mTOR (p-mTOR) and phosphorylated S6 (p-S6) on surgical specimen blocks from 98 rectal cancer patients after NACRT (cohort 1) and 80 colorectal cancer patients without NACRT (cohort 2). In addition, we investigated the association between mTOR pathway activity, affected by irradiation, and the migration ability of colorectal cancer cells in vitro. Based on the results of the clinical study, p-mTOR was significantly overexpressed in cohort 1 (with NACRT) as compared to levels in cohort 2 (without NACRT) (P < .001). High p-mTOR and p-S6 levels correlated with the development of distant metastasis only in cohort 1. Specifically, high p-S6 expression (HR 4.51, P = .002) and high pathological T-stage (HR 3.73, P = .020) after NACRT were independent predictors of the development of distant metastasis. In vitro, p-S6 levels and migration ability increased after irradiation in SW480 cells (TP53 mutation-type) but decreased in LoVo cells (TP53 wild-type), suggesting that irradiation modulates mTOR signaling and migration through cell type-dependent mechanisms. We next assessed the expression level of p53 by immunostaining in cohort 1 and demonstrated that p-S6 was overexpressed in samples with high p53 expression as compared to levels in samples with low p53 expression (P = .008). In conclusion, p-S6 levels after NACRT correlate with postoperative distant metastasis in rectal cancer patients, suggesting that chemoradiotherapy might modulate the mTOR signaling pathway, promoting metastasis.


Asunto(s)
Neoplasias del Recto/tratamiento farmacológico , Proteína S6 Ribosómica/genética , Serina-Treonina Quinasas TOR/genética , Proteína p53 Supresora de Tumor/genética , Anciano , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Quimioradioterapia Adyuvante/efectos adversos , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Neoplasias del Recto/radioterapia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación
13.
Eur Rev Med Pharmacol Sci ; 24(1): 151-163, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31957828

RESUMEN

OBJECTIVE: Esophageal cancer (EC) ranks as the sixth leading cause of cancer-related mortality worldwide. Circular RNAs (circRNAs) are involved in the pathogenesis of different cancers. However, the regulatory mechanism of circ_0006168 in EC progression is still unclear. MATERIALS AND METHODS: The expression of circ_0006168, microRNA (miR)-384, and retinoblastoma binding protein 7 (RBBP7) in tumors and cells was measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The stability of circ_0006168 was analyzed after RNase R treatment. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was conducted to evaluate cell viability. Transwell assay was applied to determine cell migration and invasion. Glucose consumption and lactate production were detected using glucose detection and lactic acid detection kits. The interaction between miR-384 and circ_0006168 or RBBP7 was certified by Dual-Luciferase reporter system. Protein expression of pyruvate kinase (PK), RBBP7, S6 ribosomal protein kinase (S6K), phosphorylated S6K (p-S6K), S6, phosphorylated S6 (p-S6) was analyzed by Western blot. RESULTS: Circ_0006168 and RBBP7 were over-expressed while miR-384 was low-expressed in EC tumors and cells. The repression of circ_0006168 attenuated cell proliferation, migration, invasion, and glycolysis in EC. Of note, circ_0006168 functioned as a sponge while RBBP7 acted as a target of miR-384 in EC. Rescue experiment revealed that miR-384 inhibitor abrogated circ_0006168 silencing-induced repression on cell proliferation, migration, and invasion in EC. Meanwhile, upregulation of RBBP7 restored the inhibition of miR-384 on EC cell progression. Moreover, circ_0006168 was able to improve RBBP7 level by interacting with miR-384. Also, circ_0006168 could activate S6K/S6 pathway by regulating RBBP7 expression. CONCLUSIONS: Abundance of circ_0006168 contributes to cell proliferation, migration, invasion, and glycolysis in EC by competitively sponging miR-384 to facilitate RBBP7 expression, representing prospective targets for EC therapy.


Asunto(s)
Neoplasias Esofágicas/metabolismo , MicroARNs/metabolismo , ARN Circular/metabolismo , Proteína 7 de Unión a Retinoblastoma/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteína S6 Ribosómica/metabolismo , Movimiento Celular , Proliferación Celular , Células Cultivadas , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Humanos , MicroARNs/genética , ARN Circular/genética , Proteína 7 de Unión a Retinoblastoma/genética , Proteína S6 Ribosómica/genética , Proteínas Quinasas S6 Ribosómicas/genética
14.
J Genet Genomics ; 47(12): 735-742, 2020 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-33612456

RESUMEN

Tuberous sclerosis complex (TSC) is a dominant genetic neurocutaneous syndrome characterized by multiple organ hamartomas. Although rodent models bearing a germline mutation in either TSC1 or TSC2 gene have been generated, they do not develop pathogenic lesions matching those seen in patients with TSC because of the significant differences between mice and humans, highlighting the need for an improved large animal model of TSC. Here, we successfully generate monoallelic TSC1-modified Bama miniature pigs using the CRISPR/Cas9 system along with somatic cell nuclear transfer (SCNT) technology. The expression of phosphorylated target ribosomal protein S6 is significantly enhanced in the piglets, indicating that disruption of a TSC1 allele activate the mechanistic target of rapamycin (mTOR) signaling pathway. Notably, differing from the mouse TSC models reported previously, the TSC1+/- Bama miniature pig developed cardiac rhabdomyoma and subependymal nodules, resembling the major clinical features that occur in patients with TSC. These TSC1+/- Bama miniature pigs could serve as valuable large animal models for further elucidation of the pathogenesis of TSC and the development of therapeutic strategies for TSC disease.


Asunto(s)
Neoplasias Cardíacas/genética , Rabdomioma/genética , Proteína S6 Ribosómica/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Esclerosis Tuberosa/genética , Alelos , Animales , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Neoplasias Cardíacas/complicaciones , Neoplasias Cardíacas/patología , Humanos , Ratones , Mutación/genética , Técnicas de Transferencia Nuclear , Rabdomioma/complicaciones , Rabdomioma/patología , Porcinos/genética , Porcinos Enanos/genética , Serina-Treonina Quinasas TOR/genética , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/patología , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética
15.
Structure ; 27(12): 1771-1781.e5, 2019 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-31676287

RESUMEN

The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5' terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the +1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.


Asunto(s)
Autoantígenos/química , Guanosina/análogos & derivados , ARN Mensajero/química , Ribonucleoproteínas/química , Proteína S6 Ribosómica/química , Secuencias de Aminoácidos , Autoantígenos/genética , Autoantígenos/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Guanosina/química , Guanosina/metabolismo , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Especificidad por Sustrato , Termodinámica , Antígeno SS-B
16.
Biol Reprod ; 101(5): 1046-1055, 2019 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-31403169

RESUMEN

Maternal inflammation (MI) is associated with many adverse perinatal outcomes. The placenta plays a vital role in mediating maternal-fetal resource allocation. Studies have shown that MI contributes to placental dysfunction, which then leads to adverse birth outcomes and high health risks throughout childhood. Placental mammalian target of rapamycin complex 1 (mTORC1) signaling pathway links maternal nutrient availability to fetal growth; however, the impact of MI on mTORC1 signaling in the placenta remains unclear. In this study, we sought to explore the changes of mTORC1 signaling in the mouse placenta at late gestation by using two models of MI employing lipopolysaccharide (LPS) and interleukin-1ß (IL-1ß) to mimic acute (aMI) and sub-chronic (cMI) inflammatory states, respectively. We determined placental mTORC1 activity by measuring the activity of mTORC1 downstream molecules, including S6k, 4Ebp1, and rpS6. In the aMI model, we found that mTORC1 activity was significantly decreased in the placental decidual and junctional zone at 2 and 6 h after LPS surgery, respectively; however, mTORC1 activity was significantly increased in the placental labyrinth zone at 2, 6, and 24 h after LPS treatment, respectively. In the cMI model, we observed that mTORC1 activity was increased only in the placental labyrinth zone after consecutive IL-1ß exposure. Our study reveals that different parts of the mouse placenta react differently to MI, leading to variable mTORC1 activity throughout the placenta. This suggests that different downstream molecules of mTORC1 from different parts of the mouse placenta may be used in clinical research to monitor the fetal well-being during MI.


Asunto(s)
Inflamación/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Placenta/metabolismo , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Interleucina-1beta/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Fosforilación , Embarazo , Distribución Aleatoria , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo
17.
Hum Mol Genet ; 28(22): 3755-3765, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31411685

RESUMEN

Single germline or somatic activating mutations of mammalian target of rapamycin (mTOR) pathway genes are emerging as a major cause of type II focal cortical dysplasia (FCD), hemimegalencephaly (HME) and tuberous sclerosis complex (TSC). A double-hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures and hypochromic skin patches, we identified the ribosomal protein S6 (RPS6) p.R232H variant, present as somatic mosaicism at ~15.1% in dysplastic brain tissue and ~11% in blood, and the MTOR p.S2215F variant, detected as ~8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.


Asunto(s)
Hemimegalencefalia/genética , Proteína S6 Ribosómica/genética , Serina-Treonina Quinasas TOR/genética , Animales , Encéfalo/metabolismo , Niño , Epilepsia Refractaria/genética , Epilepsia Refractaria/metabolismo , Epilepsia/genética , Femenino , Humanos , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I/genética , Ratones , Mosaicismo , Mutación , Neuronas/metabolismo , Proteína S6 Ribosómica/metabolismo , Serina-Treonina Quinasas TOR/metabolismo
18.
EMBO Rep ; 20(7): e47546, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31267709

RESUMEN

Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long-term hematopoietic stem cells (LT-HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT-HSC in the BM of murine xenografts with emphasis on the role of AML-derived extracellular vesicles (EV). AML-EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT-HSC quiescence. Mechanistically, AML-EV transfer a panel of miRNA, including miR-1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo-phosphorylation, which in turn impairs protein synthesis in LT-HSC. While HSC functionally recover from quiescence upon transplantation to an AML-naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double-strand breaks and evidence of a sustained DNA-damage response. In sum, AML-EV contribute to niche-dependent, reversible quiescence and elicit persisting DNA damage in LT-HSC.


Asunto(s)
Vesículas Extracelulares/metabolismo , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Nicho de Células Madre , Animales , Línea Celular Tumoral , Células Cultivadas , Roturas del ADN de Doble Cadena , Femenino , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Proteína S6 Ribosómica/genética
19.
Reprod Toxicol ; 89: 54-66, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31278979

RESUMEN

mTORC1/rpS6 signaling complex promoted Sertoli blood-testis barrier (BTB) remodeling by perturbing Sertoli cell-cell adhesion site known as the basal ectoplasmic specialization (ES). mTORC1/rpS6 complex also promoted disruption of spermatid adhesion at the Sertoli-spermatid interface called the apical ES. Herein, we performed analyses using the adjudin (a non-hormonal male contraceptive drug under development) model, wherein adjudin was known to perturb apical and basal ES function when used at high dose. Through direct administration of adjudin to the testis, adjudin at doses that failed to perturb BTB integrity per se, overexpression of an rpS6 phosphomimetic (i.e., constitutively active) mutant (i.e., p-rpS6-MT) that modified BTB function considerably potentiated adjudin efficacy. This led to disorderly spatial expression of proteins necessary to maintain the proper cytoskeletal organization of F-actin and microtubules (MTs) across the seminiferous epithelium, leading to germ cell exfoliation and aspermatogenesis. These findings yielded important insights regarding the role of mTORC1/rpS6 signaling complex in regulating BTB homeostasis.


Asunto(s)
Barrera Hematotesticular/efectos de los fármacos , Anticonceptivos Masculinos/farmacología , Hidrazinas/farmacología , Indazoles/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteína S6 Ribosómica/metabolismo , Células de Sertoli/efectos de los fármacos , Espermatogénesis/efectos de los fármacos , Animales , Barrera Hematotesticular/metabolismo , Anticonceptivos Masculinos/administración & dosificación , Relación Dosis-Respuesta a Droga , Hidrazinas/administración & dosificación , Indazoles/administración & dosificación , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratas , Ratas Sprague-Dawley , Proteína S6 Ribosómica/genética , Células de Sertoli/metabolismo , Transfección
20.
Am J Physiol Endocrinol Metab ; 317(1): E121-E138, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-31112404

RESUMEN

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.


Asunto(s)
Barrera Hematotesticular/metabolismo , Anticonceptivos Masculinos/farmacología , Hidrazinas/farmacología , Indazoles/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteína S6 Ribosómica/metabolismo , Animales , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mutagénesis Sitio-Dirigida , Ratas , Ratas Sprague-Dawley , Proteína S6 Ribosómica/genética , Epitelio Seminífero/metabolismo , Células de Sertoli/metabolismo , Transducción de Señal/efectos de los fármacos , Espermatocitos/metabolismo , Espermatogénesis/efectos de los fármacos
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