RESUMEN
Nucleostemin (NS) plays a role in liver regeneration, and aging reduces its expression in the baseline and regenerating livers following 70% partial hepatectomy (PHx). Here we interrogate the mechanism controlling NS expression during liver regeneration and aging. The NS promoter was analyzed by TRANSFAC. Functional studies were performed using cell-based luciferase assay, endogenous NS expression in Hep3B cells, mouse livers with a gain-of-function mutation of C/EBPα (S193D), and mouse livers with C/EBPα knockdown. We found a CAAT box with four C/EBPα binding sites (-1216 to -735) and a GC box with consensus binding sites for c-Myc, E2F1, and p300-associated protein complex (-633 to -1). Age-related changes in NS expression correlated positively with the expression of c-Myc, E2F1, and p300, and negatively with that of C/EBPα and C/EBPß. PHx upregulated NS expression at 1d, coinciding with an increase in E2F1 and a decrease in C/EBPα. C/EBPα bound to the consensus sequences found in the NS promoter in vitro and in vivo, inhibited its transactivational activity in a binding site-dependent manner, and decreased the expression of endogenous NS in Hep3B cells. In vivo activation of C/EBPα by the S193D mutation resulted in a 4th-day post-PHx reduction of NS, a feature shared by 16-m/o livers. Finally, C/EBPα knockdown increased its expression in aged (24-m/o) livers under both baseline and regeneration conditions. This study reports the C/EBPα suppression of NS expression in aged livers, providing a new perspective on the mechanistic orchestration of tissue homeostasis in aging.
Asunto(s)
Envejecimiento , Proteínas de Unión al GTP , Regeneración Hepática , Proteínas Nucleares , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc , Animales , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Ratones , Envejecimiento/metabolismo , Envejecimiento/fisiología , Envejecimiento/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Hepatectomía , Sitios de Unión , Hígado/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Masculino , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Ratones Endogámicos C57BL , Línea Celular Tumoral , Proteínas de Unión al ARNRESUMEN
GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-α-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of ß-arrestin-2 but GPR56 internalization was ß-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.
Asunto(s)
Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Humanos , Transducción de Señal , Células HEK293 , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Línea Celular Tumoral , Ligandos , Animales , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genéticaRESUMEN
Fish retinal ganglion cells (RGCs) can regenerate after optic nerve lesions (ONLs). We previously reported that heat shock factor 1 (HSF1) and Yamanaka factors increased in the zebrafish retina 0.5-24 h after ONLs, and they led to cell survival and the transformation of neuro-stem cells. We also showed that retinoic acid (RA) signaling and transglutaminase 2 (TG2) were activated in the fish retina, performing neurite outgrowth 5-30 days after ONLs. In this study, we found that RA signaling and TG2 increased within 0.5 h in the zebrafish retina after ONLs. We examined their interaction with the TG2-specific morpholino and inhibitor due to the significantly close initiation time of TG2 and HSF1. The inhibition of TG2 led to the complete suppression of HSF1 expression. Furthermore, the results of a ChIP assay with an anti-TG2 antibody evidenced significant anti-TG2 immunoprecipitation of HSF1 genome DNA after ONLs. The inhibition of TG2 also suppressed Yamanaka factors' gene expression. This rapid increase in TG2 expression occurred 30 min after the ONLs, and RA signaling occurred 15 min before this change. The present study demonstrates that TG2 regulates Yamanaka factors via HSF1 signals in the acute phase of fish optic nerve regeneration.
Asunto(s)
Factores de Transcripción del Choque Térmico , Regeneración Nerviosa , Nervio Óptico , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Pez Cebra , Animales , Pez Cebra/genética , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Transglutaminasas/genética , Transglutaminasas/metabolismo , Regeneración Nerviosa/genética , Nervio Óptico/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico/genética , Tretinoina/farmacología , Tretinoina/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Células Ganglionares de la Retina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/genética , Transducción de SeñalRESUMEN
Dynamin-like GTPase proteins, including myxoma (Mx) and guanylate-binding proteins (GBPs), are among the many interferon stimulated genes induced following viral infections. While studies report that human (h)GBPs inhibit different viruses in vitro, few have convincingly demonstrated that mouse (m)GBPs mediate antiviral activity, although mGBP-deficient mice have been used extensively to define their importance in immunity to diverse intracellular bacteria and protozoa. Herein, we demonstrate that individual (overexpression) or collective (knockout (KO) mice) mGBPs of the chromosome 3 cluster (mGBPchr3) do not inhibit replication of five viruses from different virus families in vitro, nor do we observe differences in virus titres recovered from wild type versus mGBPchr3 KO mice after infection with three of these viruses (influenza A virus, herpes simplex virus type 1 or lymphocytic choriomeningitis virus). These data indicate that mGBPchr3 do not appear to be a major component of cell-intrinsic antiviral immunity against the diverse viruses tested in our studies.
Asunto(s)
Proteínas de Unión al GTP , Ratones Noqueados , Animales , Ratones , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/inmunología , Modelos Animales de Enfermedad , Replicación Viral , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/genética , Ratones Endogámicos C57BL , Virus de la Coriomeningitis Linfocítica/fisiología , Virus de la Coriomeningitis Linfocítica/inmunología , Virosis/inmunología , Virosis/genéticaRESUMEN
Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.
Asunto(s)
Ácidos Cumáricos , Proteínas de Unión al GTP , Glioblastoma , Nanopartículas , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Ácidos Cumáricos/farmacología , Humanos , Transglutaminasas/metabolismo , Transglutaminasas/genética , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Línea Celular Tumoral , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Nanopartículas/química , Portadores de Fármacos/química , Apoptosis/efectos de los fármacos , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
RHOBTB2 was first described as epileptogenic when it presents a missense variant in 2016 and studied more specifically in 2018. It is a gene that causes rare, but potentially severe childhood epileptic encephalopathy. In 2021, research confirmed that heterozygous mutations of RHOBTB2 included other clinical signs besides these encephalopathies. Thus, these infantile epilepsies are mainly associated with highly variable phenotypes, with developmental delay, post-traumatic encephalitis, paroxysmal movement disorders and iconographic brain damage. In this work, after presenting a clinical case, we will recall the role of RhoGTPases on neuronal development. We will then discuss a study which highlighted the neurodevelopmental impact of mutations on the RHOBTB2 gene by carrying out work on Drosophila melanogaster flies. Finally, we will compare the presented clinical case with a literature review.
Le gène RHOBTB2 est décrit pour la première fois comme épileptogène alors qu'il présente un variant faux-sens en 2016, puis est étudié plus précisément en 2018. Il s'agit d'un gène qui est à l'origine d'encéphalopathies épileptiques infantiles rares, mais pouvant être sévères. En 2021, des recherches ont confirmé que les mutations hétérozygotes de RHOBTB2 englobaient d'autres signes cliniques que ces encéphalopathies. Ainsi, ces épilepsies infantiles sont associées, principalement, avec des phénotypes fortement variables, à un retard développemental, à des encéphalites post-traumatiques, à des troubles paroxystiques des mouvements et à des atteintes iconographiques de l'encéphale. Dans ce travail, après avoir présenté un cas clinique, nous rappellerons le rôle des RhoGTPases sur le développement neuronal. Nous discuterons ensuite d'une étude qui a mis en évidence l'impact neurodéveloppemental de mutations sur le gène RHOBTB2 en réalisant des travaux sur des mouches Drosophila melanogaster. Pour terminer, nous mettrons le cas clinique présenté en parallèle avec une revue de la littérature réalisée par rapport à ce gène.
Asunto(s)
Mutación , Humanos , Animales , Proteínas Supresoras de Tumor/genética , Proteínas de Unión al GTP/genética , Masculino , Drosophila melanogaster/genética , Femenino , LactanteRESUMEN
Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.
Asunto(s)
Microscopía por Crioelectrón , Guanosina Difosfato , Guanosina Trifosfato , Mutación , Humanos , Células HEK293 , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Unión Proteica , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genéticaRESUMEN
Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.
Asunto(s)
Epigénesis Genética , Histonas , Neoplasias , Proteína Glutamina Gamma Glutamiltransferasa 2 , Procesamiento Proteico-Postraduccional , Humanos , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/enzimología , Neoplasias/patología , Histonas/metabolismo , Animales , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Transglutaminasas/metabolismo , Transglutaminasas/genética , Regulación Neoplásica de la Expresión Génica , Transducción de SeñalRESUMEN
Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.
Asunto(s)
Supervivencia Celular , Proteínas de Unión al GTP , Conformación Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Humanos , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Transglutaminasas/metabolismo , Transglutaminasas/química , Transglutaminasas/genética , Supervivencia Celular/efectos de los fármacos , Microscopía por Crioelectrón , Línea Celular Tumoral , Muerte Celular/efectos de los fármacos , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Calcio/metabolismoRESUMEN
ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.
Asunto(s)
Proteínas de Ciclo Celular , Centrosoma , Microtúbulos , Proteínas del Tejido Nervioso , Células-Madre Neurales , Neurogénesis , Animales , Microtúbulos/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Neurogénesis/genética , Células-Madre Neurales/metabolismo , Centrosoma/metabolismo , Proliferación Celular , Movimiento Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Tubulina (Proteína)/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genéticaRESUMEN
Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.
Asunto(s)
Colesterol , Proteínas de Unión al GTP , Unión Proteica , Receptor de Colecistoquinina A , Receptor de Colecistoquinina B , Animales , Humanos , Células CHO , Colesterol/metabolismo , Cricetulus , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Células HEK293 , Ligandos , Mutación , Conformación Proteica , Receptor de Colecistoquinina A/metabolismo , Receptor de Colecistoquinina A/genética , Receptor de Colecistoquinina B/metabolismo , Receptor de Colecistoquinina B/genéticaRESUMEN
Spastic paraplegia type 3A (SPG3A) is the second most common form of hereditary spastic paraplegia (HSP). This autosomal-dominant-inherited motor disorder is caused by heterozygous variants in the ATL1 gene which usually presents as a pure childhood-onset spastic paraplegia. Affected individuals present muscle weakness and spasticity in the lower limbs, with symptom onset in the first decade of life. Individuals with SPG3A typically present a slow progression and remain ambulatory throughout their life. Here we report three unrelated individuals presenting with very-early-onset (before 7 months) complex, and severe HSP phenotypes (axial hypotonia, spastic quadriplegia, dystonia, seizures and intellectual disability). For 2 of the 3 patients, these phenotypes led to the initial diagnosis of cerebral palsy (CP). These individuals carried novel ATL1 pathogenic variants (a de novo ATL1 missense p.(Lys406Glu), a homozygous frameshift p.(Arg403Glufs*3) and a homozygous missense variant (p.Tyr367His)). The parents carrying the heterozygous frameshift and missense variants were asymptomatic. Through these observations, we increase the knowledge on genotype-phenotype correlations in SPG3A and offer additional proof for possible autosomal recessive forms of SPG3A, while raising awareness on these exceptional phenotypes. Their ability to mimic CP also implies that genetic testing should be considered for patients with atypical forms of CP, given the implications for genetic counseling.
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Proteínas de Unión al GTP , Proteínas de la Membrana , Paraplejía Espástica Hereditaria , Humanos , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/fisiopatología , Paraplejía Espástica Hereditaria/diagnóstico , Masculino , Femenino , Proteínas de la Membrana/genética , Proteínas de Unión al GTP/genética , Estudios de Asociación Genética , Fenotipo , Niño , Lactante , Adolescente , Adulto , PreescolarRESUMEN
Pulmonary hypertension (PH) is a severe cardiovascular disease characterised by pulmonary vascular remodelling. The pivotal role of cellular senescence in vascular remodelling has been acknowledged. Transglutaminase type 2 (TG2), a calcium-dependent enzyme, is intricately linked to both cellular senescence and PH. However, the precise mechanisms underlying the involvement of TG2 in PH remain unclear. In this study, we explored the expression of TG2 and the cellular senescence marker p16INK4a in the pulmonary vasculature of mice with PH induced by hypoxia combined with SU5416. Our findings revealed upregulation of both TG2 and p16INK4a expression in the pulmonary vasculature of PH mice. Additionally, a notable increase in TG2 expression was observed in senescent pulmonary artery smooth muscle cells (PASMC). To delve deeper, we employed proteomic sequencing to reveal seven genes associated with cellular senescence, with a subsequent focus on MAPK14. Our investigation revealed that TG2 regulates senescence in PASMC by modulating the phosphorylation levels of MAPK14. Additionally, in the context of hypoxia combined with SU5416, our observations revealed a noteworthy reduction in both pulmonary vascular remodelling and senescent manifestations in smooth muscle-specific TG2 knockout mice compared with their wild-type counterparts. In summary, our findings indicate that TG2 deficiency lowers the senescence levels of PASMC by inhibiting the activity of MAPK14. This inhibition of senescence in the pulmonary vasculature of PH mice helps to decelerate the progression of pulmonary vascular remodelling and consequently hinders the onset and development of PH.
Asunto(s)
Senescencia Celular , Hipertensión Pulmonar , Miocitos del Músculo Liso , Proteína Glutamina Gamma Glutamiltransferasa 2 , Arteria Pulmonar , Remodelación Vascular , Animales , Masculino , Ratones , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Hipoxia/metabolismo , Indoles , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Arteria Pulmonar/metabolismo , Pirroles , Transglutaminasas/metabolismo , Transglutaminasas/genéticaAsunto(s)
Clatrina , Endocitosis , Proteínas de Unión al GTP , Virus Hantaan , Internalización del Virus , Humanos , Endocitosis/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Clatrina/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Virus Hantaan/fisiología , Virus Hantaan/efectos de los fármacos , Interacciones Huésped-Patógeno , AnimalesRESUMEN
Inherited retinal diseases encompass a genetically diverse group of conditions caused by variants in genes critical to retinal function, including handful of ribosome-associated genes. This study focuses on the HBS1L gene, which encodes for the HBS1-like translational GTPase that is crucial for ribosomal rescue. We have reported a female child carrying biallelic HBS1L variants, manifesting with poor growth and neurodevelopmental delay. Here, we describe the ophthalmologic findings in the patient and in Hbs1ltm1a/tm1a hypomorph mice and describe the associated microscopic and molecular perturbations. The patient has impaired visual function, showing dampened amplitudes of a- and b-waves in both rod- and cone-mediated responses. Hbs1ltm1a/tm1a mice exhibited profound thinning of the entire retina, specifically of the outer photoreceptor layer, due to extensive photoreceptor cell apoptosis. Loss of Hbs1l resulted in comprehensive proteomic alterations by mass spectrometry analysis, with an increase in the levels of 169 proteins and a decrease in the levels of 480 proteins, including rhodopsin (Rho) and peripherin 2 (Prph2). Gene Ontology biological process and gene set enrichment analyses reveal that the downregulated proteins are primarily involved in phototransduction, cilium assembly and photoreceptor cell development. These findings underscore the importance of ribosomal rescue proteins in maintaining retinal health, particularly in photoreceptor cells.
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Modelos Animales de Enfermedad , Distrofias Retinianas , Animales , Distrofias Retinianas/patología , Distrofias Retinianas/genética , Femenino , Humanos , Ratones , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Apoptosis , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/deficiencia , Proteínas de Unión al GTP/genética , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , NiñoRESUMEN
BACKGROUND: Lung adenocarcinoma (LUAD) is a leading cause of tumor-associated mortality, and it is needed to find new target to combat this disease. Guanine nucleotide-binding -protein-like 3 (GNL3) mediates cell proliferation and apoptosis in several cancers, but its role in LUAD remains unclear. OBJECTIVE: To explore the expression and function of Guanine nucleotide-binding protein-like 3 (GNL3) in lung adenocarcinoma (LUAD) and its potential mechanism in inhibiting the growth of LUAD cells. METHODS: We evaluated the expression of GNL3 in LUAD tissues and its association with patient prognosis using databases and immunohistochemistry. Cell proliferation was assessed by CCK-8 assay as well as colony formation, while apoptosis was evaluated by FCM. The effect of GNL3 knockdown on the Wnt/ß-catenin axis was investigated by Immunoblot analysis. RESULTS: GNL3 is overexpressed in LUAD tissues and is correlated with poor prognosis. Knockdown of GNL3 significantly inhibited the growth as well as induced apoptosis in A549 as well as H1299 cells. Furthermore, we found that the inhibitory effect of GNL3 knockdown on LUAD cell growth is associated with the downregulation of the Wnt/ß-catenin axis. CONCLUSION: GNL3 is key in the progression of LUAD by metiating Wnt/ß-catenin axis. Targeting GNL3 may represent a novel therapeutic method for LUAD treatment.
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Adenocarcinoma del Pulmón , Apoptosis , Proliferación Celular , Técnicas de Silenciamiento del Gen , Neoplasias Pulmonares , Vía de Señalización Wnt , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Línea Celular Tumoral , Pronóstico , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , beta Catenina/metabolismo , Regulación Neoplásica de la Expresión Génica , Células A549 , Proteínas NuclearesRESUMEN
The metal-resistant beta-proteobacterium Cupriavidus metallidurans is also able to survive conditions of metal starvation. We show that zinc-starved cells can substitute some of the required zinc with cobalt but not with nickel ions. The zinc importer ZupT was necessary for this process but was not essential for either zinc or cobalt import. The cellular cobalt content was also influenced by the two COG0523-family proteins, CobW2 and CobW3. Pulse-chase experiments with radioactive and isotope-enriched zinc demonstrated that both proteins interacted with ZupT to control the cellular flow-equilibrium of zinc, a central process of zinc homeostasis. Moreover, an antagonistic interplay of CobW2 and CobW3 in the presence of added cobalt caused a growth defect in mutant cells devoid of the cobalt efflux system DmeF. Full cobalt resistance also required a synergistic interaction of ZupT and DmeF. Thus, the two transporters along with CobW2 and CobW3 interact to control cobalt homeostasis in a process that depends on zinc availability. Because ZupT, CobW2, and CobW3 also direct zinc homeostasis, this process links the control of cobalt and zinc homeostasis, which subsequently protects C. metallidurans against cadmium stress and general metal starvation.IMPORTANCEIn bacterial cells, zinc ions need to be allocated to zinc-dependent proteins without disturbance of this process by other transition metal cations. Under zinc-starvation conditions, C. metallidurans floods the cell with cobalt ions, which protect the cell against cadmium toxicity, help withstand metal starvation, and provide cobalt to metal-promiscuous paralogs of essential zinc-dependent proteins. The number of cobalt ions needs to be carefully controlled to avoid a toxic cobalt overload. This is accomplished by an interplay of the zinc importer ZupT with the COG0523-family proteins, CobW3, and CobW2. At high external cobalt concentrations, this trio of proteins additionally interacts with the cobalt efflux system, DmeF, so that these four proteins form an inextricable link between zinc and cobalt homeostasis.
Asunto(s)
Proteínas Bacterianas , Cobalto , Cupriavidus , Homeostasis , Zinc , Cobalto/metabolismo , Zinc/metabolismo , Cupriavidus/metabolismo , Cupriavidus/genética , Cupriavidus/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genéticaRESUMEN
Mathematical models of biochemical reaction networks are an important and emerging tool for the study of cell signaling networks involved in disease processes. One promising potential application of such mathematical models is the study of how disease-causing mutations promote the signaling phenotype that contributes to the disease. It is commonly assumed that one must have a thorough characterization of the network readily available for mathematical modeling to be useful, but we hypothesized that mathematical modeling could be useful when there is incomplete knowledge and that it could be a tool for discovery that opens new areas for further exploration. In the present study, we first develop a mechanistic mathematical model of a G-protein coupled receptor signaling network that is mutated in almost all cases of uveal melanoma and use model-driven explorations to uncover and explore multiple new areas for investigating this disease. Modeling the two major, mutually-exclusive, oncogenic mutations (Gαq/11 and CysLT2R) revealed the potential for previously unknown qualitative differences between seemingly interchangeable disease-promoting mutations, and our experiments confirmed oncogenic CysLT2R was impaired at activating the FAK/YAP/TAZ pathway relative to Gαq/11. This led us to hypothesize that CYSLTR2 mutations in UM must co-occur with other mutations to activate FAK/YAP/TAZ signaling, and our bioinformatic analysis uncovers a role for co-occurring mutations involving the plexin/semaphorin pathway, which has been shown capable of activating this pathway. Overall, this work highlights the power of mechanism-based computational systems biology as a discovery tool that can leverage available information to open new research areas.