RESUMO
The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAFV600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRASQ61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAFV600E PDX highlighting its effectiveness in combating the advent of drug resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Pirazóis/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Humanos , Melanoma/enzimologia , Melanoma/fisiopatologia , Proteína Fosfatase 2/antagonistas & inibidoresRESUMO
The CADM family of proteins consists of four neuronal specific adhesion molecules (CADM1, CADM2, CADM3 and CADM4) that mediate the direct contact and interaction between axons and glia. In the peripheral nerve, axon-Schwann cell interaction is essential for the structural organization of myelinated fibres and is primarily mediated by the binding of CADM3, expressed in axons, to CADM4, expressed by myelinating Schwann cells. We have identified-by whole exome sequencing-three unrelated families, including one de novo patient, with axonal Charcot-Marie-Tooth disease (CMT2) sharing the same private variant in CADM3, Tyr172Cys. This variant is absent in 230 000 control chromosomes from gnomAD and predicted to be pathogenic. Most CADM3 patients share a similar phenotype consisting of autosomal dominant CMT2 with marked upper limb involvement. High resolution mass spectrometry analysis detected a newly created disulphide bond in the mutant CADM3 potentially modifying the native protein conformation. Our data support a retention of the mutant protein in the endoplasmic reticulum and reduced cell surface expression in vitro. Stochastic optical reconstruction microscopy imaging revealed decreased co-localization of the mutant with CADM4 at intercellular contact sites. Mice carrying the corresponding human mutation (Cadm3Y170C) showed reduced expression of the mutant protein in axons. Cadm3Y170C mice showed normal nerve conduction and myelin morphology, but exhibited abnormal axonal organization, including abnormal distribution of Kv1.2 channels and Caspr along myelinated axons. Our findings indicate the involvement of abnormal axon-glia interaction as a disease-causing mechanism in CMT patients with CADM3 mutations.
Assuntos
Moléculas de Adesão Celular/genética , Doença de Charcot-Marie-Tooth/genética , Imunoglobulinas/genética , Adulto , Axônios/patologia , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neuroglia/patologia , Linhagem , FenótipoRESUMO
Transactivating DNA-binding protein-43 (TDP-43) inclusions and the accumulation of phosphorylated and ubiquitinated tau proteins (p-tau) have been identified in postmortem brain specimens from patients with chronic traumatic encephalopathy (CTE). To examine whether these proteins contribute to the development of CTE, we utilized an in vitro trauma system known to reproduce many of the findings observed in humans and experimental animals with traumatic brain injury. Accordingly, we examined the role of TDP-43 and Tau in an in vitro model of trauma, and determined whether these proteins contribute to the defective neuronal integrity associated with CNS trauma. Single or multiple episodes of trauma to cultured neurons resulted in a time-dependent increase in cytosolic levels of phosphorylated TDP-43 (p-TDP-43). Trauma to cultured neurons also caused an increase in levels of casein kinase 1 epsilon (CK1ε), and ubiquitinated p-TDP-43, along with a decrease in importin-ß (all factors known to mediate the "TDP-43 proteinopathy"). Defective neuronal integrity, as evidenced by a reduction in levels of the NR1 subunit of the NMDA receptor, and in PSD95, along with increased levels of phosphorylated tau were also observed. Additionally, increased levels of intra- and extracellular thrombospondin-1 (TSP-1) (a factor known to regulate neuronal integrity) were observed in cultured astrocytes at early stages of trauma, while at later stages decreased levels were identified. The addition of recombinant TSP-1, conditioned media from cultured astrocytes at early stages of trauma, or the CK1ε inhibitor PF4800567 hydrochloride to traumatized cultured neurons reduced levels of p-TDP-43, and reversed the trauma-induced decline in NR1 subunit of the NMDA receptor and PSD95 levels. These findings suggest that a trauma-induced increase in TDP-43 phosphorylation contributes to defective neuronal integrity, and that increasing TSP-1 levels may represent a useful therapeutic approach for the prevention of the neuronal TDP-43 proteinopathy associated with CTE. Read the Editorial Highlight for this article on page 531.
Assuntos
Astrócitos/metabolismo , Encefalopatia Traumática Crônica/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/fisiologia , Proteinopatias TDP-43/metabolismo , Trombospondina 1/biossíntese , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Masculino , Ratos , Ratos Endogâmicos F344 , Trombospondina 1/metabolismoRESUMO
Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.
Assuntos
Amônia/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Trombospondina 1/metabolismo , Amônia/metabolismo , Animais , Antioxidantes/farmacologia , Feminino , Encefalopatia Hepática/metabolismo , NF-kappa B/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-myc/farmacologia , Ratos , Sinaptofisina/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Tirzepatide, a glucose-dependent insulinotropic polypeptide/glucagon-like peptide 1 receptor (GIPR/GLP-1R) agonist, has, in clinical trials, demonstrated greater reductions in glucose, body weight, and triglyceride levels compared with selective GLP-1R agonists in people with type 2 diabetes (T2D). However, cellular mechanisms by which GIPR agonism may contribute to these improved efficacy outcomes have not been fully defined. Using human adipocyte and mouse models, we investigated how long-acting GIPR agonists regulate fasted and fed adipocyte functions. In functional assays, GIPR agonism enhanced insulin signaling, augmented glucose uptake, and increased the conversion of glucose to glycerol in a cooperative manner with insulin; however, in the absence of insulin, GIPR agonists increased lipolysis. In diet-induced obese mice treated with a long-acting GIPR agonist, circulating triglyceride levels were reduced during oral lipid challenge, and lipoprotein-derived fatty acid uptake into adipose tissue was increased. Our findings support a model for long-acting GIPR agonists to modulate both fasted and fed adipose tissue function differentially by cooperating with insulin to augment glucose and lipid clearance in the fed state while enhancing lipid release when insulin levels are reduced in the fasted state.
Assuntos
Adipócitos , Polipeptídeo Inibidor Gástrico , Receptores dos Hormônios Gastrointestinais , Animais , Humanos , Masculino , Camundongos , Adipócitos/metabolismo , Adipócitos/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 2 , Glucose/metabolismo , Insulina/metabolismo , Lipólise/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Nutrientes/metabolismo , Obesidade/metabolismo , Obesidade/tratamento farmacológico , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/agonistas , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/metabolismoRESUMO
Background: Optic neuropathy (ON) is a major cause of irreversible blindness, yet the molecular determinants that contribute to neuronal demise have not been fully elucidated. Several studies have identified 'ephrin signaling' as one of the most dysregulated pathways in the early pathophysiology of ON with varied etiologies. Developmentally, gradients in ephrin signaling coordinate retinotopic mapping via repulsive modulation of cytoskeletal dynamics in neuronal membranes. Little is known about the role ephrin signaling played in the post-natal visual system and its correlation with the onset of optic neuropathy. Methods: Postnatal mouse retinas were collected for mass spectrometry analysis for Eph receptors. Optic nerve crush (ONC) model was employed to induce optic neuropathy, and proteomic changes during the acute phase of neuropathic onset were analyzed. Confocal and super-resolution microscopy determined the cellular localization of activated Eph receptors after ONC injury. Eph receptor inhibitors assessed the neuroprotective effect of ephrin signaling modulation. Results: Mass spectrometry revealed expression of seven Eph receptors (EphA2, A4, A5, B1, B2, B3, and B6) in postnatal mouse retinal tissue. Immunoblotting analysis indicated a significant increase in phosphorylation of these Eph receptors 48 hours after ONC. Confocal microscopy demonstrated the presence of both subclasses of Eph receptors in the inner retinal layers. STORM super-resolution imaging combined with optimal transport colocalization analysis revealed a significant co-localization of activated Eph receptors with injured neuronal processes, compared to uninjured neuronal and/or injured glial cells, 48 hours post-ONC. Eph receptor inhibitors displayed notable neuroprotective effects after 6 days of ONC injury. Conclusions: Our findings demonstrate the functional presence of diverse Eph receptors in the postnatal mammalian retina, capable of modulating multiple biological processes. Pan-Eph receptor activation contributes to the onset of neuropathy in ONs, with preferential activation of Eph receptors on neuronal processes in the inner retina following optic nerve injury. Notably, Eph receptor activation precedes neuronal loss. We observed neuroprotective effects upon inhibiting Eph receptors. Our study highlights the importance of investigating this repulsive pathway in early optic neuropathies and provides a comprehensive characterization of the receptors present in the developed retina of mice, relevant to both homeostasis and disease processes.
RESUMO
BACKGROUND: Optic neuropathy is a major cause of irreversible blindness, yet the molecular determinants that contribute to neuronal demise have not been fully elucidated. Several studies have identified 'ephrin signaling' as one of the most dysregulated pathways in the early pathophysiology of optic neuropathy with varied etiologies. Developmentally, gradients in ephrin signaling coordinate retinotopic mapping via repulsive modulation of cytoskeletal dynamics in neuronal membranes. Little is known about the role ephrin signaling plays in the post-natal visual system and its correlation with the onset of optic neuropathy. METHODS: Postnatal mouse retinas were collected for mass spectrometry analysis for erythropoietin-producing human hepatocellular (Eph) receptors. Optic nerve crush (ONC) model was employed to induce optic neuropathy, and proteomic changes during the acute phase of neuropathic onset were analyzed. Confocal and super-resolution microscopy determined the cellular localization of activated Eph receptors after ONC injury. Eph receptor inhibitors assessed the neuroprotective effect of ephrin signaling modulation. RESULTS: Mass spectrometry revealed expression of seven Eph receptors (EphA2, A4, A5, B1, B2, B3, and B6) in postnatal mouse retinal tissue. Immunoblotting analysis indicated a significant increase in phosphorylation of these Eph receptors 48 h after ONC. Confocal microscopy demonstrated the presence of both subclasses of Eph receptors within the retina. Stochastic optical reconstruction microscopy (STORM) super-resolution imaging combined with optimal transport colocalization analysis revealed a significant co-localization of activated Eph receptors with injured neuronal cells, compared to uninjured neuronal and/or injured glial cells, 48 h post-ONC. Eph receptor inhibitors displayed notable neuroprotective effects for retinal ganglion cells (RGCs) after six days of ONC injury. CONCLUSIONS: Our findings demonstrate the functional presence of diverse Eph receptors in the postnatal mammalian retina, capable of modulating multiple biological processes. Pan-Eph receptor activation contributes to the onset of neuropathy in optic neuropathies, with preferential activation of Eph receptors on neuronal processes in the inner retina following optic nerve injury. Notably, Eph receptor activation precedes neuronal loss. We observed a neuroprotective effect on RGCs upon inhibiting Eph receptors. Our study highlights the importance of investigating this repulsive pathway in early optic neuropathies and provides a comprehensive characterization of the receptors present in the developed retina of mice, relevant to both homeostasis and disease processes.
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MicroRNA (miRNA) homeostasis is crucial for the posttranscriptional regulation of their target genes during development and in disease states. miRNAs are derived from primary transcripts and are processed from a hairpin precursor intermediary to a mature 22-nucleotide duplex RNA. Loading of the duplex into the Argonaute (AGO) protein family is pivotal to miRNA abundance and its posttranscriptional function. The Integrator complex plays a key role in protein coding and noncoding RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination. Here, we report that loss of Integrator results in global destabilization of mature miRNAs. Enhanced ultraviolet cross-linking and immunoprecipitation of Integrator uncovered an association with duplex miRNAs before their loading onto AGOs. Tracing miRNA fate from biogenesis to stabilization by incorporating 4-thiouridine in nascent transcripts pinpointed a critical role for Integrator in miRNA assembly into AGOs.
Assuntos
MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica , Núcleo Celular/metabolismoRESUMO
The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Proteínas RGS/metabolismo , Frações Subcelulares/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cricetinae , Cricetulus , Subunidades beta da Proteína de Ligação ao GTP/deficiência , Gânglios Espinais/citologia , Regulação da Expressão Gênica/genética , Imageamento Tridimensional , Imunoprecipitação , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Mutação/genética , Neurônios/citologia , Conformação Proteica , Proteínas RGS/genética , Retina/citologia , Retina/metabolismo , TransfecçãoRESUMO
BACKGROUND: Mast cells are implicated in the pathogenesis of obesity and insulin resistance. Here, we explored the effects of leptin deficiency-induced obesity on the density of mast cells in metabolic (abdominal fat depots, skeletal muscle, and liver) and lymphatic (abdominal lymph nodes, spleen, and thymus) organs. Fourteen-week-old male leptin-deficient ob/ob mice and their controls fed a standard chow were studied. Tissue sections were stained with toluidine blue to determine the density of mast cells. CD117/c-kit protein expression analysis was also carried out. Furthermore, mast cells containing immunoreactive tumor necrosis factor-α (TNF-α), a proinflammatory cytokine involved in obesity-linked insulin resistance, were identified by immunostaining. RESULTS: ob/ob mice demonstrated adiposity and insulin resistance. In abdominal fat depots, mast cells were distributed differentially. While most prevalent in subcutaneous fat in controls, mast cells were most abundant in epididymal fat in ob/ob mice. Leptin deficiency-induced obesity was accompanied by a 20-fold increase in the density of mast cells in epididymal fat, but a 13-fold decrease in subcutaneous fat. This finding was confirmed by CD117/c-kit protein expression analysis. Furthermore, we found that a subset of mast cells in epididymal and subcutaneous fat were immunoreactive for TNF-α. The proportion of mast cells immunoreactive for TNF-α was higher in epididymal than in subcutaneous fat in both ob/ob and control mice. Mast cells were also distributed differentially in retroperitoneal, mesenteric, and inguinal lymph nodes. In both ob/ob mice and lean controls, mast cells were more prevalent in retroperitoneal than in mesenteric and inguinal lymph nodes. Leptin deficiency-induced obesity was accompanied by increased mast cell density in all lymph node stations examined. No significant difference in the density of mast cells in skeletal muscle, liver, spleen, and thymus was noted between ob/ob and control mice. CONCLUSIONS: This study demonstrates that leptin deficiency-induced obesity is accompanied by alterations in the density of mast cells in abdominal fat depots. The divergent distribution of mast cells in subcutaneous versus visceral fat might partially account for their differential biological behavior. Mast cells might also play a role in adaptive immune response occurring in regional lymph nodes in obesity.
Assuntos
Gordura Abdominal/patologia , Leptina/deficiência , Linfonodos/patologia , Mastócitos/fisiologia , Obesidade/patologia , Adiposidade , Animais , Glicemia , Contagem de Células , Colesterol/sangue , Epididimo/imunologia , Epididimo/patologia , Fígado/patologia , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Obesos , Músculo Esquelético/patologia , Obesidade/sangue , Obesidade/etiologia , Especificidade de Órgãos , Proteínas Proto-Oncogênicas c-kit , Baço/patologia , Gordura Subcutânea/imunologia , Gordura Subcutânea/patologia , Timo/patologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The ATPase Family AAA Domain Containing 3A (ATAD3A), is a mitochondrial inner membrane protein conserved in metazoans. ATAD3A has been associated with several mitochondrial functions, including nucleoid organization, cholesterol metabolism, and mitochondrial translation. To address its primary role, we generated a neuronal-specific conditional knockout (Atad3 nKO) mouse model, which developed a severe encephalopathy by 5 months of age. Pre-symptomatic mice showed aberrant mitochondrial cristae morphogenesis in the cortex as early as 2 months. Using a multi-omics approach in the CNS of 2-to-3-month-old mice, we found early alterations in the organelle membrane structure. We also show that human ATAD3A associates with different components of the inner membrane, including OXPHOS complex I, Letm1, and prohibitin complexes. Stochastic Optical Reconstruction Microscopy (STORM) shows that ATAD3A is regularly distributed along the inner mitochondrial membrane, suggesting a critical structural role in inner mitochondrial membrane and its organization, most likely in an ATPase-dependent manner.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Fosforilação Oxidativa , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Encefalopatias/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Deleção de Sequência , TranscriptomaRESUMO
The activity of Rho family GTPase protein, RAC1, which plays important normal physiological functions, is dysregulated in multiple cancers. RAC1 is expressed in both estrogen receptor alpha (ER)-positive and ER-negative breast cancer (BC) cells. However, ER-positive BC is more sensitive to RAC1 inhibition. We have determined that reducing RAC1 activity, using siRNA or EHT 1864 (a small molecule Rac inhibitor), leads to rapid ER protein degradation. RAC1 interacts with ER within the ER complex and RAC1 localizes to chromatin binding sites for ER upon estrogen treatment. RAC1 activity is important for RNA Pol II function at both promoters and enhancers of ER target genes and ER-regulated gene transcription is blocked by EHT 1864, in a dose-dependent manner. Having identified that RAC1 is an essential ER cofactor for ER protein stability and ER transcriptional activity, we report that RAC1 inhibition could be an effective therapeutic approach for ER-positive BC.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Feminino , Humanos , TransfecçãoRESUMO
RNA 3' end processing provides a source of transcriptome diversification which affects various (patho)-physiological processes. A prime example is the transcript isoform switch that leads to the read-through expression of the long non-coding RNA NEAT1_2, at the expense of the shorter polyadenylated transcript NEAT1_1. NEAT1_2 is required for assembly of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy. Searching for proteins that modulate this event, we identified factors involved in the 3' end processing of polyadenylated RNA and components of the Integrator complex. Perturbation experiments established that, by promoting the cleavage of NEAT1_2, Integrator forces NEAT1_2 to NEAT1_1 isoform switching and, thereby, restrains PS assembly. Consistently, low levels of Integrator subunits correlated with poorer prognosis of cancer patients exposed to chemotherapeutics. Our study establishes that Integrator regulates PS biogenesis and a link between Integrator, cancer biology, and chemosensitivity, which may be exploited therapeutically.
RESUMO
Transcription by RNA polymerase II (RNAPII) is pervasive in the human genome. However, the mechanisms controlling transcription at promoters and enhancers remain enigmatic. Here, we demonstrate that Integrator subunit 11 (INTS11), the catalytic subunit of the Integrator complex, regulates transcription at these loci through its endonuclease activity. Promoters of genes require INTS11 to cleave nascent transcripts associated with paused RNAPII and induce their premature termination in the proximity of the +1 nucleosome. The turnover of RNAPII permits the subsequent recruitment of an elongation-competent RNAPII complex, leading to productive elongation. In contrast, enhancers require INTS11 catalysis not to evict paused RNAPII but rather to terminate enhancer RNA transcription beyond the +1 nucleosome. These findings are supported by the differential occupancy of negative elongation factor (NELF), SPT5, and tyrosine-1-phosphorylated RNAPII. This study elucidates the role of Integrator in mediating transcriptional elongation at human promoters through the endonucleolytic cleavage of nascent transcripts and the dynamic turnover of RNAPII.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Elongação da Transcrição Genética , Biocatálise , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Nucleossomos/metabolismo , Fosforilação , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Terminação da Transcrição GenéticaRESUMO
BACKGROUND: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. METHODS AND RESULTS: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. CONCLUSIONS: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Demência Frontotemporal/genética , Proteínas Nucleares/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Linhagem Celular , Expansão das Repetições de DNA/genética , Dipeptídeos , Demência Frontotemporal/metabolismo , Humanos , NucleofosminaRESUMO
Purpose: Although normal function of the lacrimal gland is essential for vision (and thus for human well-being), the lacrimal gland remains rather poorly understood at a molecular level. The purpose of this study was to identify new genes and signaling cascades involved in lacrimal gland development. Methods: To identify these genes, we used microarray analysis to compare the gene expression profiles of developing (embryonic) and adult lacrimal glands. Differential data were validated by quantitative RT-PCR, and several corresponding proteins were confirmed by immunohistochemistry and Western blot analysis. To evaluate the role of NOTCH signaling in lacrimal gland (LG) development, we used the NOTCH inhibitor DAPT and conditional Notch1 knockouts. Results: Our microarray data and an in silico reconstruction of cellular networks revealed significant changes in the expression patterns of genes from the NOTCH, WNT, TGFß, and Hedgehog pathways, all of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Our study also revealed new putative lacrimal gland stem cell/progenitor markers. We found that inhibiting Notch signaling both increases the average number of lacrimal gland lobules and reduces the size of each lobule. Conclusions: Our findings suggest that NOTCH-, WNT-, TGFß-, and Hedgehog-regulated EMT transition are critical mechanisms in lacrimal gland development and morphogenesis. Our data also supports the hypothesis that NOTCH signaling regulates branching morphogenesis in the developing lacrimal gland by suppressing cleft formation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Aparelho Lacrimal/crescimento & desenvolvimento , Morfogênese , RNA/genética , Receptores Notch/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Western Blotting , Células Cultivadas , Transição Epitelial-Mesenquimal , Proteínas Hedgehog/biossíntese , Imuno-Histoquímica , Aparelho Lacrimal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fator de Crescimento Transformador beta/biossíntese , Proteínas Wnt/biossíntese , Proteínas Wnt/genéticaRESUMO
BACKGROUND: Efficient techniques to noninvasively monitor stem cell transplants will accelerate the development of stem cell therapies. Magnetic resonance (MR) imaging of labeled stem cells is a noninvasive approach that can provide images with high spatial resolution. In this report, we have evaluated the ability of a commercially available, FDA-approved contrast agent to allow for the monitoring of therapeutic stem cell transplants in murine dystrophic muscle. METHODS: Multipotent, muscle-derived stem cells were labeled by incubation with ferumoxide:polycation complexes. Labeled stem cells were evaluated for the ability to differentiate into mature myotubes and be detected by high resolution MR images in vitro and in vivo. RESULTS: Endosomal accumulation of superparamagnetic iron-oxide resulted in changes in the MR contrast T(2) and T(2)*, allowing for three dimensional, noninvasive detection of labeled cells. Relaxivity measurements on cell phantoms indicate that less than eight labeled cells could be detected by MR imaging. Furthermore, therapeutic cellular grafts transplanted into dystrophic muscle could be imaged sequentially and these noninvasive images correlated with histological images obtained by conventional microscopy. Additional studies revealed that MR imaging is applicable to tracking the migration of a small number of labeled cells following arterial delivery. CONCLUSIONS: MR monitoring is a highly sensitive technique that is applicable to muscle stem cell transplantation. We anticipate that it will enhance stem cell investigations by reducing the need for invasive tissue harvests and biopsies.
Assuntos
Músculos/citologia , Distrofia Muscular Animal/terapia , Transplante de Células-Tronco , Animais , Artérias/citologia , Morte Celular , Diferenciação Celular , Células Cultivadas , Imageamento por Ressonância Magnética , CamundongosRESUMO
We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis.