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1.
Nat Cell Biol ; 22(2): 187-199, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932738

RESUMO

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Autofagossomos/química , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Biotinilação , Vesículas Extracelulares/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteômica/métodos , Células RAW 264.7 , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo
2.
Nat Cell Biol ; 21(10): 1273-1285, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548606

RESUMO

Chromosome translocation is a major cause of the onset and progression of diverse types of cancers. However, the mechanisms underlying this process remain poorly understood. Here, we identified a non-homologous end-joining protein, IFFO1, which structurally forms a heterotetramer with XRCC4. IFFO1 is recruited to the sites of DNA damage by XRCC4 and promotes the repair of DNA double-strand breaks in a parallel pathway with XLF. Interestingly, IFFO1 interacts with lamin A/C, forming an interior nucleoskeleton. Inactivating IFFO1 or its interaction with XRCC4 or lamin A/C leads to increases in both the mobility of broken ends and the frequency of chromosome translocation. Importantly, the destruction of this nucleoskeleton accounts for the elevated frequency of chromosome translocation in many types of cancer cells. Our results reveal that the lamin A/C-IFFO1-constituted nucleoskeleton prevents chromosome translocation by immobilizing broken DNA ends during tumorigenesis.


Assuntos
Carcinogênese/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Lamina Tipo A/metabolismo , Translocação Genética , Animais , Carcinoma/genética , Cromossomos Humanos , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteínas de Filamentos Intermediários/genética , Camundongos , Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/fisiologia
3.
Cell Host Microbe ; 26(3): 369-384.e8, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31513772

RESUMO

Pathogen pattern recognition receptors (PRRs) trigger innate immune responses to invading pathogens. All known PRRs for viral RNA have extranuclear localization. However, for many viruses, replication generates dsRNA in the nucleus. Here, we show that the nuclear matrix protein SAFA (also known as HnRNPU) functions as a nuclear viral dsRNA sensor for both DNA and RNA viruses. Upon recognition of viral dsRNA, SAFA oligomerizes and activates the enhancers of antiviral genes, including IFNB1. Moreover, SAFA is required for the activation of super-enhancers, which direct vigorous immune gene transcription to establish the antiviral state. Myeloid-specific SAFA-deficient mice were more susceptible to lethal HSV-1 and VSV infection, with decreased type I IFNs. Thus, SAFA functions as a nuclear viral RNA sensor and trans-activator to bridge innate sensing with chromatin remodeling and potentiate robust antiviral responses.


Assuntos
Antivirais/imunologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/imunologia , Proteínas Associadas à Matriz Nuclear/imunologia , RNA Viral/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Adenosina Trifosfatases/genética , Animais , Proteínas Cromossômicas não Histona/genética , DNA Topoisomerases Tipo I/genética , Vírus de DNA , Células HEK293 , Células HeLa , Herpesvirus Humano 1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon , Fator Regulador 7 de Interferon , Camundongos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Serina-Treonina Quinases , Vírus de RNA , RNA de Cadeia Dupla , Vírus
4.
Nat Commun ; 10(1): 2208, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101817

RESUMO

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.


Assuntos
Antígenos Nucleares/metabolismo , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/isolamento & purificação , Células CACO-2 , Cristalografia por Raios X , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
5.
Cell Physiol Biochem ; 52(4): 787-801, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30946555

RESUMO

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) represents the second most common cause of cancer-related deaths worldwide, not least due to its high chemoresistance. The long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), localised in nuclear paraspeckles, has been shown to enhance chemoresistance in several cancer types. Since data on NEAT1 in HCC chemosensitivity are completely lacking and chemoresistance is linked to poor prognosis, we aimed to study NEAT1 expression in HCC chemoresistance and its link to HCC prognosis. METHODS: NEAT1 expression was determined in either sensitive, or sorafenib, or doxorubicin resistant HepG2, PLC/PRF/5, and Huh7 cells by qPCR. Paraspeckles were detected by immunostaining of paraspeckle component 1 (PSPC1) in cell culture and in a cohort of HCC patients. PSPC1 expression was correlated with clinical data. The expression of transcript variants of NEAT1 and transcripts encoding the paraspeckle-associated proteins was analysed in the TCGA liver cancer data set. RESULTS: NEAT1 was overexpressed in all three sorafenib and doxorubicin resistant cell lines. Paraspeckles were present in all chemoresistant cells, whereas no signal was detected in the sensitive cells. Expression of NEAT1 transcripts as well as transcripts encoding PSPC1, NONO, and RBM14 was increased in tumour tissue. Expression of PSPC1, NONO, and RBM14 transcripts was significantly associated with poor survival, whereas NEAT1 expression was not. Immunohistochemical analysis revealed that nuclear and cytoplasmic PSPC1-positivity was significantly associated with shorter overall survival of HCC patients. CONCLUSION: Our data show an induction of NEAT1 in HCC chemoresistance and a high correlation of transcripts encoding paraspeckle-associated proteins with poor survival in HCC. Therefore, NEAT1, PSPC1, NONO, and RBM14 might be promising targets for novel HCC therapies, and the paraspeckle-associated proteins might be clinical markers and predictors for poor survival in HCC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Antineoplásicos/uso terapêutico , Área Sob a Curva , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Estimativa de Kaplan-Meier , Fígado/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Curva ROC , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico
6.
Nucleic Acids Res ; 47(11): 5465-5479, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31034558

RESUMO

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.


Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleotídeos Antissenso/química , Organofosfonatos/química , Oligonucleotídeos Fosforotioatos/química , Células 3T3-L1 , Animais , Caspases/metabolismo , Linhagem Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células HeLa , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Fosforotioatos/administração & dosagem , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo
7.
Nat Commun ; 10(1): 1001, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824709

RESUMO

In vertebrates, the telomere repeat containing long, non-coding RNA TERRA is prone to form RNA:DNA hybrids at telomeres. This results in the formation of R-loop structures, replication stress and telomere instability, but also contributes to alternative lengthening of telomeres (ALT). Here, we identify the TERRA binding proteins NONO and SFPQ as novel regulators of RNA:DNA hybrid related telomere instability. NONO and SFPQ locate at telomeres and have a common role in suppressing RNA:DNA hybrids and replication defects at telomeres. NONO and SFPQ act as heterodimers to suppress fragility and homologous recombination at telomeres, respectively. Combining increased telomere fragility with unleashing telomere recombination upon NONO/SFPQ loss of function causes massive recombination events, involving 35% of telomeres in ALT cells. Our data identify the RNA binding proteins SFPQ and NONO as novel regulators at telomeres that collaborate to ensure telomere integrity by suppressing telomere fragility and homologous recombination triggered by RNA:DNA hybrids.


Assuntos
DNA/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Hibridização de Ácido Nucleico , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Telômero/metabolismo , Animais , Linhagem Celular Tumoral , Replicação do DNA , Recombinação Homóloga , Humanos , Camundongos , RNA não Traduzido , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo
8.
Cell ; 176(4): 716-728.e18, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30712871

RESUMO

Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.


Assuntos
Células Matadoras Naturais/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Animais , Axônios , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Regeneração Nervosa , Neurônios/citologia , Neurônios Aferentes/imunologia , Neurônios Aferentes/metabolismo , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Dor , Traumatismos dos Nervos Periféricos/imunologia , Doenças do Sistema Nervoso Periférico , Nervo Isquiático , Células Receptoras Sensoriais/metabolismo
9.
Blood ; 133(14): 1560-1571, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30755420

RESUMO

Hematopoietic stem cell (HSC) homeostasis is controlled by cytokine receptor-mediated Janus kinase 2 (JAK2) signaling. We previously found that JAK2 is promptly ubiquitinated upon cytokine stimulation. Whether a competing JAK2 deubiquitination activity exists is unknown. LNK is an essential adaptor protein that constrains HSC expansion through dampening thrombopoietin (TPO)-induced JAK2 signaling. We show here that a LNK-associated lysine-63 (K63)-deubiquitinating enzyme complex, Brcc36 isopeptidase complex (BRISC), attenuates HSC expansion through control of JAK2 signaling. We pinpoint a direct interaction between the LNK SH2 domain and a phosphorylated tyrosine residue in KIAA0157 (Abraxas2), a unique and defining BRISC component. Kiaa0157 deficiency in mice led to an expansion of phenotypic and functional HSCs. Endogenous JAK2 and phospho-JAK2 were rapidly K63-ubiquitinated upon TPO stimulation, and this action was augmented in cells depleted of the BRISC core components KIAA0157, MERIT40, or BRCC36. This increase in JAK2 ubiquitination after BRISC knockdown was associated with increased TPO-mediated JAK2 activation and protein levels, and increased MPL receptor presence at the cell surface. In addition, BRISC depletion promoted membrane proximal association between the MPL receptor and pJAK2/JAK2, thus enhancing activated JAK2/MPL at the cell membrane. These findings define a novel pathway by which K63-ubiquitination promotes JAK2 stability and activation in a proteasome-independent manner. Moreover, mutations in BRCC36 are found in clonal hematopoiesis in humans. This research may shed light on the mechanistic understanding of a potential role of BRCC36 in human HSCs.


Assuntos
Proliferação de Células , Enzimas Desubiquitinantes/fisiologia , Células-Tronco Hematopoéticas/citologia , Janus Quinase 2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Enzimas Desubiquitinantes/genética , Humanos , Camundongos , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Trombopoetina/metabolismo , Transdução de Sinais , Trombopoetina/farmacologia , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Domínios de Homologia de src
10.
J Biol Chem ; 294(14): 5549-5561, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30782847

RESUMO

In Sonic hedgehog (SHH) signaling, GLI family zinc finger (GLI)-mediated diverse gene transcription outcomes are strictly regulated and are important for SHH function in both development and disease. However, how the GLI factors differentially regulate transcription in response to variable SHH activities is incompletely understood. Here, using a newly generated, tagged Gli3 knock-in mouse (Gli3TAP ), we performed proteomic analyses and identified the chromatin-associated SAFB-like transcription modulator (SLTM) as a GLI-interacting protein that context-dependently regulates GLI activities. Using immunoprecipitation and immunoblotting, RT-quantitative PCR, and ChIP assays, we show that SLTM interacts with all three GLI proteins and that its cellular levels are regulated by SHH. We also found that SLTM enhances GLI3 binding to chromatin and increases GLI3 repressor (GLI3R) form protein levels. In a GLI3-dependent manner, SLTM promoted the formation of a repressive chromatin environment and functioned as a GLI3 co-repressor. In the absence of GLI3 or in the presence of low GLI3 levels, SLTM co-activated GLI activator (GLIA)-mediated target gene activation and cell differentiation. Moreover, in vivo Sltm deletion generated through CRISPR/Cas9-mediated gene editing caused perinatal lethality and SHH-related abnormal ventral neural tube phenotypes. We conclude that SLTM regulates GLI factor binding to chromatin and contributes to the transcriptional outcomes of SHH signaling via a novel molecular mechanism.


Assuntos
Proteínas Hedgehog/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Transdução de Sinais , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Sistemas CRISPR-Cas , Cromatina , Edição de Genes , Técnicas de Introdução de Genes , Proteínas Hedgehog/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteômica , Proteína Gli3 com Dedos de Zinco/genética
11.
Cell Mol Life Sci ; 76(10): 2015-2030, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30725116

RESUMO

Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20 µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.


Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteínas Associadas à Matriz Nuclear/genética , Fatores de Transcrição de Octâmero/genética , Fator de Processamento Associado a PTB/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Interferência de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
12.
Mol Biol Cell ; 30(8): 1037-1049, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30726174

RESUMO

Mitogen-activated protein kinases (MAPKs) mediate numerous eukaryotic signaling responses. They also can modulate their own signaling output via positive or negative feedback loops. In the yeast pheromone response pathway, the MAPK Fus3 triggers negative feedback that dampens its own activity. One target of this feedback is Ste5, a scaffold protein that promotes Fus3 activation. Binding of Fus3 to a docking motif (D motif) in Ste5 causes signal dampening, which was proposed to involve a central cluster of phosphorylation sites in Ste5. Here, we reanalyzed the role of these central sites. Contrary to prior claims, phosphorylation-mimicking mutations at these sites did not impair signaling. Also, the hyperactive signaling previously observed when these sites were mutated to nonphosphorylatable residues arose from their replacement with valine residues and was not observed with other substitutes. Instead, a cluster of N-terminal sites in Ste5, not the central sites, is required for the rapid dampening of initial responses. Further results suggest that the role of the Fus3 D motif is most simply explained by a tethering effect that promotes Ste5 phosphorylation, rather than an allosteric effect proposed to regulate Fus3 activity. These findings substantially revise our understanding of how MAPK feedback attenuates scaffold-mediated signaling in this model pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Feromônios/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Feromônios/metabolismo , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais
13.
Oncol Rep ; 41(1): 333-340, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30320347

RESUMO

Licochalcone H (LCH) is a chemical compound that is a positional isomer of licochalcone C (LCC), a chalconoid isolated from the root of Glycyrrhiza inflata, which has various pharmacological properties including anti­inflammatory, antioxidant, antitumor, and anticancer effects. However, the efficacy of LCH on cancer cells has not been investigated. The present study examined the effects of LCH on cell proliferation, induction of apoptosis, and the regulation of matrin 3 (Matr3) protein in oral squamous cell carcinoma (OSCC) cells by Annexin V/propidium iodide (PI) staining and western blot analysis. LCH reduced cell viability and colony forming ability, and induced cell cycle arrest and apoptosis in HSC2 and HSC3 cells through the suppression of Matr3. It was also found that LCH directly bound to Matr3 in a Sepharose 4B pull­down assay. Consequently, the results of the present study suggest that LCH may be used as an anticancer drug in combination with conventional chemotherapy for the treatment of OSCC, and that Matr3 may be a potential effective therapeutic target.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Chalconas/farmacologia , Neoplasias Bucais/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética
14.
Nucleic Acids Res ; 47(2): 762-778, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30445466

RESUMO

RNF8 plays a critical role in DNA damage response (DDR) to initiate ubiquitination-dependent signaling. To better characterize the role of RNF8 in UV-induced DDR, we searched for novel substrates of RNF8 and identified NONO as one intriguing substrate. We found that: (i) RNF8 ubiquitinates NONO and (ii) UV radiation triggers NONO ubiquitination and its subsequent degradation. Depletion of RNF8 inhibited UV-induced degradation of NONO, suggesting that RNF8 targets NONO for degradation in response to UV damage. In addition, we found that 3 NONO lysine residues (positions 279, 290 and 295) are important for conferring its instability in UV-DDR. Depletion of RNF8 or expression of NONO with lysine to arginine substitutions at positions 279, 290 and 295 prolonged CHK1 phosphorylation over an extended period of time. Furthermore, expression of the stable mutant, but not wild-type NONO, induced a prolonged S phase following UV exposure. Stable cell lines expressing the stable NONO mutant showed increased UV sensitivity in a clonogenic survival assay. Since RNF8 recruitment to the UV-damaged sites is dependent on ATR, we propose that RNF8-mediated NONO degradation and subsequent inhibition of NONO-dependent chromatin loading of TOPBP1, a key activator of ATR, function as a negative feedback loop critical for turning off ATR-CHK1 checkpoint signaling in UV-DDR.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem/metabolismo , Humanos , Lisina/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Fatores de Transcrição de Octâmero/química , Proteínas de Ligação a RNA/química , Fase S , Transdução de Sinais , Ubiquitinação , Raios Ultravioleta
15.
Acta Neuropathol Commun ; 6(1): 137, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30563574

RESUMO

Mutations in MATR3 have been associated with amyotrophic lateral sclerosis (ALS) as well as a form of distal myopathy termed vocal cord pharyngeal distal myopathy (VCPDM). To begin to understand how mutations in MATR3 may cause disease, here we provide initial characterization of transgenic (Tg) mice expressing human wild-type (WT) MATR3 (MATR3WT) and ALS-mutant F115C MATR3 (MATR3F115C) proteins under the control of the mouse prion promoter (MoPrP). For each construct, we established multiple independent lines of mice that stably transmitted the transgene. Unexpectedly, for all stably-transmitting lines examined, MATR3 transgenic mRNA expression was more robust in muscle, with minimal expression in spinal cord. The levels of transgenic mRNA in muscle did not differ between mice from our lead MATR3F115C line and lead MATR3WT line, but mice from the lead MATR3F115C line had significantly higher levels of MATR3 protein in muscle over the lead MATR3WT line. Mice from the three independent, established lines of MATR3F115C mice developed weakness in both fore- and hind-limbs as early as < 1 months of age; whereas, MATR3WT mice aged to > 20 months were not overtly distinguishable from non-transgenic (NT) littermates based on basic motor phenotype. Muscle of both MATR3WT and MATR3F115C mice showed vacuoles by 2 months of age which worsened by ~ 10 months, but vacuolation was noticeably more severe in MATR3F115C mice. Overall, our results indicate that increasing the levels of MATR3 in muscle can cause pathologic changes associated with myopathy, with MATR3F115C expression causing overt muscle atrophy and a profound motor phenotype. The findings suggest that analysis of muscle pathology in individuals harboring ALS-linked MATR3 mutations should be routinely considered.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Músculo Esquelético/patologia , Mutação/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Ligação a RNA/genética , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Análise de Variância , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas da Gravidez/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
16.
Viruses ; 10(12)2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30572664

RESUMO

Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells.


Assuntos
Antígenos Nucleares/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Células A549 , Antígenos Nucleares/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Vírus da Influenza A/genética , Proteínas Associadas à Matriz Nuclear/genética , Interferência de RNA , Proteínas não Estruturais Virais/genética , Replicação Viral
17.
Cell ; 175(7): 1902-1916.e13, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30550788

RESUMO

Nuclear architecture has never been carefully examined during early mammalian development at the stages leading to establishment of the embryonic and extra-embryonic lineages. Heterogeneous activity of the methyltransferase CARM1 during these stages results in differential methylation of histone H3R26 to modulate establishment of these two lineages. Here we show that CARM1 accumulates in nuclear granules at the 2- to 4-cell stage transition in the mouse embryo, with the majority corresponding to paraspeckles. The paraspeckle component Neat1 and its partner p54nrb are required for CARM1's association with paraspeckles and for H3R26 methylation. Conversely, CARM1 also influences paraspeckle organization. Depletion of Neat1 or p54nrb results in arrest at the 16- to 32-cell stage, with elevated expression of transcription factor Cdx2, promoting differentiation into the extra-embryonic lineage. This developmental arrest occurs at an earlier stage than following CARM1 depletion, indicating that paraspeckles act upstream of CARM1 but also have additional earlier roles in fate choice.


Assuntos
Blastocisto/metabolismo , Diferenciação Celular , Linhagem da Célula , Desenvolvimento Embrionário , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Blastocisto/citologia , Pontos de Checagem do Ciclo Celular , Camundongos , Proteínas Associadas à Matriz Nuclear/genética , Proteína-Arginina N-Metiltransferases/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética
18.
Cell ; 175(2): 488-501.e22, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30270045

RESUMO

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.


Assuntos
Proteínas do Capsídeo/imunologia , Proteínas Associadas à Matriz Nuclear/imunologia , Proteínas Associadas à Matriz Nuclear/fisiologia , Fatores de Transcrição de Octâmero/imunologia , Fatores de Transcrição de Octâmero/fisiologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/fisiologia , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/fisiologia , Núcleo Celular/metabolismo , DNA Viral/genética , DNA Viral/imunologia , Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/imunologia , HIV-2/genética , HIV-2/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata/imunologia , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/fisiologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/imunologia
19.
Sci Rep ; 8(1): 13412, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194346

RESUMO

To investigate the mechanisms underlying the maintenance of neural stem cells, we performed two-dimensional fluorescence-difference gel electrophoresis (2D-DIGE) targeting the nuclear phosphorylated proteins. Nuclear phosphorylated protein Matrin-3 was identified in neural stem cells (NSCs) after stimulation using fibroblast growth factor 2 (FGF2). Matrin-3 was expressed in the mouse embryonic subventricular and ventricular zones. Small interfering RNA (siRNA)-mediated knockdown of Matrin-3 caused neuronal differentiation of NSCs in vitro, and altered the cerebral layer structure of foetal brain in vivo. Transfection of Matrin-3 plasmids in which the serine 208 residue was point-mutated to alanine (Ser208Ala mutant Matrin3) and inhibition of Ataxia telangiectasia mutated kinase (ATM kinase), which phosphorylates Matrin-3 Ser208 residue, caused neuronal differentiation and decreased the proliferation of neurosphere-forming stem cells. Thus, our proteomic approach revealed that Matrin-3 phosphorylation was essential for FGF2-dependent maintenance of NSCs in vitro and in vivo.


Assuntos
Diferenciação Celular , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Substituição de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos ICR , Mutação de Sentido Incorreto , Células-Tronco Neurais/citologia , Proteínas Associadas à Matriz Nuclear/genética , Fosforilação/genética , Proteínas de Ligação a RNA/genética
20.
Endocrinology ; 159(11): 3615-3630, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30204866

RESUMO

Among their pleiotropic functions, scaffold proteins are required for the accurate coordination of signaling pathways. It has only been within the past 10 years that their roles in glucose homeostasis and metabolism have emerged. It is well appreciated that changes in the expression or function of signaling effectors, such as receptors or kinases, can influence the development of chronic diseases such as diabetes and obesity. However, little is known regarding whether scaffolds have similar roles in the pathogenesis of metabolic diseases. In general, scaffolds are often underappreciated in the context of metabolism or metabolic diseases. In the present review, we discuss various scaffold proteins and their involvement in signaling pathways related to metabolism and metabolic diseases. The aims of the present review were to highlight the importance of scaffold proteins and to raise awareness of their physiological contributions. A thorough understanding of how scaffolds influence metabolism could aid in the discovery of novel therapeutic approaches to treat chronic conditions, such as diabetes, obesity, and cardiovascular disease, for which the incidence of all continue to increase at alarming rates.


Assuntos
Doenças Cardiovasculares/metabolismo , Diabetes Mellitus/metabolismo , Resistência à Insulina/fisiologia , Secreção de Insulina/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Obesidade/metabolismo , Transdução de Sinais/fisiologia , Adiposidade/fisiologia , Animais , Apoptose/fisiologia , Glicemia/metabolismo , Metabolismo Energético , Gluconeogênese/fisiologia , Homeostase , Humanos , Doenças Metabólicas/metabolismo , Proteínas Associadas à Matriz Nuclear/fisiologia
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