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1.
Proc Natl Acad Sci U S A ; 117(32): 19190-19200, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723828

RESUMO

The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.


Assuntos
Macroautofagia , Organelas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Humanos , Organelas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
2.
Exp Anim ; 69(4): 395-406, 2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-32493884

RESUMO

Gender and menopause influence the severity and development manner of nonalcoholic steatohepatitis (NASH). Male p62/Sqstm1 and nuclear factor E2-related factor-2 (p62 and Nrf2) double-knockout (DKO) mice exhibit severe steatohepatitis caused by hyperphagia-induced obesity, overload of lipopolysaccharide (LPS) into the liver, and potentiation of the inflammatory response in Kupffer cells. However, the pathogenetic phenotype of steatohepatitis in female DKO mice remains unknown. Phenotypic changes of steatohepatitis in DKO mice were compared in terms of gender differences. Compared with DKO male mice, DKO female mice exhibited later onset of steatohepatitis with obesity after 30 weeks of age, as well as milder severity of hepatic inflammation and fibrosis. Serum estradiol was higher in female than male mice, with levels increasing up to 30 weeks of age before decreasing until 50 weeks of age (corresponding to the post-menopausal period). Fecal and serum LPS were lower in female mice than male mice, and inflammatory signaling in the liver was attenuated in female compared with male mice. Correlating with LPS levels, the composition of intestinal microbiota in female mice was different from male mice. Gender differences were observed for the development of steatohepatitis in DKO mice. Low-grade inflammatory hit in the liver under in vivo conditions of high estradiol may be attributable to the milder pathological features of steatohepatitis in female mice.


Assuntos
Estradiol/fisiologia , Fígado Gorduroso/genética , Menopausa/fisiologia , Camundongos Knockout/genética , Fator 2 Relacionado a NF-E2/genética , Proteína Sequestossoma-1/genética , Caracteres Sexuais , Animais , Fígado Gorduroso/etiologia , Fígado Gorduroso/patologia , Feminino , Fibromialgia , Hiperfagia/complicações , Inflamação , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/metabolismo , Fígado/patologia , Masculino , Obesidade/complicações
3.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32513711

RESUMO

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
4.
Nat Commun ; 11(1): 2306, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385399

RESUMO

During ß-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62Δ69-251 mice, global p62-/- and Ucp1-Cre p62flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Proteína Sequestossoma-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adipogenia/fisiologia , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/diagnóstico por imagem , Tecido Adiposo Branco/metabolismo , Animais , Núcleo Celular/metabolismo , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Desacopladora 1/metabolismo
5.
Mol Carcinog ; 59(8): 967-979, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32424979

RESUMO

Drug resistance is the leading cause for rapid progression and relapse in small-cell lung cancer (SCLC) patients. Thus overcoming drug resistance still remains to be urgently resolved during SCLC treatment. Here, we found p62/SQSTM1 was enriched in SCLC spheroids, a subpopulation possessing cancer stem-like properties, which is responsible for cancer relapse and metastasis. Subsequent functional assays in vitro showed that short hairpin RNA (shRNA)-mediated p62 knockdown increased sensitivity of SCLC cell lines to cisplatin (DDP), whereas lentivirus-mediated p62 ectopic overexpression diminished DDP-induced cytotoxicity in both NCI-H446 and NCI-H1688 cell lines. Moreover, ectopic p62 overexpression promoted DDP resistance of NCI-H446 cells-derived tumor xenografts in immunodeficient mice in vivo, as indicated by accelerated tumor growth rate and reduced fluorescent activity of cleaved caspase-3. Gene expression profiling analysis revealed that p62 was positively correlated with neuronal precursor cell-expressed, developmentally downregulated gene 9 (NEDD9) expression level. Consistently, NEDD9 messenger RNA (mRNA) level was decreased upon p62 suppression by small interfering RNA (siRNA) and increased with p62 transient overexpression in SCLC cell lines, suggesting that p62 positively regulated NEDD9 mRNA. Depletion of NEDD9 by siRNA, to a large extent, reversed p62-overexpressed SCLC cells to DDP-induced cytotoxicity, implying NEDD9 might act as a downstream target which was in charge of p62-mediated DDP resistance. Taken together, our findings uncovered a previously unknown role of p62 in the regulation of SCLC drug resistance, assigning p62 as an attractive target for SCLC treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Sequestossoma-1/antagonistas & inibidores , Carcinoma de Pequenas Células do Pulmão/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Interferente Pequeno/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
PLoS One ; 15(4): e0232036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343715

RESUMO

The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the phenylalanine-glycine (FG) repeat motifs of the NUP moiety that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we took advantage of the Sequestosome-1 (SQSTM1)-NUP214 fusion. SQSTM1-NUP214 retains only a short C-terminal portion of NUP214 which contains FG motifs that mediate interaction with Crm1. We introduced point mutations targeting these FG motifs and found that the ability of the resulting SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 impaired myeloid maturation and conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was considerably diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice, whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia. These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impaired SQSTM1-NUP214 interaction with Crm1 correlated with impaired binding of the fusion protein to Hoxa and Meis1 genes. These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.


Assuntos
Proteínas de Homeodomínio/genética , Carioferinas/metabolismo , Leucemia/metabolismo , Proteína Meis1/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Sequestossoma-1/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Leucemia/patologia , Camundongos , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Mutação Puntual , Regulação para Cima
7.
PLoS Biol ; 18(2): e3000620, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32053600

RESUMO

Primary cilia are lost during cancer development, but the mechanism regulating cilia degeneration is not determined. While transcription factor nuclear factor-erythroid 2-like 2 (NRF2) protects cells from oxidative, proteotoxic, and metabolic stress in normal cells, hyperactivation of NRF2 is oncogenic, although the detailed molecular mechanisms by which uncontrolled NRF2 activation promotes cancer progression remain unclear. Here, we report that NRF2 suppresses hedgehog (Hh) signaling through Patched 1 (PTCH1) and primary ciliogenesis via p62/sequestosome 1 (SQSTM1). PTCH1, a negative regulator of Hh signaling, is an NRF2 target gene, and as such, hyperactivation of NRF2 impairs Hh signaling. NRF2 also suppresses primary cilia formation through p62-dependent inclusion body formation and blockage of Bardet-Biedl syndrome 4 (BBS4) entrance into cilia. Simultaneous ablation of PTCH1 and p62 completely abolishes NRF2-mediated inhibition of both primary ciliogenesis and Hh signaling. Our findings reveal a previously unidentified role of NRF2 in controlling a cellular organelle, the primary cilium, and its associated Hh signaling pathway and also uncover a mechanism by which NRF2 hyperactivation promotes tumor progression via primary cilia degeneration and aberrant Hh signaling. A better understanding of the crosstalk between NRF2 and primary cilia/Hh signaling could not only open new avenues for cancer therapeutic discovery but could also have significant implications regarding pathologies other than cancer, including developmental disorders, in which improper primary ciliogenesis and Hh signaling play a major role.


Assuntos
Cílios/fisiologia , Proteínas Hedgehog/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Elementos de Resposta Antioxidante , Células Cultivadas , Cílios/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
8.
Gene ; 738: 144454, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32035240

RESUMO

Parkinson's disease (PD) is a common neurodegenerative disorder which affects dopaminergic neurons leading to alteration of numerous cellular pathways. Several reports highlight that PD disturbs also other cells than CNS neurons including PBMCs, which could lead, among other things, to dysfunctions of immune functions. Because autophagy could be altered in PD, a monocentric pilot study was performed to quantify the transcripts levels of several autophagy genes in blood cells. MAP1LC3B, GABARAP, GABARAPL1, GABARAPL2 and P62/SQSTM1 were found to be overexpressed in patients. On the contrary, transcripts for HSPA8 and GAPDH were both decreased. Expression of MAP1LC3B and GABARAP was able to successfully segregate PD patients from healthy controls. The accuracy of this segregation was substantially increased when combined expressions of MAP1LC3B and GAPDH or GABARAP and GAPDH were used as categorical variables. This pilot study suggests that autophagy genes expression is dysregulated in PD patients and may open new perspectives for the characterisation of prediction markers.


Assuntos
Autofagia/genética , Doença de Parkinson/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/genética , Biomarcadores/sangue , Neurônios Dopaminérgicos/metabolismo , Feminino , França , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Humanos , Leucócitos Mononucleares , Aprendizado de Máquina , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Doença de Parkinson/sangue , Projetos Piloto , Proteína Sequestossoma-1/genética
9.
Life Sci ; 248: 117466, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101760

RESUMO

AIMS: Nanoparticles (NPs)-based drugs have been recently introduced to improve the efficacy of current therapeutic strategies for the treatment of cancer; however, the molecular mechanisms by which a NP interacts with cellular systems still need to be delineated. Here, we utilize the autophagic potential of TiO2 NPs for improving chemotherapeutic effects of 5-fluorouracil (5-FU) in human AGS gastric cells. MATERIALS AND METHODS: Cell growth and viability were determined by trypan blue exclusion test and MTT assay, respectively. Vesicular organelles formation was evaluated by acridine orange staining of cells. Cell cycle and apoptosis were monitored by flow cytometry. Reactive oxygen species (ROS) level were measured by DCHF-DA staining. Autophagy was examined by q-PCR and western blotting. Molecular docking was used for studying NP interaction with autophagic proteins. KEY FINDINGS: TiO2 NPs increase ROS production, impair lysosomal function and subsequently block autophagy flux in AGS cells. In addition, the autophagy blockade induced by non-toxic concentrations of TiO2 NPs (1 µg/ml) can promote cytotoxic and apoptotic effects of 5-FU in AGS cells. SIGNIFICANCE: These results confirm the beneficial effects of TiO2 NPs in combination with chemotherapy in in vitro model of gastric cancer, which may pave the way to develop a possible solution to circumvent chemoresistance in cancer.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Nanopartículas/química , Titânio/farmacologia , Antimetabólitos Antineoplásicos/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fluoruracila/química , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Nanopartículas/ultraestrutura , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/antagonistas & inibidores , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Titânio/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
10.
Aging Cell ; 19(2): e13096, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31944526

RESUMO

Necroptosis is crucially involved in severe cardiac pathological conditions. However, whether necroptosis contributes to age-related intolerance to ischemia/reperfusion (I/R) injury remains elusive. In addition, metformin as a potential anti-aging related injury drug, how it interacts with myocardial necroptosis is not yet clear. Male C57BL/6 mice at 3-4- (young) and 22-24 months of age (aged) and RIPK3-deficient (Ripk3-/- ) mice were used to investigate aging-related I/R injury in vivo. Metformin (125 µg/kg, i.p.), necrostatin-1 (3.5 mg/kg), and adenovirus vector encoding p62-shRNAs (Ad-sh-p62) were used to treat aging mice. I/R-induced myocardial necroptosis was exaggerated in aged mice, which correlated with autophagy defects characterized by p62 accumulation in aged hearts or aged human myocardium. Functionally, blocking autophagic flux promoted H/R-evoked cardiomyocyte necroptosis in vitro. We further revealed that p62 forms a complex with RIP1-RIP3 (necrosome) and promotes the binding of RIP1 and RIP3. In mice, necrostatin-1 treatment (a RIP1 inhibitor), RIP3 deficiency, and cardiac p62 knockdown in vivo demonstrated that p62-RIP1-RIP3-dependent myocardial necroptosis contributes to aging-related myocardial vulnerability to I/R injury. Notably, metformin treatment disrupted p62-RIP1-RIP3 complexes and effectively repressed I/R-induced necroptosis in aged hearts, ultimately reducing mortality in this model. These findings highlight previously unknown mechanisms of aging-related myocardial ischemic vulnerability: p62-necrosome-dependent necroptosis. Metformin acts as a cardioprotective agent that inhibits this unfavorable chain mechanism of aging-related I/R susceptibility.


Assuntos
Envelhecimento/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Metformina/uso terapêutico , Necroptose/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Envelhecimento/patologia , Animais , Autofagia/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Masculino , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Necroptose/genética , Ligação Proteica , RNA Interferente Pequeno , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/mortalidade , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo
11.
BMC Cancer ; 20(1): 46, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959131

RESUMO

BACKGROUND: Breast (BCa) and prostate (PCa) cancers are hormone receptor (HR)-driven cancers. Thus, BCa and PCa patients are given therapies that reduce hormone levels or directly block HR activity; but most patients eventually develop treatment resistance. We have previously reported that interleukin-1 (IL-1) inflammatory cytokine downregulates ERα and AR mRNA in HR-positive (HR+) BCa and PCa cell lines, yet the cells can remain viable. Additionally, we identified pro-survival proteins and processes upregulated by IL-1 in HR+ BCa and PCa cells, that are basally high in HR- BCa and PCa cells. Therefore, we hypothesize that IL-1 confers a conserved gene expression pattern in HR+ BCa and PCa cells that mimics conserved basal gene expression patterns in HR- BCa and PCa cells to promote HR-independent survival and tumorigenicity. METHODS: We performed RNA sequencing (RNA-seq) for HR+ BCa and PCa cell lines exposed to IL-1 and for untreated HR- BCa and PCa cell lines. We confirmed expression patterns of select genes by RT-qPCR and used siRNA and/or drug inhibition to silence select genes in the BCa and PCa cell lines. Finally, we performed Ingenuity Pathway Analysis (IPA) and used the gene ontology web-based tool, GOrilla, to identify signaling pathways encoded by our RNA-seq data set. RESULTS: We identified 350 genes in common between BCa and PCa cells that are induced or repressed by IL-1 in HR+ cells that are, respectively, basally high or low in HR- cells. Among these genes, we identified Sequestome-1 (SQSTM1/p62) and SRY (Sex-Determining Region Y)-Box 9 (SOX9) to be essential for survival of HR- BCa and PCa cell lines. Analysis of publicly available data indicates that p62 and SOX9 expression are elevated in HR-independent BCa and PCa sublines generated in vitro, suggesting that p62 and SOX9 have a role in acquired hormone receptor independence and treatment resistance. We also assessed HR- cell line viability in response to the p62-targeting drug, verteporfin, and found that verteporfin is cytotoxic for HR- cell lines. CONCLUSIONS: Our 350 gene set can be used to identify novel therapeutic targets and/or biomarkers conserved among acquired (e.g. due to inflammation) or intrinsic HR-independent BCa and PCa.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Ativação Transcricional
12.
Nat Commun ; 11(1): 440, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974402

RESUMO

p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/química , Autofagia/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lisossomos/metabolismo , Polimerização , Conformação Proteica , Domínios Proteicos , Proteína Sequestossoma-1/genética
13.
Biochim Biophys Acta Mol Cell Res ; 1867(2): 118617, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31785334

RESUMO

Sequestration of misfolded proteins into distinct cellular compartments plays a pivotal role in proteostasis and proteopathies. Cytoplasmic ubiquitinated proteins are sequestered by p62/SQSTM1 to deposit in sequestosomes or aggresome-like induced structures (ALIS). Most aggresome or ALIS regulators identified thus far are recruiters, while little is known about the disaggregases or dissolvers. In this research, we showed that lanosterol synthase and its enzymatic product lanosterol effectively reduced the number and/or size of sequestosomes/ALIS/aggresomes formed by endogenous proteins in the HeLa and HEK-293A cells cultured under both non-stressed and stressed conditions. Supplemented lanosterol did not affect the proteasome and autophagic activities, but released the trapped proteins from the p62-positive inclusions accompanied with the activation of HSF1 and up-regulation of various heat shock proteins. Our results suggested that the coordinated actions of disaggregation by lanosterol and refolding by heat shock proteins might facilitate the cells to recycle proteins from aggregates. The disaggregation activity of lanosterol was not shared by cholesterol, indicating that lanosterol possesses additional cellular functions in proteostasis regulation. Our findings highlight that besides protein modulators, the cells also possess endogenous low-molecular-weight compounds as efficient proteostasis regulators.


Assuntos
Lanosterol/farmacologia , Proteostase/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Células HEK293 , Células HeLa , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Leupeptinas/farmacologia , Agregados Proteicos/efeitos dos fármacos , Redobramento de Proteína , Proteína Sequestossoma-1/genética , Temperatura , Regulação para Cima/efeitos dos fármacos
14.
Int J Cancer ; 146(6): 1652-1666, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31180579

RESUMO

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T-antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T-antigens induce miR-375, miR-30a-3p and miR-30a-5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV-positive tumors. Ectopic expression of MCPyV small T-antigen and truncated large T-antigen (LT), but not the wild-type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV-mediated tumorigenesis. Torin-1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan-caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV-mediated tumorigenesis and potential MCC treatment.


Assuntos
Carcinoma de Célula de Merkel/virologia , Poliomavírus das Células de Merkel/metabolismo , MicroRNAs/biossíntese , Neoplasias Cutâneas/virologia , Antígenos Virais de Tumores/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/biossíntese , Proteína 7 Relacionada à Autofagia/genética , Proteína Beclina-1/biossíntese , Proteína Beclina-1/genética , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Linhagem Celular Tumoral , Humanos , Macrolídeos/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Naftiridinas/farmacologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/patologia , Infecções por Polyomavirus/virologia , Processamento Pós-Transcricional do RNA , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia
15.
J Biol Chem ; 295(5): 1240-1260, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31857374

RESUMO

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.


Assuntos
Autofagia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Sequestossoma-1/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Cromatografia Líquida de Alta Pressão , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mutação , Quinases Relacionadas a NIMA/genética , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/genética , Espectrometria de Massas em Tandem , Treonina/metabolismo
16.
Neurobiol Aging ; 87: 139.e9-139.e15, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31859009

RESUMO

Several studies reported amyotrophic lateral sclerosis (ALS)-linked mutations in TBK1, OPTN, VCP, UBQLN2, and SQSTM1 genes encoding proteins involved in autophagy. SQSTM1 was originally identified by a candidate gene approach because it encodes p62, a multifunctional protein involved in protein degradation both through proteasomal regulation and autophagy. Both p62 and optineurin (encoded by OPTN) are direct interaction partners and substrates of TBK1, and these 3 proteins form the core of a genetic and functional network that may connect autophagy with ALS. Considering the molecular and conceptual relevance of the TBK1/OPTN/SQSTM1 "triangle," we here performed a targeted screen for SQSTM1 variants in 486 patients with familial ALS from Germany and Sweden by analyzing whole-exome sequencing data. We report 9 novel and 5 previously reported rare variants in SQSTM1 and discuss the current evidence for SQSTM1 as a primary disease gene for ALS. We conclude that the evidence for causality remains vague for SQSTM1 and is weaker than for the other autophagy genes, for example, TBK1 and OPTN.


Assuntos
Esclerose Amiotrófica Lateral/genética , Variação Genética , Proteína Sequestossoma-1/genética , Feminino , Alemanha , Humanos , Masculino , Suécia
17.
Neurobiol Aging ; 85: 154.e1-154.e3, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31530427

RESUMO

Paget's disease of bone (PDB) is a focal bone disorder affecting the skeleton segmentally. A strong genetic component has been shown in PDB, and variants in several genes, such as SQSTM1, VCP, and OPTN, have been associated with the disease. Mutations in the same genes have also been reported in patients with frontotemporal dementia and amyotrophic lateral sclerosis. Hexanucleotide repeat expansions in the C9ORF72 gene have been shown to be responsible for both familial and sporadic frontotemporal dementia/amyotrophic lateral sclerosis. Thence, we evaluated the frequency of the C9ORF72 hexanucleotide repeat expansions in a cohort of 191 Italian PDB patients and in 106 controls. The pathogenic repeat expansion was detected in 2 PDB patients (1.0%). During the follow-up period, both PDB patients did not develop any sign of mental decline and/or motor neuron disease. Our study suggests that repeat expansions in the C9ORF72 gene are rare in patients with PDB.


Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA , Osteíte Deformante/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Seguimentos , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Proteína Sequestossoma-1/genética
18.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31852783

RESUMO

Nuclear factor erythroid 2-related factor 2 (Nrf2) dissociates from its inhibitor, Keap1, upon stress signals and subsequently induces an antioxidant response that critically controls the viral life cycle and pathogenesis. Besides intracellular Fc receptor function, tripartite motif 21 (TRIM21) E3 ligase plays an essential role in the p62-Keap1-Nrf2 axis pathway for redox homeostasis. Specifically, TRIM21-mediated p62 ubiquitination abrogates p62 oligomerization and sequestration activity and negatively regulates the Keap1-Nrf2-mediated antioxidant response. A number of viruses target the Nrf2-mediated antioxidant response to generate an optimal environment for their life cycle. Here we report that a nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) interacts with and inhibits TRIM21 to activate the Nrf2 antioxidant signal pathway. Mass spectrometry identified TRIM21 to be a binding protein for NSs. NSs bound to the carboxyl-terminal SPRY subdomain of TRIM21, enhancing p62 stability and oligomerization. This facilitated p62-mediated Keap1 sequestration and ultimately increased Nrf2-mediated transcriptional activation of antioxidant genes, including those for heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, and CD36. Mutational analysis found that the NSs-A46 mutant, which no longer interacted with TRIM21, was unable to increase Nrf2-mediated transcriptional activation. Functionally, the NS wild type (WT), but not the NSs-A46 mutant, increased the surface expression of the CD36 scavenger receptor, resulting in an increase in phagocytosis and lipid uptake. A combination of reverse genetics and assays with Ifnar -/- mouse models revealed that while the SFTSV-A46 mutant replicated similarly to wild-type SFTSV (SFTSV-WT), it showed weaker pathogenic activity than SFTSV-WT. These data suggest that the activation of the p62-Keap1-Nrf2 antioxidant response induced by the NSs-TRIM21 interaction contributes to the development of an optimal environment for the SFTSV life cycle and efficient pathogenesis.IMPORTANCE Tick-borne diseases have become a growing threat to public health. SFTSV, listed by the World Health Organization as a prioritized pathogen, is an emerging phlebovirus, and fatality rates among those infected with this virus are high. Infected Haemaphysalis longicornis ticks are the major source of human SFTSV infection. In particular, the recent spread of this tick to over 12 states in the United States has increased the potential for outbreaks of this disease beyond Far East Asia. Due to the lack of therapies and vaccines against SFTSV infection, there is a pressing need to understand SFTSV pathogenesis. As the Nrf2-mediated antioxidant response affects viral life cycles, a number of viruses deregulate Nrf2 pathways. Here we demonstrate that the SFTSV NSs inhibits the TRIM21 function to upregulate the p62-Keap1-Nrf2 antioxidant pathway for efficient viral pathogenesis. This study not only demonstrates the critical role of SFTSV NSs in viral pathogenesis but also suggests potential future therapeutic approaches to treat SFTSV-infected patients.


Assuntos
Infecções por Bunyaviridae/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Phlebovirus/metabolismo , Ribonucleoproteínas/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismo , Animais , Infecções por Bunyaviridae/genética , Infecções por Bunyaviridae/patologia , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Phlebovirus/genética , Ribonucleoproteínas/genética , Proteína Sequestossoma-1/genética , Proteínas não Estruturais Virais/genética
19.
Int J Mol Sci ; 20(23)2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31757021

RESUMO

Mucopolysaccharidosis type II (MPS II) is a rare lysosomal storage disease (LSD) involving a genetic error in iduronic acid-2-sulfatase (IDS) metabolism that leads to accumulation of glycosaminoglycans within intracellular lysosomes. The primary treatment for MPS II, enzyme replacement therapy, is not effective for central nervous system (CNS) symptoms, such as intellectual disability, because the drugs do not cross the blood-brain barrier. Recently, autophagy has been associated with LSDs. In this study, we examined the morphologic relationship between neuronal damage and autophagy in IDS knockout mice using antibodies against subunit c of mitochondrial adenosine triphosphate (ATP) synthetase and p62. Immunohistological changes suggesting autophagy, such as vacuolation, were observed in neurons, microglia, and pericytes throughout the CNS, and the numbers increased over postnatal development. Oral administration of chloroquine, which inhibits autophagy, did not suppress damage to microglia and pericytes, but greatly reduced neuronal vacuolation and eliminated neuronal cells with abnormal inclusions. Thus, decreasing autophagy appears to prevent neuronal degeneration. These results suggest that an autophagy modulator could be used in addition to conventional enzyme replacement therapy to preserve the CNS in patients with MPS II.


Assuntos
Autofagia , Mucopolissacaridose II/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cloroquina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mucopolissacaridose II/patologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo
20.
PLoS One ; 14(9): e0222802, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31539400

RESUMO

Recent studies have begun to elucidate a role for E3 ubiquitin ligases as important mediators of the innate immune response. Our previous work defined a role for the ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) in mouse and human innate immunity. Here, we present novel data describing a role for NKLAM in regulating the immune response to Sendai virus (SeV), a murine model of paramyxoviral pneumonia. NKLAM expression was significantly upregulated by SeV infection. SeV-infected mice that are deficient in NKLAM demonstrated significantly less weight loss than wild type mice. In vivo, Sendai virus replication was attenuated in NKLAM-/- mice. Autophagic flux and the expression of autophagy markers LC3 and p62/SQSTM1 were also less in NKLAM-/- mice. Using flow cytometry, we observed less neutrophils and macrophages in the lungs of NKLAM-/- mice during SeV infection. Additionally, phosphorylation of STAT1 and NFκB p65 was lower in NKLAM-/- than wild type mice. The dysregulated phosphorylation profile of STAT1 and NFκB in NKLAM-/- mice correlated with decreased expression of numerous proinflammatory cytokines that are regulated by STAT1 and/or NFκB. The lack of NKLAM and the resulting attenuated immune response is favorable to NKLAM-/- mice receiving a low dose of SeV; however, at a high dose of virus, NKLAM-/- mice succumbed to the infection faster than wild type mice. In conclusion, our novel results indicate that NKLAM plays a role in regulating the production of pro-inflammatory cytokines during viral infection.


Assuntos
Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/deficiência , Pneumonia/metabolismo , Infecções por Respirovirus/metabolismo , Animais , Citocinas/genética , Humanos , Imunidade Inata/genética , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Membrana/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Pneumonia/genética , Pneumonia/virologia , Infecções por Respirovirus/genética , Infecções por Respirovirus/virologia , Vírus Sendai/fisiologia , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
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