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1.
Nat Commun ; 11(1): 4150, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811819

RESUMO

The systemic decline in autophagic activity with age impairs homeostasis in several tissues, leading to age-related diseases. A mechanistic understanding of adipocyte dysfunction with age could help to prevent age-related metabolic disorders, but the role of autophagy in aged adipocytes remains unclear. Here we show that, in contrast to other tissues, aged adipocytes upregulate autophagy due to a decline in the levels of Rubicon, a negative regulator of autophagy. Rubicon knockout in adipocytes causes fat atrophy and hepatic lipid accumulation due to reductions in the expression of adipogenic genes, which can be recovered by activation of PPARγ. SRC-1 and TIF2, coactivators of PPARγ, are degraded by autophagy in a manner that depends on their binding to GABARAP family proteins, and are significantly downregulated in Rubicon-ablated or aged adipocytes. Hence, we propose that age-dependent decline in adipose Rubicon exacerbates metabolic disorders by promoting excess autophagic degradation of SRC-1 and TIF2.


Assuntos
Adipócitos/metabolismo , Envelhecimento/fisiologia , Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Metabólicas/metabolismo , Adipócitos/patologia , Adipogenia/genética , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Adiposidade/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Técnicas de Inativação de Genes , Glucose/genética , Glucose/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , PPAR gama/metabolismo
2.
Nat Commun ; 11(1): 4012, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782388

RESUMO

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, a Ca2+ channel-like protein, is highly up-regulated in several cancer types. Here, we show that TMBIM6 is closely associated with survival in patients with cervical, breast, lung, and prostate cancer. TMBIM6 deletion or knockdown suppresses primary tumor growth. Further, mTORC2 activation is up-regulated by TMBIM6 and stimulates glycolysis, protein synthesis, and the expression of lipid synthesis genes and glycosylated proteins. Moreover, ER-leaky Ca2+ from TMBIM6, a unique characteristic, is shown to affect mTORC2 assembly and its association with ribosomes. In addition, we identify that the BIA compound, a potentialTMBIM6 antagonist, prevents TMBIM6 binding to mTORC2, decreases mTORC2 activity, and also regulates TMBIM6-leaky Ca2+, further suppressing tumor formation and progression in cancer xenograft models. This previously unknown signaling cascade in which mTORC2 activity is enhanced via the interaction with TMBIM6 provides potential therapeutic targets for various malignancies.


Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Indenos/farmacologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Ribossomos/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
3.
PLoS One ; 15(8): e0233818, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857777

RESUMO

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling was used to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-κB (NF-κB) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, common gene expression changes were identified that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level in M1-polarized macrophages. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes.


Assuntos
Cryptococcus neoformans/patogenicidade , Macrófagos/metabolismo , Macrófagos/microbiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cryptococcus neoformans/imunologia , Ontologia Genética , Redes Reguladoras de Genes , Imunidade Inata/genética , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Transativadores/genética , Transativadores/metabolismo , Transcriptoma
4.
Proc Natl Acad Sci U S A ; 117(35): 21391-21402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817423

RESUMO

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.


Assuntos
Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Escherichia coli , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo
5.
Anticancer Res ; 40(7): 3723-3732, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620611

RESUMO

BACKGROUND/AIM: Skin melanoma belongs to the most invasive malignancies with no cure for a progressing disease. Personalized therapy would allow for the selection of patients that will benefit from treatment. For this purpose, proper predictive biomarkers must be defined. MATERIALS AND METHODS: Allogeneic whole-cell gene-modified therapeutic melanoma vaccine (AGI-101H) was applied in advanced melanoma patients. Humoral responses were analyzed using SEREX, and in silico gene expression analysis in TCGA melanoma patients was performed. RESULTS: A specific antibody response was raised against an antigen identified as BNIP3L, which correlated with a good prognosis. Moreover, AGI-101H directs an immune response against autophagy, as BNIP3L is a marker of this process. Medium and high expression of BNIP3L was also linked with longer overall survival. CONCLUSION: BNIP3L is a candidate prognostic marker of clinical outcome of melanoma patients treated with AGI-101H, and may be considered as a prediction marker for patient survival.


Assuntos
Autofagia/fisiologia , Biomarcadores Tumorais/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Vacinas Anticâncer/imunologia , Feminino , Humanos , Masculino , Melanoma/imunologia , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Neoplasias Cutâneas/imunologia
6.
Nat Commun ; 11(1): 3301, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620849

RESUMO

Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação/genética , Células Cultivadas , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Homologia de Sequência de Aminoácidos , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética
7.
Arterioscler Thromb Vasc Biol ; 40(9): 2171-2186, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640906

RESUMO

OBJECTIVE: Cerebral cavernous malformations (CCM), consisting of dilated capillary channels formed by a single layer of endothelial cells lacking surrounding mural cells. It is unclear why CCM lesions are primarily confined to brain vasculature, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in brain mural cell in CCM pathogenesis. Approach and Results: SM22α-Cre was used to drive a specific deletion of Ccm3 in mural cells, including pericytes and smooth muscle cells (Ccm3smKO). Ccm3smKO mice developed CCM lesions in the brain with onset at neonatal stages. One-third of Ccm3smKO mice survived upto 6 weeks of age, exhibiting seizures, and severe brain hemorrhage. The early CCM lesions in Ccm3smKO neonates were loosely wrapped by mural cells, and adult Ccm3smKO mice had clustered and enlarged capillary channels (caverns) formed by a single layer of endothelium lacking mural cell coverage. Importantly, CCM lesions throughout the entire brain in Ccm3smKO mice, which more accurately mimicked human disease than the current endothelial cell-specific CCM3 deletion models. Mechanistically, CCM3 loss in brain pericytes dramatically increased paxillin stability and focal adhesion formation, enhancing ITG-ß1 (integrin ß1) activity and extracellular matrix adhesion but reducing cell migration and endothelial cell-pericyte associations. Moreover, CCM3-wild type, but not a paxillin-binding defective mutant, rescued the phenotypes in CCM3-deficient pericytes. CONCLUSIONS: Our data demonstrate for the first time that deletion of a CCM gene in the brain mural cell induces CCM pathogenesis.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Deleção de Genes , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Microvasos/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Comunicação Celular , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/patologia , Feminino , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/patologia , Predisposição Genética para Doença , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Microvasos/anormalidades , Miócitos de Músculo Liso/patologia , Paxilina/metabolismo , Pericitos/patologia , Fenótipo , Estabilidade Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
10.
Nucleic Acids Res ; 48(13): 7027-7040, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542340

RESUMO

Methylation of miRNAs at the 2'-hydroxyl group on the ribose at 3'-end (2'-O-methylation, 2'Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC-MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3'-terminal 2'Ome. Predominant 3'-terminal 2'Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/ß-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3'-terminal 2'Ome. We further identify HENMT1 as the methyltransferase responsible for 3'-terminal 2'Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3'→5' exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3'-terminal 2'Ome of mammalian miRNAs and highlight its role in enhancing miRNA's stability and function.


Assuntos
Proteínas Argonauta/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Exorribonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Proteínas de Ligação a RNA/metabolismo
11.
Gene ; 755: 144897, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32561323

RESUMO

The integrity of the intestinal barrier is critical for protecting the host against the intestinal lumen and pathogens. The roles of circRNAs in the intestinal barrier dysfunction in sepsis remained unclear. The present study aims to investigate the role of circ_0001105 in the intestinal mucosal permeability, oxidative damage and morphological changes during sepsis. We found that upregulation of circ_0001105 decreased the levels of serum D-lactic acid, diamine oxidase and fluorescence isothiocyanate dextran in septic rats. Upregulation of circ_0001105 also decreased the malondialdehyde content but enhanced superoxide dismutase activity in the intestinal tissues. Upregulation of circ_0001105 reduced the production of tumor necrosis factor α, interleukin (IL)-6, and IL-1ß and the expression of YAP1. Furthermore, upregulation of circ_0001105 improved the survival of rats with sepsis. In summary, our findings showed that circ_0001105 protects the intestinal barrier function of septic rats by inhibiting intestinal inflammation, oxidative damage and YAP1 expression. Our results provide a novel insight for developing sepsis treatment.


Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Mucosa Intestinal/metabolismo , RNA Circular/metabolismo , Sepse/metabolismo , Amina Oxidase (contendo Cobre)/sangue , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Regulação para Baixo , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Perfuração Intestinal/metabolismo , Ácido Láctico/sangue , Masculino , Estresse Oxidativo/fisiologia , Permeabilidade , RNA Circular/biossíntese , RNA Circular/genética , Ratos , Ratos Sprague-Dawley , Sepse/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
Environ Toxicol ; 35(10): 1058-1069, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32485087

RESUMO

Glioblastoma is the most common primary brain tumor with poor survival rate and without effective treatment strategy. Notably, amplification and active mutation of epidermal growth factor receptor (EGFR) occur frequently in glioblastoma patient that may be a potential treatment target. Several studies indicated that various type of herbal compounds not only regulate anti-depressant effect but also shown capacity to suppress glioblastoma growth via inducing apoptosis and inhibiting oncogene signaling transduction. Hyperforin, an herb compound derived from St. John's wort was used to treat depressive disorder by inhibiting neuronal reuptake of several neurotransmitters. Although hyperforin can reduce matrix metallopeptidases-2 (MMPs) and -9-mediated metastasis of glioblastoma, the detail mechanism of hyperforin on glioblastoma is remaining unclear. Here, we suggested that hyperforin may induce extrinsic/intrinsic apoptosis and suppress anti-apoptotic related proteins expression of glioblastoma. We also indicated that hyperforin-mediated anti-apoptotic potential of glioblastoma was correlated to inactivation of EGFR/extracellular signal-regulated kinases (ERK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Floroglucinol/análogos & derivados , Terpenos/farmacologia , Fator de Transcrição RelA/metabolismo , Antineoplásicos Fitogênicos/isolamento & purificação , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Hypericum/química , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Transdução de Sinais , Terpenos/isolamento & purificação , Fator de Transcrição RelA/genética
13.
Vascul Pharmacol ; 131: 106761, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32585189

RESUMO

AIMS: Diabetes-induced retinal vascular cell death aggravates diabetic retinopathy (DR) to the proliferative stage and blindness. Pericytes play a crucial role in retinal capillaries survival, stability, and angiogenesis. Ephrin-B2 is a tyrosine kinase that regulates pericytes/endothelial cells communication during angiogenesis; yet, its role in DR is still unclear. We hypothesize that diabetes increases Ephrin-B2 signaling in pericytes, which contributes to inflammation and retinal vascular cell death. METHODS: Selective inhibition of the Ephrin-B2 expression in the retinal pericytes was achieved using an intraocular injection of adeno-associated virus (AAV) with a specific pericyte promotor. Vascular death was determined by retinal trypsin digest. Pathological angiogenesis was assessed using the oxygen-induced retinopathy model in pericyte-Ephrin-B2 knockout mice, wild type, and wild type injected with AAV. Angiogenic properties, inflammatory, and apoptotic markers were measured in human retinal pericytes (HRP) grown under diabetic conditions. KEY FINDING: Diabetes significantly increased the expression of Ephrin-B2, inflammatory, and apoptotic markers in the diabetic retinas and HRP. These effects were prevented by silencing Ephrin-B2 in HRP. Moreover, Ephrin-B2 silencing in retinal pericytes decreased pathological angiogenesis and acellular capillaries formation in diabetic retinas. SIGNIFICANCE: Increased Ephrin-B2 expression in the pericytes contributed to diabetes-induced retinal inflammation and vascular death. These results identify pericytes-Ephrin-B2 as a therapeutic target for DR.


Assuntos
Apoptose , Retinopatia Diabética/metabolismo , Efrina-B2/metabolismo , Pericitos/metabolismo , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/etiologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Efrina-B2/deficiência , Efrina-B2/genética , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pericitos/patologia , Ratos Wistar , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Vasos Retinianos/patologia , Transdução de Sinais , Estreptozocina
14.
J Cancer Res Clin Oncol ; 146(7): 1751-1764, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32377840

RESUMO

PURPOSE: Although important for apoptosis, the signaling pathway involving MOAP-1(Modulator of Apoptosis 1), RASSF1A (RAS association domain family 1A), and Bax (Bcl-2 associated X protein) is likely to be dysfunctional in many types of human cancers due to mechanisms associated with gene mutation and DNA hyper-methylation. The purpose of the present study was to assess the potential impact of generating physiologically relevant signaling pathway mediated by MOAP-1, Bax, and RASSF1A (MBR) in cancer cells and chemo-drug resistant cancer cells. METHODS: The tricistronic expression construct that encodes MOAP-1, Bax, and RASSF1A (MBR) or its mutant, MOAP-1∆BH3L, Bax and RASSF1A (MBRX) was expressed from an IRES (Internal Ribosome Entry Site)-based tricistronic expression vector in human breast cancer cells, including MCF-7, MCF-7-CR (cisplatin resistant) and triple negative breast cancer cells, BMET05, for functional characterization through in vitro and in vivo models. RESULTS: Transient expression of MBR potently promoted dose-dependent apoptotic signaling and chemo-sensitization in the cancer cells, as evidenced by loss of cell viability, nuclei condensation and Annexin-V positive staining while stable expression of MBR in MCF-7 cells significantly reduced the number of MBR stable clone by 86% and the stable clone exhibited robust chemo-drug sensitivity. In contrast, MBRX stable clone exhibited chemo-drug resistance while transiently over-expressed MOAP-1ΔBH3L inhibited the apoptotic activity of MBR. Moreover, the spheroids derived from the MBR stable clone displayed enhanced chemo-sensitivity and apoptotic activity. In mouse xenograft model, the tumors derived from MBR stable clone showed relatively high level of tumor growth retardation associated with the increase in apoptotic activity, leading to the decreases in both tumor weight and volume. CONCLUSIONS: Expression of MBR in cancer cells induces apoptotic cell death with enhanced chemo-sensitization requiring the BH3L domain of MOAP-1. In animal model, the expression of MBR significantly reduces the growth of tumors, suggesting that MBR is a potent apoptotic sensitizer with potential therapeutic benefits for cancer treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Domínios e Motivos de Interação entre Proteínas , Proteínas Supressoras de Tumor/genética , Proteína X Associada a bcl-2/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Genes Reporter , Humanos , Camundongos , Modelos Biológicos , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
15.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(1): 87-92, 2020 Jan 30.
Artigo em Chinês | MEDLINE | ID: mdl-32376565

RESUMO

OBJECTIVE: To observe the expression of NLRP1 inflammasomes in myocardial tissues in rats with a high-fat and highsugar diet and in diabetic rats analyze the role of NLRP1 inflammasomes in the pathogenesis of diabetic cardiomyopathy. METHODS: Male SD rats were divided into normal control group, high-sugar and high-fat diet (HC) group and diabetes group. Rat models of diabetes were established by intraperitoneal injection of streptozotocin (STZ; 30 mg/kg). Serum levels of cholesterol (TC), triglyceride (TG), and fasting insulin (FINS) were measured after 8 weeks of feeding, and the insulin resistance index (IRI) and insulin sensitivity index (ISI) were calculated; Echocardiographic evaluation of cardiac structure and function was performed, and Western blotting and real-time fluorescent quantitative PCR (RT-qRCP) were used to detect the protein and mRNA expressions of NLRP1, ASC, and caspase 1 in the myocardial tissue. RESULTS: Compared with the control rats, the rats in the HC group had significantly increased body weight (BW), serum levels of TG and TC, mRNA expressions of NLRP1 and caspase 1, and the protein expression of NLRP1 (P < 0.01) without significant changes in FINS, IRI, ISI, or cardiac ultrasound findings (P > 0.05) or in myocardial ASC and caspase 1 protein expressions or serum levels of IL-1ß and IL-18 (P > 0.05). In the diabetic rats, TC, TG, and FBG levels increased and FINS, ISI decreased significantly (P < 0.01); the left ventricular end-diastolic diameter (LVID) and the left ventricular end-systolic diameter (LVSD) increased while the ejection fraction (LVEF), short axis shortening rate (FS), and E/A ratio all decreased. The expressions of NLRP1/ASC/caspase 1 pathway mRNA and NLRP1 and caspase 1 proteins also increased but myocardium ASC protein expression did not show significant changes in the diabetic rats (P > 0.05). IL-1ß and IL-18 levels were also significantly higher in the diabetic rats than in the control group (P < 0.05). Compared with those in HC group, the diabetic rats showed significantly increased serum FBG and decreased FINS, ISI and BW (P < 0.01) with decreased LVSD, LVEF and E/A ratio and increased levels of NLRP1 and caspase 1 protein expressions and serum L-1ß and IL-18 levels (P < 0.01). CONCLUSIONS: Diabetes can cause abnormal changes in cardiac structure and functions and induce inflammatory response in the myocardium, which may be related to the activation of NLRP1/ASC/ caspase 1 inflammasomes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Diabetes Mellitus Experimental/metabolismo , Inflamassomos/metabolismo , Miocárdio/metabolismo , Animais , Masculino , Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Estreptozocina
16.
Cardiovasc Ther ; 2020: 8584763, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32426037

RESUMO

Background: Although many studies have been performed to elucidate the molecular mechanisms of heart failure, an effective pharmacological therapy to protect cardiac tissues from severe loss of contractile function associated with heart failure after acute myocardial infarction (MI) has yet to be developed. Methods: We examined the cardioprotective effects of (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid, a new compound with potent antioxidant and antiapoptotic activities in a rat model of heart failure. (Z)-2-Acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid was systemically delivered to rats 6 weeks after MI at different doses (15, 30, and 60 mg/kg). Cardiac function was assessed by hemodynamic measurements. The expression of proinflammatory cytokines, apoptosis-related molecules, and markers of adverse ventricular remodeling was measured using RT-PCR and Western blot. Results: Treatment with (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid significantly improved cardiac function, in particular by increasing dP/dt. Simultaneously, the expression of the proinflammatory cytokines TNF-α and IL-1ß was markedly reduced in the treatment group compared with the MI group. In addition, (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid-treated tissues displayed decreased expression of Bax, caspase-3, and caspase-9 and increased expression of Bcl-2, which was in part due to the promotion of Akt phosphorylation. Conclusion: These data demonstrated that (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl) acrylic acid possesses potent cardioprotective effects against cardiac injury in a rat model of heart failure, which is mediated, at least in part, by suppression of the inflammatory and cell apoptosis responses.


Assuntos
Acrilatos/farmacologia , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Infarto do Miocárdio/complicações , Miócitos Cardíacos/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Mediadores da Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos
17.
Nat Commun ; 11(1): 2591, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444641

RESUMO

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0-/- enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Microbioma Gastrointestinal/fisiologia , Intestinos/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Ataxina-1/metabolismo , Diferenciação Celular , Colite/induzido quimicamente , Colite/patologia , Enterócitos/patologia , Feminino , Técnicas de Silenciamento de Genes , Homeostase , Intestinos/irrigação sanguínea , Intestinos/patologia , Intestinos/efeitos da radiação , Isquemia/genética , Isquemia/patologia , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/patologia , Regeneração/fisiologia , Transdução de Sinais , Nicho de Células-Tronco , Células-Tronco/metabolismo
18.
Nat Commun ; 11(1): 2598, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451402

RESUMO

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.


Assuntos
Mutação , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reparo de DNA por Recombinação/genética , Aneuploidia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Fertilização , Genes bcl-2 , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
19.
Nucleic Acids Res ; 48(11): 6340-6352, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383752

RESUMO

API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified, the function related to the resulting complex is unknown. Here, we determined the crystal structure of the API5-FGF2 complex and identified critical residues driving the protein interaction. These findings provided a structural basis for the nuclear localization of the FGF2 isoform lacking a canonical nuclear localization signal and identified a cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was important for mRNA nuclear export through both the TREX and eIF4E/LRPPRC mRNA export complexes, thus regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC and cyclin D1. These data show the newly identified molecular function of API5 and nuclear FGF2, and provide a clue to understanding the dynamic regulation of mRNA export.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , Ciclina D1/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo
20.
Nucleic Acids Res ; 48(11): 5891-5906, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421830

RESUMO

Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , DNA Ribossômico/genética , Genes de RNAr/genética , RNA Polimerase I/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Genética , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Dano ao DNA , DNA Ribossômico/metabolismo , Homeostase , Humanos , Fosforilação , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Ribossomos/metabolismo
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