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1.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206257

RESUMO

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that modulate innate immune responses and play essential roles in the pathogenesis of heart diseases. Although important, the molecular mechanisms controlling cardiac TLR genes expression have not been clearly addressed. This study examined the expression pattern of Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, and Tlr9 in normal and disease-stressed mouse hearts. Our results demonstrated that the expression levels of cardiac Tlr3, Tlr7, Tlr8, and Tlr9 increased with age between neonatal and adult developmental stages, whereas the expression of Tlr5 decreased with age. Furthermore, pathological stress increased the expression levels of Tlr2, Tlr4, Tlr5, Tlr7, Tlr8, and Tlr9. Hippo-YAP signaling is essential for heart development and homeostasis maintenance, and YAP/TEAD1 complex is the terminal effector of this pathway. Here we found that TEAD1 directly bound genomic regions adjacent to Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, and Tlr9. In vitro, luciferase reporter data suggest that YAP/TEAD1 repression of Tlr4 depends on a conserved TEAD1 binding motif near Tlr4 transcription start site. In vivo, cardiomyocyte-specific YAP depletion increased the expression of most examined TLR genes, activated the synthesis of pro-inflammatory cytokines, and predisposed the heart to lipopolysaccharide stress. In conclusion, our data indicate that the expression of cardiac TLR genes is associated with age and activated by pathological stress and suggest that YAP/TEAD1 complex is a default repressor of cardiac TLR genes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunidade Inata , Miócitos Cardíacos/metabolismo , Receptores Toll-Like/genética , Fatores de Transcrição/metabolismo , Fatores Etários , Animais , Citocinas/metabolismo , Regulação da Expressão Gênica , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais
2.
Nat Commun ; 12(1): 3412, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099696

RESUMO

De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms ß-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Materiais Biocompatíveis/metabolismo , Proteínas Oncogênicas/metabolismo , Peptídeos/metabolismo , Actinas/metabolismo , Animais , Materiais Biocompatíveis/química , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeos/química , Conformação Proteica em Folha beta , Domínios Proteicos , Multimerização Proteica , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
3.
Nat Commun ; 12(1): 3705, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140493

RESUMO

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with poor prognosis. Up to 30% of PTCL lack distinctive features and are classified as PTCL, not otherwise specified (PTCL-NOS). To further improve our understanding of the genetic landscape and biology of PTCL-NOS, we perform RNA-sequencing of 18 cases and validate results in an independent cohort of 37 PTCL cases. We identify FYN-TRAF3IP2, KHDRBS1-LCK and SIN3A-FOXO1 as new in-frame fusion transcripts, with FYN-TRAF3IP2 as a recurrent fusion detected in 8 of 55 cases. Using ex vivo and in vivo experiments, we demonstrate that FYN-TRAF3IP2 and KHDRBS1-LCK activate signaling pathways downstream of the T cell receptor (TCR) complex and confer therapeutic vulnerability to clinically available drugs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Linfoma de Células T Periférico/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas de Ligação a RNA/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Estudos de Coortes , Proteínas de Ligação a DNA/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Transdução de Sinais/genética , Complexo Correpressor Histona Desacetilase e Sin3/genética , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismo
4.
Nat Commun ; 12(1): 3575, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117258

RESUMO

An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatia Restritiva/genética , Cardiomiopatia Restritiva/metabolismo , Animais , Autofagia , Sítios de Ligação , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/terapia , Cardiomiopatia Restritiva/terapia , Criança , Modelos Animais de Doenças , Regulação da Expressão Gênica , Terapia Genética , Coração , Proteínas de Choque Térmico , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Proteômica , Sarcômeros/metabolismo
5.
Aging (Albany NY) ; 13(12): 16341-16352, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34126594

RESUMO

Breast cancer is a prevalent malignancy with high mortality and poor prognosis. Ropivacaine is a widely used local anesthetic and presents potential anti-tumor activity. Nevertheless, the function of ropivacaine in breast cancer development remains elusive. Here, we tried to investigate the impact of ropivacaine on breast cancer progression and the underlying mechanism. Significantly, we revealed that ropivacaine was able to reduce the proliferation and induce the apoptosis of breast cancer cells. Ropivacaine could attenuate the invasion and migration in the cells. Mechanically, ropivacaine could enhance the miR-27b-3p expression and miR-27b-3p inhibited breast cancer progression in breast cancer cells. MiR-27b-3p targeted YAP in the breast cancer cells. Ropivacaine decreased the breast cancer progression by modulating miR-27b-3p/YAP axis in vitro. Ropivacaine could inhibit tumor growth in vivo. In conclusion, we discovered that the local anesthetic ropivacaine inhibits the progression of breast cancer via the miR-27b-3p/YAP axis. Our finding presents novel insights into the mechanism of ropivacaine inhibiting the development of breast cancer. Ropivacaine may potentially serve as an anti-tumor candidate in the therapeutic strategy of breast cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Anestésicos Locais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Ropivacaina/uso terapêutico , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anestésicos Locais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , Invasividade Neoplásica , Ropivacaina/farmacologia , Fatores de Transcrição/metabolismo
6.
Life Sci ; 280: 119729, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34146553

RESUMO

AIMS: To study 5-desmethylsinensetin exhibiting potential anticancer activity against breast cancer stem cells and the related molecular mechanism. MAIN METHODS: In this study, isolation of a cancer stem cell (CSC) inhibitor of Artemisia princeps was performed using a silica gel column, a Sephadex gel column, and high-performance liquid chromatography. A single compound was purified via activity-based isolation using mammosphere formation assays. An MTS was used to examine the proliferation of breast cancer cells, and flow cytometry was used to analyze apoptosis and cancer stem cell markers. Western blotting was used to detect the signaling pathway. RESULTS: The isolated compound was identified as 5-desmethylsinensetin using nuclear magnetic resonance and mass spectrometry. 5-Desmethylsinensetin suppresses the proliferation and mammosphere formation of breast cancer cells, reduces the subpopulations of CD44+/CD24- and ALDH1+ cancer cells, and reduces the transcription of the stemness markers Oct4, c-Myc, Nanog and CD44 in Breast CSCs. 5-Desmethylsinensetin inhibits the total and nuclear expression of Stat3 and p-Stat3, as well as the translocation of YAP1. Additionally, 5-desmethylsinensetin reduces the mRNA and protein levels of IL-6. CONCLUSION: Our results show that 5-desmethylsinensetin exhibits potential anticancer activity against breast cancer stem cells via Stat3-IL-6 and Stat3-YAP1 signaling.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Artemisia , Neoplasias da Mama/tratamento farmacológico , Flavonoides/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Artemisia/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Flavonoides/química , Humanos , Interleucina-6/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/metabolismo
7.
Nat Commun ; 12(1): 3624, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131132

RESUMO

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Artrite/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Artrite/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Caderinas/metabolismo , Proteínas do Citoesqueleto/genética , Feminino , Proteínas de Homeodomínio , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos , beta Catenina/metabolismo
8.
PLoS One ; 16(6): e0253458, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34185793

RESUMO

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , Dopa Descarboxilase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Enzima de Conversão de Angiotensina 2/genética , Área Sob a Curva , COVID-19/virologia , Dopa Descarboxilase/genética , Regulação para Baixo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Curva ROC , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Regulação para Cima , Carga Viral
9.
Nat Commun ; 12(1): 3910, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162877

RESUMO

Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.


Assuntos
Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Processamento de Proteína Pós-Traducional , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , Citrulinação , Citomegalovirus/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Resistência a Myxovirus/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células Vero , Proteínas Virais/metabolismo
10.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070186

RESUMO

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Neovascularização Retiniana/etiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Eletrorretinografia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/patologia , Vasos Retinianos/fisiopatologia , Transdução de Sinais
11.
Anticancer Res ; 41(6): 2805-2815, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083270

RESUMO

BACKGROUND/AIM: Copper metabolism MURR1 domain-containing 5 (COMMD5) is mainly expressed in renal tubules (RTs), where it facilitates re-differentiation of injured RTs. We reported that COMMD5 regulates the expression of epidermal growth factor receptor by participating in its endocytic membrane trafficking, thus inhibiting tumor growth. Here we aimed to determine the role of COMMD5 in malignant phenotypes of renal cell carcinoma (RCC). MATERIALS AND METHODS: The associations between COMMD5 levels in RTs adjacent to RCC tumors in patients and their clinicopathologic characteristics were evaluated, and the effects of COMMD5 on cancer stemness in RCC cells were investigated. RESULTS: Low COMMD5 levels in RTs correlated with high tumorigenesis and poor patient outcomes. COMMD5 overexpression in RCC cells reduced the proportion of cancer stem cell-like cells and their malignant phenotypes, including proliferation, invasion and sphere formation. Secreted COMMD5 from RT cells also reduced malignant phenotypes. CONCLUSION: COMMD5 might suppress malignant phenotypes of RCC, thus inhibiting tumor development and improving patient prognosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Proteínas Nucleares/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/metabolismo , Proliferação de Células/fisiologia , Progressão da Doença , Feminino , Humanos , Masculino , Invasividade Neoplásica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prognóstico , RNA Interferente Pequeno/genética
12.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068565

RESUMO

Pancreatic cancer is one of the most malignant cancers with high mortality. Therefore, it is of great urgency to develop new agents that could improve the prognosis of Pancreatic cancer patients. Chinese propolis (CP), a flavonoid-rich beehive product, has been reported to have an anticancer effect. In this study, we applied CP to the human Pancreatic cancer cell line Panc-1 to verify its impact on tumor development. CP induced apoptosis in Panc-1 cells from 12.5 µg/mL in a time- and dose-dependent manner with an IC50 value of approximately 50 µg/mL. Apoptosis rate induced by CP was examined by Annexing FITC/PI assay. We found that 48 h treatment with 50 µg/mL CP resulted in 34.25 ± 3.81% apoptotic cells, as compared to 9.13 ± 1.76% in the control group. We further discovered that the Panc-1 cells tended to be arrested at G2/M phase after CP treatment, which is considered to contribute to the anti-proliferation effect of CP. Furthermore, our results demonstrated that CP suppressed Panc-1 cell migration by regulating epithelial-mesenchymal transition (EMT). Interestingly, the Hippo pathway was activated in Panc-1 cells after CP treatment, serving as a mechanism for the anti-pancreatic cancer effect of CP. These findings provide a possibility of beehive products as an alternative treatment for pancreatic cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Própole/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Padrões de Referência , Transdução de Sinais/efeitos dos fármacos
13.
Nat Commun ; 12(1): 3340, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099657

RESUMO

Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Gluconeogênese/fisiologia , Fígado/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Glicemia/metabolismo , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Glucagon/metabolismo , Gluconeogênese/genética , Glucose/metabolismo , Hepatócitos/metabolismo , Homeostase , Humanos , Hiperglicemia , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Regulação para Cima
14.
Nat Commun ; 12(1): 3333, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099704

RESUMO

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2-5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II-mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Leucócitos/metabolismo , Lisossomos/metabolismo , Miosina Tipo II/metabolismo , Actomiosina/efeitos dos fármacos , Animais , Linhagem Celular , Células Dendríticas , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatase de Miosina-de-Cadeia-Leve , Neutrófilos , Transdução de Sinais
15.
Nat Commun ; 12(1): 3464, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103493

RESUMO

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFß signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFß-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFß-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.


Assuntos
Carcinogênese/metabolismo , Neoplasias do Colo/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinogênese/patologia , Diferenciação Celular , Sobrevivência Celular , Colo/patologia , Neoplasias do Colo/genética , Células Epiteliais/metabolismo , Feto/patologia , Inflamação/patologia , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Mutação , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
16.
J Med Chem ; 64(11): 7839-7852, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34038131

RESUMO

Inspired by the success of dual-targeting drugs, especially bispecific antibodies, we propose to combine the concept of proteolysis targeting chimera (PROTAC) and dual targeting to design and synthesize dual PROTAC molecules with the function of degrading two completely different types of targets simultaneously. A library of novel dual-targeting PROTAC molecules has been rationally designed and prepared. A convergent synthetic strategy has been utilized to achieve high synthetic efficiency. These dual PROTAC structures are characterized using trifunctional natural amino acids as star-type core linkers to connect two independent inhibitors and E3 ligands together. In this study, gefitinib, olaparib, and CRBN or VHL E3 ligands were used as substrates to synthesize novel dual PROTACs. They successfully degraded both the epidermal growth factor receptor (EGFR) and poly(ADP-ribose) polymerase (PARP) simultaneously in cancer cells. Being the first successful example of dual PROTACs, this technique will greatly widen the range of application of the PROTAC method and open up a new field for drug discovery.


Assuntos
Desenho de Fármacos , Receptores ErbB/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gefitinibe/química , Humanos , Ligantes , Ftalazinas/química , Piperazinas/química , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
17.
J Med Chem ; 64(11): 7296-7311, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34042448

RESUMO

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Fatores de Terminação de Peptídeos/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Administração Oral , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Meia-Vida , Humanos , Fator de Transcrição Ikaros/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Estudos Retrospectivos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Talidomida/metabolismo , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
18.
Cancer Sci ; 112(7): 2625-2641, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33931921

RESUMO

Bladder cancer (BLCA) remains the leading cause of cancer-related mortality among genitourinary malignancies worldwide. BLCA metastasis represents the primary reason for its poor prognosis. In this study, we report that decreased expression of partitioning defective 3 (Par3), a polarity protein (encoded by PARD3), is associated with tumor aggressive phenotypes and poor prognosis in BLCA patients. Consistently, ablation of Par3 promotes the metastasis and invasion of BLCA cells in vitro and in vivo. Further studies reveal that zinc finger protein Snail represses the expression of Par3 by binding to E2-box (CAGGTG) of PARD3 promoter-proximal. Inhibition of GSK-3ß promotes the expression and nuclear localization of Snail and then reduces the expression of Par3, resulting in the metastasis and invasion of BLCA cells. Moreover, we detected the interaction between Par3 (936-1356 aa) and ZO-1 (1372-1748 aa), which is involved in the maintenance of tight junction. Together, our results demonstrate that the GSK-3ß/Snail/Par3/ZO-1 axis regulates BLCA metastasis, and Snail is a major regulator for Par3 protein expression in BLCA.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Neoplasias Pulmonares/secundário , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Polaridade Celular/fisiologia , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , Fosforilação , Prognóstico , Distribuição Aleatória , Fatores de Transcrição da Família Snail/genética , Junções Íntimas/fisiologia , Proteína da Zônula de Oclusão-1/metabolismo
19.
Cancer Sci ; 112(7): 2714-2727, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33939216

RESUMO

Breast cancer is one of the most commonly diagnosed malignancies worldwide, while the triple negative breast cancer (TNBC) is the most aggressive and virulent subtype in breast cancers. Compared with luminal type breast cancers, which could be well controlled by endocrine treatment, TNBC is worse in prognosis and lack of effective targeted therapy. Thus, it would be interesting and meaningful to identify novel therapeutic targets for TNBC treatments. Recent genomic data showed the activation of Hippo/YAP signaling in TNBC, indicating its critical roles in TNBC carcinogenesis and cancer progression. Hippo/YAP signaling could subject to several kinds of protein modifications, including ubiquitination and phosphorylation. Quite a few studies have demonstrated these modifications, which controlled YAP protein stability and turnover, played critical role in Hippo signaling activation In our current study, we identified ZNF213 as a negative modifier for Hippo/YAP axis. ZNF213 depletion promoted TNBC cell migration and invasion, which could be rescued by further YAP silencing. ZNF213 knocking down facilitated YAP protein stability and Hippo target gene expression, including CTGF and CYR61. Further mechanism studies demonstrated that ZNF213 associated with YAP and facilitated YAP K48-linked poly-ubiquitination at several YAP lysine sites (K252, K254, K321 and K497). Besides, the clinical data showed that ZNF213 negatively correlated with YAP protein level and Hippo target gene expression in TNBC samples. ZNF213 expression correlated with good prognosis in TNBC patients. Our data provided novel insights in YAP proteolytic regulation and TNBC progression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Linhagem Celular Tumoral , Movimento Celular , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Imunofluorescência , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fosforilação , Prognóstico , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição/deficiência , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ubiquitinação
20.
Biochem J ; 478(10): 1853-1859, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: covidwho-1232077

RESUMO

The current SARS-CoV-2 pandemic has spurred new interest in interferon signaling in response to viral pathogens. Much of what we know about the signaling molecules and associated signal transduction induced during the host cellular response to viral pathogens has been gained from research conducted from the 1990's to the present day, but certain intricacies of the mechanisms involved, still remain unclear. In a recent study by Vaughn et al. the authors examine one of the main mechanisms regulating interferon induction following viral infection, the RIG-I/MAVS/IRF3 pathway, and find that similar to PKR both DICER interacting proteins, PACT and TRBP, regulate RIG-I signaling in an opposing manner. More specifically, the reported findings demonstrate, like others, that PACT stimulates RIG-I-mediated signaling in a manner independent of PACT dsRNA-binding ability or phosphorylation at sites known to be important for PACT-dependent PKR activation. In contrast, they show for the first time that TRBP inhibits RIG-I-mediated signaling. RIG-I inhibition by TRBP did not require phosphorylation of sites shown to be important for inhibiting PKR, nor did it involve PACT or PKR, but it did require the dsRNA-binding ability of TRBP. These findings open the door to a complex co-regulation of RIG-I, PKR, MDA5, miRNA processing, and interferon induction.


Assuntos
COVID-19/imunologia , Interferons/metabolismo , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , COVID-19/virologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferons/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
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