RESUMO
2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.
Assuntos
Proteínas Serina-Treonina Quinases , Transdução de Sinais , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , DNA , Imunidade InataRESUMO
After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Endossomos/metabolismo , SARS-CoV-2 , Nexinas de Classificação/química , COVID-19/virologia , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Endocitose , Perfilação da Expressão Gênica , Células HEK293 , Células HeLa , Homeostase , Humanos , Lentivirus , Lisossomos/metabolismo , Peptídeos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Nexinas de Classificação/metabolismo , Internalização do VírusRESUMO
Oestrogen receptor α (ERα) is overexpressed in two-thirds of all breast cancer cases and is involved in breast cancer development and progression. Although ERα -positive breast cancer can be effectively treated by endocrine therapy, endocrine resistance is an urgent clinical problem. Thus, further understanding of the underlying mechanisms involved in ERα signalling is critical in dealing with endocrine resistance in patients with breast cancer. In the present study, unbiased RNA sequence analysis was conducted between the MCF-7 and MCF-7 tamoxifen-resistant (LCC2) cell lines in order to identify differentially expressed genes. The whole transcriptomic data indicated that the JAK-STAT pathway is markedly up-regulated, particularly the ISGF3 complex. As the critical effectors, STAT1 and IRF9 were up-regulated 5- and 20-fold, respectively, in LCC2 cells. The biological experiments indicated that STAT1 is important for ERα signalling. Depletion of STAT1 or inhibition of STAT1 function significantly decreased levels of ERα protein, ERα -target gene expression and cell proliferation in both the MCF-7 and LCC2 cell lines. Chromatin immunoprecipitation revealed that ERα transcription is associated with STAT1 recruitment to the ERα promoter region, suggesting that transcriptional regulation is one mechanism by which STAT1 regulates ERα mRNA levels and ERα signalling in breast cancer cells. The present study reveals a possible endocrine-resistant mechanism by which STAT1 modulates ERα signalling and confers tamoxifen resistance. Targeting of STAT1 is a potential treatment strategy for endocrine-resistant breast cancers.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Fator de Transcrição STAT1/genética , Transcrição Gênica , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Gênico 3 Estimulado por Interferon, Subunidade alfa/genética , Células MCF-7 , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
Oestrogen receptor É (ERÉ) is overexpressed in two-thirds of all breast cancers and involves in development and breast cancer progression. Although ERÉ-positive breast cancer could be effective treated by endocrine therapy, the endocrine resistance is still an urgent clinical problem. Thus, further understanding of the underlying mechanisms ERÉ signalling is critical in dealing with endocrine resistance in breast cancer patients. MCF-7 and T47D breast cancer cell lines are used to carry out the molecular biological experiments. Western blot is used to assess the relative protein level of ERÉ, RNF168 and actin. Real-time PCR is used the measure the relative ERÉ-related gene mRNA level. Luciferase assay is used to measure the relative ERÉ signalling activity. Chromatin immunoprecipitation is used to measure the RNF168 binding affinity to ERÉ promoter regions. WST assay and flow cytometry are used to measure the cell proliferation capacity. We use Student's t test and one-way ANOVA test for statistical data analysis. Here, we report an important role in ERÉ-positive breast cancer cells for RNF168 protein in supporting cell proliferation by driving the transcription of ERÉ. RNF168 is highly expressed in breast cancer samples, compared with normal breast tissue. In patients with breast cancer, RNF168 expression level is correlated with poor endocrine treatment outcome. Depletion of RNF168 causes decreased cell proliferation in MCF-7 and T47D cells. Besides, depletion RNF168 reduced mRNA level of ERÉ and its target genes, such as PS2 and GREB1. Chromatin immunoprecipitation revealed that ERÉ transcription is associated with RNF168 recruitment to ERÉ promoter region, suggesting that transcriptional regulation is one mechanism by which RNF168 regulates ERÉ mRNA level and ERÉ signalling in breast cancer cells. RNF168 is required for ERÉ-positive breast cancer cell proliferation and facilitate ERÉ signalling activity possibly through promoting transcription of ERÉ.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Ubiquitina-Proteína Ligases/genética , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7 , Proteínas de Membrana , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Transcrição Gênica , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Estrogen receptor alpha (ER alpha) is expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression. ER alpha positive breast cancer can be well controlled by ER alpha modulators, such as tamoxifen. However, tamoxifen resistance is commonly observed by altered ER alpha signaling. Thus, further understanding of the molecular mechanisms, which regulates ER alpha signaling, is important to improve breast cancer therapy. METHODS: SMURF1 and ER alpha protein expression levels were measured by western blot, while the ER alpha target genes were measured by real-time PCR. WST-1 assay was used to measure cell viability; the xeno-graft tumor model were used for in vivo study. RNA sequencing was analyzed by Ingenuity Pathway Analysis. Identification of ER alpha signaling was accomplished with luciferase assays, real-time RT-PCR and Western blotting. Protein stability assay and ubiquitin assay was used to detect ER alpha protein degradation. Immuno-precipitation based assays were used to detect the interaction domain between ER alpha and SMURF1. The ubiquitin-based Immuno-precipitation based assays were used to detect the specific ubiquitination manner happened on ER alpha. RESULTS: Here, we identify the E3 ligase SMURF1 facilitates ER alpha signaling. We show that depletion SMURF1 decreases ER alpha positive cell proliferation in vitro and in vivo. SMURF1 depletion based RNA-sequence data shows SMURF1 is necessary for ER alpha target gene expression in the transcriptomic scale. Immunoprecipitation indicates that SMURF1 associates with the N-terminal of ER alpha in the cytoplasm via its HECT domain. SMURF1 increases ER alpha stability, possibly by inhibiting K48-specific poly-ubiquitination process on ER alpha protein. Interestingly, SMURF1 expression could be induced via estradiol treatment. CONCLUSIONS: Our study reveals a novel positive feedback between SMURF1 and ER alpha signaling in supporting breast cancer growth. Targeting SMURF1 could be one promising strategy for ER alpha positive breast cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genes Reporter , Xenoenxertos , Humanos , Modelos Biológicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The ubiquitin binding protein SHAPRIN is highly expressed in human breast cancer, one of the most frequent female malignancies worldwide. Here, we perform SHARPIN depletion in breast cancer cells together with RNA sequencing. The global expression profiling showed p53 signaling as a potential SHARPIN target. SHARPIN depletion decreased cell proliferation, which effect could be rescue by p53 knocking down. Depletion SHARPIN significantly increases p53 protein level and its target genes in multiple breast cancer cell lines. Further experiment revealed that SHARPIN could facilitate p53 poly-ubiquitination and degradation in MDM2 dependent manner. Immuno-precipitation assay showed that SHARPIN associated with MDM2 and prolonged MDM2 protein stability. Analysis of public available database showed SHARPIN correlated with poor prognosis specifically in p53 wild-type breast cancer patients. Together, our finding revealed a novel modifier for p53/MDM2 complex and suggested SHARPIN as a promising target to restore p53 function in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Prognóstico , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Ubiquitinação , Ubiquitinas/genéticaRESUMO
Estrogen receptor α is expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression. In our study, we identified the novel E3 ubiquitin ligase SHARPIN function to facilitate ERα signaling. SHARPIN is highly expressed in human breast cancer and correlates with ERα protein level by immunohistochemistry. SHARPIN expression level correlates with poor prognosis in ERα positive breast cancer patients. SHARPIN depletion based RNA-sequence data shows that ERα signaling is a potential SHARPIN target. SHARPIN depletion significantly decreases ERα protein level, ERα target genes expression and estrogen response element activity in breast cancer cells, while SHARPIN overexpression could reverse these effects. SHARPIN depletion significantly decreases estrogen stimulated cell proliferation in breast cancer cells, which effect could be further rescued by ERα overexpression. Further mechanistic study reveals that SHARPIN mainly localizes in the cytosol and interacts with ERα both in the cytosol and the nuclear. SHARPIN regulates ERα signaling through protein stability, not through gene expression. SHARPIN stabilizes ERα protein via prohibiting ERα protein poly-ubiquitination. Further study shows that SHARPIN could facilitate the mono-ubiquitinaiton of ERα at K302/303 sites and facilitate ERE luciferase activity. Together, our findings propose a novel ERα modulation mechanism in supporting breast cancer cell growth, in which SHARPIN could be one suitable target for development of novel therapy for ERα positive breast cancer.