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1.
J Chem Phys ; 151(12): 125101, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31575173

RESUMO

Gene regulation is one of the most important fundamental biological processes in living cells. It involves multiple protein molecules that locate specific sites on DNA and assemble gene initiation or gene repression multimolecular complexes. While the protein search dynamics for DNA targets has been intensively investigated, the role of intermolecular interactions during the genetic activation or repression remains not well quantified. Here, we present a simple one-dimensional model of target search for two interacting molecules that can reversibly form a dimer molecular complex, which also participates in the search process. In addition, the proteins have finite residence times on specific target sites, and the gene is activated or repressed when both proteins are simultaneously present at the target. The model is analyzed using first-passage analytical calculations and Monte Carlo computer simulations. It is shown that the search dynamics exhibit a complex behavior depending on the strength of intermolecular interactions and on the target residence times. We also found that the search time shows a nonmonotonic behavior as a function of the dissociation rate for the molecular complex. Physical-chemical arguments to explain these observations are presented. Our theoretical approach highlights the importance of molecular interactions in the complex process of gene activation/repression by multiple transcription factor proteins.


Assuntos
DNA/química , Modelos Químicos , Simulação por Computador , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Cinética , Método de Monte Carlo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
2.
J Cancer Res Clin Oncol ; 145(9): 2273-2283, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31428934

RESUMO

OBJECTIVES: Recent research has classified lung adenocarcinoma patients with KRAS mutation into three subtypes by co-occurring genetic events in TP53 (KP subgroup), STK11/LKB1 (KL subgroup) and CDKN2A/B inactivation plus TTF-1 low expression (KC subgroup). The aim of this study was to identify valuable biomarkers by searching the candidate molecules that contribute to lung adenocarcinoma pathogenesis, especially KC subtype. MATERIALS AND METHODS: We analyzed the publicly available database and identified the candidate REG4 using the E-GEOD-31210 dataset, and then confirmed by TCGA dataset. In addition, an independent cohort of 55 clinical samples was analyzed by quantitative real-time PCR analysis. Functional studies and RNA sequencing were performed after silencing the REG4 expression. RESULTS: REG4, an important regulator of gastro-intestinal carcinogenesis, was highly expressed in KRAS mutant lung adenocarcinoma with low expression of TTF-1 (KC subtype). The results were validated both by gene expression analysis and immunohistochemistry study in an independent 55 clinical samples from Fudan University Shanghai Cancer Center. Further in vitro and in vivo functional assays revealed silencing REG4 expression significantly reduces cancer cell proliferation and tumorigenesis. Moreover, RNA sequencing and GSEA analysis displayed that REG4 knockdown might induce cell cycle arrest by regulating G2/M checkpoint and E2F targets. CONCLUSION: Our results indicate that REG4 plays an important role in KRAS-driven lung cancer pathogenesis and is a novel biomarker of lung adenocarcinoma subtype. Future studies are required to clarify the underlying mechanisms of REG4 in the division and proliferation of KC tumors and its potential therapeutic value.


Assuntos
Adenocarcinoma de Pulmão/diagnóstico , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/diagnóstico , Proteínas Associadas a Pancreatite/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fatores de Transcrição/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/patologia , Estudos de Coortes , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/metabolismo
4.
J Biol Regul Homeost Agents ; 33(4): 1085-1095, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31389223

RESUMO

The purpose of this study was to explore the effect of Allograft Inflammatory Factor 1 (AIF-1) on the regulation of proliferation of breast cancer cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), cell culture and counting, and mass spectrometry were performed. The biologically active high-purity recombinant protein rhAIF-1 was obtained by optimizing the rhAIF-1 protein purification system, and MDA-MB-231 and MDA-MB-361 breast cancer cell lines were used. After adding to the culture medium, rhAIF-1 was found to promote cell proliferation in dose-dependent and time-dependent manners. The purified protein rhAIF-1 was marked with rhodamine and incubated with the cells. Confocal imaging analysis revealed that the foreign protein was localized in the cytoplasm, and rhAIF-1 was unevenly distributed in the cytoplasm. Although AIF-1 accumulates around the nucleus, it can not enter the nucleus, suggesting that other factors might be involved in the regulation of cell proliferation. In order to find the possible interacting protein of rhAIF-1, protein immunoprecipitation technique and mass spectrometry were employed, and it was indicated that ADAM28m was the possible interacting protein of rhAIF-1. The interaction between rhAIF-1 and ADAM28m was validated by immunoprecipitation along with Western blotting. It was found that rhAIF-1 could precipitate ADAM28m protein by immunoprecipitation. The results indicated that IF-1 participates in the development of breast cancer by interacting with ADAM28m and activating downstream signaling pathways. It was concluded that AIF-1 provides a new idea for the molecular mechanism of breast cancer cell proliferation and acts as a new target for the prevention and treatment of breast cancer in the future.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Proteínas ADAM/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Recombinantes/metabolismo
5.
Gene ; 715: 144015, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31357025

RESUMO

Tripartite Motif Containing 13 (TRIM13), a member of TRIM proteins, is deleted in multiple tumor types, especially in B-cell chronic lymphocytic leukemia and multiple myeloma. The present study explored the expression and potential role of TRIM13 in non-small-cell lung carcinoma (NSCLC). We found that TRIM13 mRNA and protein expression was reduced in NSCLC tissues and cell lines in comparison to paired non-cancerous tissues and a human normal bronchial epithelial cell line, respectively. Overexpression of TRIM13 in NCI-H1975 and SPC-A-1 cells hampered cell proliferation. Additionally, TRIM13 overexpression increased the levels of cleaved caspase-3. TRIM13-induced NSCLC cell apoptosis was attenuated by a caspase-3 inhibitor Ac-DEVD-CHO, suggesting that TRIM13 induced cell apoptosis partially through a caspase-3-dependent pathway. Moreover, it has been reported that TRIM13 can regulate nuclear factor kappaB (NF-κB) activity. Our data showed that TRIM13 overexpression inactivated NF-κB as indicated by the increased cytosolic NF-κB and decreased nuclear NF-κB. Exposure to an NF-κB inhibitor PDTC significantly blocked the impact of TRIM13 knockdown on cell proliferation and apoptosis, indicating the functions of TRIM13 in NSCLC cells were mediated by the NF-κB pathway. Finally, we demonstrated that TRIM13 overexpression suppressed tumor growth and induced cell apoptosis in vivo by using a xenograft mouse model. Collectively, our results indicate that TRIM13 behaves as a tumor suppressor in NSCLC through regulating NF-κB pathway. Our findings may offer a promising therapeutic target for NSCLC.


Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ligação a DNA/genética , Feminino , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , NF-kappa B/genética , Transplante de Neoplasias , Transdução de Sinais , Proteínas Supressoras de Tumor/genética
6.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 44(5): 485-490, 2019 May 28.
Artigo em Chinês | MEDLINE | ID: mdl-31303610

RESUMO

OBJECTIVE: To explore the magnetic resonance imaging (MRI) characteristics of glioma with Brg/Brm-associated factor 53a (BAF53a) expression.
 Methods: A total of 121 patients with glioma was divided into a BAF53a high expression group (n=79) and a low expression group (n=42) according to the results of immunohistochemistry. Then the MRI characteristics, including lesion location, number, boundary, maximum diameter, peripheral edema, midline structure shift, homogeneity, cystic necrosis, hemorrhage, strengthening degree, ependymal strengthening, pia mater enhancement, deep white matter invasion and lesion across the midline (total 14 items), were analyzed.
 Results: The results showed that there were significance difference in lesion border, lesion edema, enhancement of the lesion, and deep white matter invasion between the 2 groups (all P<0.05).
 Conclusion: The MRI characteristics, such as lesion border, lesion edema degree, enhancement degree of the lesion and deep white matter invasion, might be associated with BAF53a expression in gliomas.


Assuntos
Actinas/metabolismo , Neoplasias Encefálicas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glioma , Humanos , Imagem por Ressonância Magnética , Necrose
7.
Chem Commun (Camb) ; 55(61): 8959-8962, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290487

RESUMO

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Ciclização , Cisteína/química , Hidrocarbonetos Bromados/química , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/química
8.
Nat Commun ; 10(1): 2936, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270323

RESUMO

ß-Arrestins are major regulators of G protein-coupled receptor-mediated signaling processes. Their potential roles in regulating adipocyte function in vivo remain unexplored. Here we report the novel finding that mice lacking ß-arrestin-2 (barr2) selectively in adipocytes show significantly reduced adiposity and striking metabolic improvements when consuming excess calories. We demonstrate that these beneficial metabolic effects are due to enhanced signaling through adipocyte ß3-adrenergic receptors (ß3-ARs), indicating that barr2 represents a potent negative regulator of adipocyte ß3-AR activity in vivo. Interestingly, essentially all beneficial metabolic effects caused by adipocyte barr2 deficiency are absent in adipocyte barr2-PRDM16 double KO mice, indicating that the metabolic improvements caused by the lack of barr2 in adipocytes are mediated by the browning/beiging of white adipose tissue. Our data support the novel concept that 'G protein-biased' ß3-AR agonists that do not promote ß3-AR/barr2 interactions may prove useful for the treatment of obesity and related metabolic disorders.


Assuntos
Adipócitos/metabolismo , Metabolismo Energético , Glucose/metabolismo , beta-Arrestina 2/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta-Arrestina 2/genética
9.
Nat Commun ; 10(1): 2958, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273195

RESUMO

RNAi-based bone anabolic gene therapy has demonstrated initial success, but many practical challenges are still unmet. Here, we demonstrate that a recombinant adeno-associated virus 9 (rAAV9) is highly effective for transducing osteoblast lineage cells in the bone. The adaptor protein Schnurri-3 (SHN3) is a promising therapeutic target for osteoporosis, as deletion of shn3 prevents bone loss in osteoporotic mice and short-term inhibition of shn3 in adult mice increases bone mass. Accordingly, systemic and direct joint administration of an rAAV9 vector carrying an artificial-microRNA that targets shn3 (rAAV9-amiR-shn3) in mice markedly enhanced bone formation via augmented osteoblast activity. Additionally, systemic delivery of rAAV9-amiR-shn3 in osteoporotic mice counteracted bone loss and enhanced bone mechanical properties. Finally, we rationally designed a capsid that exhibits improved specificity to bone by grafting the bone-targeting peptide motif (AspSerSer)6 onto the AAV9-VP2 capsid protein. Collectively, our results identify a bone-targeting rAAV-mediated gene therapy for osteoporosis.


Assuntos
Reabsorção Óssea/complicações , Reabsorção Óssea/prevenção & controle , Osso e Ossos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dependovirus/metabolismo , Inativação Gênica , Osteoporose/complicações , Animais , Reabsorção Óssea/patologia , Osso e Ossos/virologia , Capsídeo/metabolismo , Cartilagem/virologia , Modelos Animais de Doenças , Deleção de Genes , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Sorogrupo
10.
Cancer Sci ; 110(9): 2760-2772, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31325400

RESUMO

Long noncoding RNAs (lncRNAs) are emerging as key regulators in cancer initiation and progression. TP53TG1 is a recently identified lncRNA and several studies have shown that TP53TG1 may play the role of tumor suppressor gene or oncogene in different tumors. Nevertheless, the involvement of TP53TG1 in carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) has not been characterized. In our studies, we identified that TP53TG1 was highly expressed in PDAC and was a novel regulator of PDAC development. Knockdown of TP53TG1 inhibited proliferation, induced apoptosis, and decreased migration and invasion in PDAC cells, whereas enhanced expression of TP53TG1 had the opposite effects. Mechanistically, TP53TG1 could directly bind to microRNA (miR)-96 and effectively function as a sponge for miR-96, thus antagonizing the functions of miR-96 and leading to derepression of its endogenous target KRAS, which is a core oncogene in the initiation and maintenance of PDAC. Taken together, these observations imply that TP53TG1 contributes to the growth and progression of PDAC by acting as a competing endogenous RNA (ceRNA) to competitively bind to miR-96 and regulate KRAS expression, which highlights the importance of the complicated miRNA-lncRNA network in modulating the progression of PDAC.


Assuntos
Carcinoma Ductal Pancreático/genética , Proteínas de Ligação a DNA/metabolismo , MicroRNAs/antagonistas & inibidores , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/metabolismo , Carcinogênese/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Longo não Codificante/genética
11.
Anticancer Res ; 39(6): 2845-2853, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31177122

RESUMO

BACKGROUND/AIM: Recent studies have shown a marked increase of AT-rich interactive domain 3A (ARID3A) in colon cancer tissue compared to normal colon mucosa. However, the role of ARID3A has not yet been determined in rectal cancer. We, therefore, investigated the clinical relevance of ARID3A expression in patients with residual rectal cancer after neoadjuvant chemoradiotherapy (NACRT). MATERIALS AND METHODS: One hundred thirty-four patients who underwent surgical resection for residual rectal cancer after NACRT were analyzed. ARID3A expression was evaluated using immunohistochemistry on whole-tissue sections. KRAS exon 2 (codons 12 and 13) and BRAF V600E mutation status were determined using polymerase chain reaction. RESULTS: ARID3A positivity was found in 91 cases (64.5%), and it correlated with absence of perineural invasion (p=0.031), longer disease-free survival (DFS) (p=0.048) and cancer-specific survival (CSS) (p=0.006). However, ARID3A positivity was not correlated with KRAS (p=0.231) or BRAF mutation status (p=0.577). In multivariate analysis, ARID3A positivity was independently associated with a favorable CSS (p=0.035), but not DFS (p=0.051). CONCLUSION: ARID3A positivity can predict favorable prognosis in patients with residual rectal cancer after NACRT.


Assuntos
Quimiorradioterapia Adjuvante/métodos , Proteínas de Ligação a DNA/metabolismo , Neoplasia Residual/cirurgia , Neoplasias Retais/terapia , Fatores de Transcrição/metabolismo , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Humanos , Leucovorina/administração & dosagem , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Mutação , Terapia Neoadjuvante , Neoplasia Residual/genética , Neoplasia Residual/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Retais/genética , Neoplasias Retais/metabolismo , Estudos Retrospectivos , Resultado do Tratamento
12.
Nat Commun ; 10(1): 2420, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160570

RESUMO

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Endonucleases Flap/metabolismo , Humanos , Hidrólise , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Nat Commun ; 10(1): 2757, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227697

RESUMO

Inappropriate expansion of the adipose cells in the subcutaneous adipose tissue (SAT) is a characteristic of hypertrophic obesity and of individuals with genetic predisposition for T2D (first-degree relatives; FDR). It is associated with insulin resistance, a dysfunctional, adipose tissue and reduced adipogenesis. We examined the regulation of adipogenesis in human SAT precursor cells and found ZNF521 to be a critical regulator of early adipogenic commitment and precursor cells leaving the cell cycle. However, neither altered upstream signalling nor lack of SAT progenitor cells could explain the reduced adipogenesis in hypertrophic obesity. Instead, we show that progenitor cells undergoing poor differentiation are characterized by senescence, inability to suppress p53/P16INK4 and secretion of factors reducing adipogenesis in non-senescent cells. We found aging, FDR and established T2D to be associated with increased progenitor cell senescence, reduced adipogenesis and hypertrophic expansion of the SAT adipose cells.


Assuntos
Adipogenia , Senescência Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Obesidade/patologia , Gordura Subcutânea/patologia , Adipócitos , Adulto , Idoso , Envelhecimento/fisiologia , Biópsia por Agulha , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Feminino , Predisposição Genética para Doença , Humanos , Hipertrofia/patologia , Masculino , Pessoa de Meia-Idade , Obesidade/etiologia , Cultura Primária de Células , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Gordura Subcutânea/citologia , Proteína Supressora de Tumor p53/metabolismo , Adulto Jovem
14.
Nat Commun ; 10(1): 2885, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253769

RESUMO

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.


Assuntos
Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Adenosina Trifosfatases , Animais , Linhagem Celular , Clonagem Molecular , Microscopia Crioeletrônica , DNA/química , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli , Regulação da Expressão Gênica , Humanos , Insetos , Modelos Químicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
15.
Cancer Sci ; 110(8): 2471-2484, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31187548

RESUMO

Endoplasmic reticulum stress (ERS) plays a key role in the pathogenesis and development of tumors and protects tumor cells from radiation damage and drug-induced stress. We previously demonstrated that EGFR confers radioresistance in human papillomavirus (HPV)-negative human oropharyngeal carcinoma by activating ERS signaling through PERK and IRE1α. In addition, PERK confers radioresistance by activating the inflammatory cytokine NF-κB. However, the effect of IRE1 on radiosensitivity has not yet been fully elucidated. Here, we clarified that IRE1 overexpression was associated with poor outcome in HPV-negative patients treated with radiotherapy (P = 0.0001). In addition, a significantly higher percentage of radioresistant HPV-negative patients than radiosensitive HPV-negative patients exhibited high IRE expression (66.7% vs 27.8%, respectively; P = 0.001). Silencing IRE1 and XBP1 increased DNA double-strand break (DSB) and radiation-induced apoptosis, thereby increasing the radiosensitivity of HPV-negative oropharyngeal carcinoma cells. IRE1-XBP1 silencing also inhibited radiation-induced IL-6 expression at both the RNA and protein levels. The regulatory effect of IRE1-XBP1 silencing on DNA DSB-induced and radiation-induced apoptosis was inhibited by pretreatment with IL-6. These data indicate that IRE1 regulates radioresistance in HPV-negative oropharyngeal carcinoma through IL-6 activation, enhancing X-ray-induced DNA DSB and cell apoptosis.


Assuntos
Endorribonucleases/metabolismo , Interleucina-6/metabolismo , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Tolerância a Radiação/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Apoptose/fisiologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Humanos , NF-kappa B/metabolismo , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
16.
Nat Commun ; 10(1): 2860, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253784

RESUMO

Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer. In this study, we reveal that Death Effector Domain-containing DNA-binding protein (DEDD), which is overexpressed in > 60% of TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71 kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with TNBC.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Lapatinib/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Receptores ErbB/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptor ErbB-2/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo
17.
Microbiol Res ; 223-225: 79-87, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178055

RESUMO

Vibrio parahaemolyticus is a seafood-borne Gram-negative bacteria causing diarrheal diseases in humans world wide. ToxR is a membrane-associated transcriptional factor which plays an important role in acid stress tolerance and regulates the expression of virulence genes including type III secretion system 1 (T3SS1) and type VI secretion system 1 (T6SS1) in V. parahaemolyticus. However, possible mechanisms of ToxR mediating virulence gene expression have not been fully understood. In this study, we demonstrated that ToxR is essential for V. parahaemolyticus to tolerate acid stress by constructing a ToxR deletion mutant (ΔtoxR) and its complemented strain (toxR+). Quantitative PCR showed that the expression of toxR was up regulated under acid stress condition. RNA-seq analysis showed that ompU encoding one of outer membrane proteins was dramatically down regulated in ΔtoxR. Furthermore, the mutation of ompU also led to a significant reduction in tolerating acid stress indicating that ToxR mediated acid stress through regulating ompU expression. RNA-seq results further confirmed that acid stress condition could alter multiple signaling pathways either depending on ToxR (e.g., quorum sensing, fatty acid metabolism) or independent of ToxR (e.g., biosynthesis of secondary metabolites, microbial metabolism in diverse environment, biosynthesis of antibiotics, biosynthesis of amino acids and carbon metabolism pathways). We also for the first time demonstrated that ToxR positively regulated the expression of T6SS2 gene and the interbacteria killing activity. Our study provides comprehensive understanding of signaling pathways which are regulated by both acid stress and ToxR.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Vibrio parahaemolyticus/metabolismo , Adesinas Bacterianas/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Ácidos Graxos/metabolismo , Percepção de Quorum , Metabolismo Secundário , Análise de Sequência de RNA , Deleção de Sequência , Fatores de Transcrição/genética , Transcriptoma , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crescimento & desenvolvimento , Virulência/genética
18.
Chem Commun (Camb) ; 55(52): 7466-7469, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31184647
19.
Subcell Biochem ; 92: 169-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214987

RESUMO

The periplasm of Gram-negative bacteria contains a specialized chaperone network that facilitates the transport of unfolded membrane proteins to the outer membrane as its primary functional role. The network, involving the chaperones Skp and SurA as key players and potentially additional chaperones, is indispensable for the survival of the cell. Structural descriptions of the apo forms of these molecular chaperones were initially provided by X-ray crystallography. Subsequently, a combination of experimental biophysical methods including solution NMR spectroscopy provided a detailed understanding of full-length chaperone-client complexes . The data showed that conformational changes and dynamic re-organization of the chaperones upon client binding, as well as client dynamics on the chaperone surface are crucial for function. This chapter gives an overview of the structure-function relationship of the dynamic conformational rearrangements that regulate the functional cycles of the periplasmic molecular chaperones Skp and SurA.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Periplasma/metabolismo , Bactérias Gram-Negativas/enzimologia
20.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 871-879, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223005

RESUMO

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.


Assuntos
Proteínas de Escherichia coli , Peptídeos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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