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1.
J Cell Mol Med ; 28(12): e18492, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38890795

RESUMO

Intervertebral disc degeneration (IVDD) severely affects the work and the quality of life of people. We previously demonstrated that silencing activation transcription factor 3 (ATF3) blocked the IVDD pathological process by regulating nucleus pulposus cell (NPC) ferroptosis, apoptosis, inflammation, and extracellular matrix (ECM) metabolism. Nevertheless, whether miR-874-3p mediated the IVDD pathological process by targeting ATF3 remains unclear. We performed single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis to identify ATF3 as a key ferroptosis gene in IVDD. Then, Western blotting, flow cytometry, ELISA, and animal experiments were performed to validate the roles and regulatory mechanisms of miR-874-3p/ATF3 signalling axis in IVDD. ATF3 was highly expressed in IVDD patients and multiple cell types of IVDD rat, as revealed by scRNA-seq and bioinformatics analysis. GO analysis unveiled the involvement of ATF3 in regulating cell apoptosis and ECM metabolism. Furthermore, we verified that miR-874-3p might protect against IVDD by inhibiting NPC ferroptosis, apoptosis, ECM degradation, and inflammatory response by targeting ATF3. In vivo experiments displayed the protective effect of miR-874-3p/ATF3 axis on IVDD. These findings propose the potential of miR-874-3p and ATF3 as biomarkers of IVDD and suggest that targeting the miR-874-3p/ATF3 axis may be a therapeutic target for IVDD.


Assuntos
Fator 3 Ativador da Transcrição , Ferroptose , Degeneração do Disco Intervertebral , MicroRNAs , Núcleo Pulposo , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Humanos , Ratos , Ferroptose/genética , Masculino , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Análise de Célula Única/métodos , Apoptose/genética , Transdução de Sinais , Feminino , Pessoa de Meia-Idade , Ratos Sprague-Dawley , Análise de Sequência de RNA/métodos , Matriz Extracelular/metabolismo , Adulto , Regulação da Expressão Gênica , Modelos Animais de Doenças , Biologia Computacional/métodos
2.
Chem Biol Drug Des ; 103(6): e14565, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38862254

RESUMO

Ferroptosis is a novel form of programmed cell death that is triggered by iron-dependent lipid peroxidation. Brusatol (BRU), a natural nuclear factor erythroid 2-related factor 2 inhibitor, exhibits potent anticancer effects in various types of cancer. However, the exact mechanism of BRU in the treatment of hepatocellular carcinoma (HCC) remains unknown. The anticancer effects of BRU in HCC were detected using cell counting kit-8 and colony formation assays and a xenograft model. RNA sequencing (RNA-seq) and bioinformatics analyses of HCC cells were utilized to elucidate the mechanism underlying the effects of BRU in HCC. The levels of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and Fe2+ were measured using assay kits. The expression of activating transcription factor 3 (ATF3) was tested using RT-qPCR, western blotting, and immunofluorescence staining. The role of ATF3 in BRU-induced ferroptosis was examined using siATF3. BRU significantly inhibited HCC cell proliferation, both in vitro and in vivo. BRU activated the ferroptosis signaling pathway and increased ATF3 expression. Furthermore, ATF3 knockdown impeded BRU-induced ferroptosis. BRU suppressed HCC growth through ATF3-mediated ferroptosis, supporting BRU as a promising therapeutic agent for HCC.


Assuntos
Fator 3 Ativador da Transcrição , Carcinoma Hepatocelular , Ferroptose , Neoplasias Hepáticas , Quassinas , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Ferroptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Animais , Quassinas/farmacologia , Quassinas/química , Quassinas/uso terapêutico , Linhagem Celular Tumoral , Camundongos , Proliferação de Células/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos
3.
Sci Rep ; 14(1): 14669, 2024 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918575

RESUMO

Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b, and Tnfa, and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection.


Assuntos
Fator 3 Ativador da Transcrição , Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Camundongos Endogâmicos NOD , Transdução de Sinais , Animais , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Camundongos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Feminino , Inflamação/genética , Inflamação/patologia , Inflamação/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Perfilação da Expressão Gênica , Modelos Animais de Doenças
4.
BMC Musculoskelet Disord ; 25(1): 331, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38725009

RESUMO

BACKGROUND: The development of neuropathic pain (NP) is one of the reasons why the pain is difficult to treat, and microglial activation plays an important role in NP. Recently, platelet-rich plasma (PRP) has emerged as a novel therapeutic method for knee osteoarthritis (KOA). However, it's unclarified whether PRP has analgesic effects on NP induced by KOA and the underlying mechanisms unknown. PURPOSE: To observe the analgesic effects of PRP on NP induced by KOA and explore the potential mechanisms of PRP in alleviating NP. METHODS: KOA was induced in male rats with intra-articular injections of monosodium iodoacetate (MIA) on day 0. The rats received PRP or NS (normal saline) treatment at days 15, 17, and 19 after modeling. The Von Frey and Hargreaves tests were applied to assess the pain-related behaviors at different time points. After euthanizing the rats with deep anesthesia at days 28 and 42, the corresponding tissues were taken for subsequent experiments. The expression of activating transcription factor 3 (ATF3) in dorsal root ganglia (DRG) and ionized-calcium-binding adapter molecule-1(Iba-1) in the spinal dorsal horn (SDH) was detected by immunohistochemical staining. In addition, the knee histological assessment was performed by hematoxylin-eosin (HE) staining. RESULTS: The results indicated that injection of MIA induced mechanical allodynia and thermal hyperalgesia, which could be reversed by PRP treatment. PRP downregulated the expression of ATF3 within the DRG and Iba-1 within the SDH. Furthermore, an inhibitory effect on cartilage degeneration was observed in the MIA + PRP group only on day 28. CONCLUSION: These results indicate that PRP intra-articular injection therapy may be a potential therapeutic agent for relieving NP induced by KOA. This effect could be attributed to downregulation of microglial activation and reduction in nerve injury.


Assuntos
Regulação para Baixo , Microglia , Neuralgia , Osteoartrite do Joelho , Plasma Rico em Plaquetas , Ratos Sprague-Dawley , Animais , Masculino , Neuralgia/terapia , Neuralgia/metabolismo , Microglia/metabolismo , Ratos , Osteoartrite do Joelho/terapia , Fator 3 Ativador da Transcrição/metabolismo , Gânglios Espinais/metabolismo , Modelos Animais de Doenças , Injeções Intra-Articulares , Proteínas de Ligação ao Cálcio/metabolismo , Ácido Iodoacético/toxicidade , Proteínas dos Microfilamentos
5.
Artigo em Inglês | MEDLINE | ID: mdl-38777778

RESUMO

BACKGROUND: Aristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephropathy caused by Aristolochic acid (AA). AAN is associated with the development of nephropathy and urothelial carcinoma. It is estimated that more than 100 million people worldwide are at risk of developing AAN. However, the underlying mechanisms driving renal deterioration in AAN remain poorly understood, and the treatment options are limited. METHODS: We obtained GSE27168 and GSE136276 series matrix data from the Gene Expression Omnibus (GEO) related to AAN. Using the R Studio environment, we applied the limma package and WGCNA package to identify co-differently expressed genes (co-DEGs). By GO/KEGG/GSVA analysis, we revealed common biological pathways. Subsequently, co-DEGs were subjected to the String database to construct a protein-protein interaction (PPI) network. The MCC algorithms implemented in the Cytohubba plugin were employed to identify hub genes. The hub genes were cross-referenced with the transcription factor (TF) database to identify hub TFs. Immune infiltration analysis was performed to identify key immune cell groups by utilizing CIBERSORT. The expressions of AAN-associated hub TFs were verified in vivo and in vitro. Finally, siRNA intervention was performed on the two TFs to verify their regulatory effect in AAN. RESULTS: Our analysis identified 88 co-DEGs through the "limma" and "WGCNA" R packages. A PPI network comprising 53 nodes and 34 edges was constructed with a confidence level >0.4. ATF3 and c-JUN were identified as hub TFs potentially linked to AAN. Additionally, expressions of ATF3 and c-JUN positively correlated with monocytes, basophils, and vessels, and negatively correlated with eosinophils and endothelial cells. We observed a significant increase in protein and mRNA levels of these two hub TFs. Furthermore, it was found that siRNA intervention targeting ATF3, but not c-JUN, alleviated cell damage induced by AA. The knockdown of ATF3 protects against oxidative stress and inflammation in the AAN cell model. CONCLUSION: This study provides novel insights into the role of ATF3 in AAN. The comprehensive analysis sheds light on the molecular mechanisms and identifies potential biomarkers and drug targets for AAN treatment.


Assuntos
Ácidos Aristolóquicos , Nefropatias , Fatores de Transcrição , Ácidos Aristolóquicos/toxicidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/genética , Animais , Camundongos , Humanos , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Mapas de Interação de Proteínas
6.
Cell Death Dis ; 15(5): 318, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710703

RESUMO

Glioblastoma stem cells (GSCs) play a key role in glioblastoma (GBM) resistance to temozolomide (TMZ) chemotherapy. With the increase in research on the tumour microenvironment, exosomes secreted by GSCs have become a new focus in GBM research. However, the molecular mechanism by which GSCs affect drug resistance in GBM cells via exosomes remains unclear. Using bioinformatics analysis, we identified the specific expression of ABCB4 in GSCs. Subsequently, we established GSC cell lines and used ultracentrifugation to extract secreted exosomes. We conducted in vitro and in vivo investigations to validate the promoting effect of ABCB4 and ABCB4-containing exosomes on TMZ resistance. Finally, to identify the transcription factors regulating the transcription of ABCB4, we performed luciferase assays and chromatin immunoprecipitation-quantitative PCR. Our results indicated that ABCB4 is highly expressed in GSCs. Moreover, high expression of ABCB4 promoted the resistance of GSCs to TMZ. Our study found that GSCs can also transmit their highly expressed ABCB4 to differentiated glioma cells (DGCs) through exosomes, leading to high expression of ABCB4 in these cells and promoting their resistance to TMZ. Mechanistic studies have shown that the overexpression of ABCB4 in GSCs is mediated by the transcription factor ATF3. In conclusion, our results indicate that GSCs can confer resistance to TMZ in GBM by transmitting ABCB4, which is transcribed by ATF3, through exosomes. This mechanism may lead to drug resistance and recurrence of GBM. These findings contribute to a deeper understanding of the mechanisms underlying drug resistance in GBM and provide novel insights into its treatment.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Fator 3 Ativador da Transcrição , Neoplasias Encefálicas , Resistencia a Medicamentos Antineoplásicos , Exossomos , Glioblastoma , Células-Tronco Neoplásicas , Temozolomida , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Exossomos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Animais , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Camundongos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Nus
7.
Neurosci Lett ; 832: 137806, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38714229

RESUMO

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.3 , Ratos Sprague-Dawley , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Receptor 4 Toll-Like , Animais , Receptor 4 Toll-Like/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Masculino , Canal de Sódio Disparado por Voltagem NAV1.3/metabolismo , Transdução de Sinais/fisiologia , NF-kappa B/metabolismo , Neuralgia do Trigêmeo/metabolismo , Ratos , Modelos Animais de Doenças , Fator de Transcrição RelA/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Limiar da Dor/fisiologia
8.
Environ Toxicol Pharmacol ; 108: 104469, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38759848

RESUMO

We analyzed gene expression in THP-1 cells exposed to metal-based nanomaterials (NMs) [TiO2 (NM-100), ZnO (NM-110), SiO2 (NM-200), Ag (NM-300 K)]. A functional enrichment analysis of the significant differentially expressed genes (DEGs) identified the key modulated biological processes and pathways. DEGs were used to construct protein-protein interaction networks. NM-110 and NM-300 K induced changes in the expression of genes involved in oxidative and genotoxic stress, immune response, alterations of cell cycle, detoxification of metal ions and regulation of redox-sensitive pathways. Both NMs shared a number of highly connected protein nodes (hubs) including CXCL8, ATF3, HMOX1, and IL1B. NM-200 induced limited transcriptional changes, mostly related to the immune response; however, several hubs (CXCL8, ATF3) were identical with NM-110 and NM-300 K. No effects of NM-100 were observed. Overall, soluble nanomaterials NM-110 and NM-300 K exerted a wide variety of toxic effects, while insoluble NM-200 induced immunotoxicity; NM-100 caused no detectable changes on the gene expression level.


Assuntos
Mapas de Interação de Proteínas , Prata , Titânio , Humanos , Titânio/toxicidade , Células THP-1 , Mapas de Interação de Proteínas/efeitos dos fármacos , Prata/toxicidade , Nanoestruturas/toxicidade , Nanopartículas Metálicas/toxicidade , Óxido de Zinco/toxicidade , Óxido de Zinco/química , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Transcriptoma/efeitos dos fármacos , Dióxido de Silício/toxicidade , Interleucina-8/metabolismo , Interleucina-8/genética , Heme Oxigenase-1
9.
J Ethnopharmacol ; 330: 118228, 2024 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-38643863

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer (PCa) is the most common malignancy of the male genitourinary system and currently lacks effective treatment. Semen Impatientis, the dried ripe seed of Impatiens balsamina L., is described by the Chinese Pharmacopoeia as a traditional Chinese medicine (TCM) and is used in clinical practice to treat tumors, abdominal masses, etc. In our previous study, the ethyl acetate extracts of Semen Impatientis (EAESI) was demonstrated to be the most effective extract against PCa among various extracts. However, the biological effects of EAESI against PCa in vivo and the specific antitumor mechanisms involved remain unknown. AIM OF THE STUDY: In this study, we aimed to investigate the antitumor effect of EAESI on PCa in vitro and in vivo by performing network pharmacology analysis, transcriptomic analysis, and experiments to explore and verify the underlying mechanisms involved. MATERIALS AND METHODS: The antitumor effect of EAESI on PCa in vitro and in vivo was investigated via CCK-8, EdU, flow cytometry, and wound healing assays and xenograft tumor models. Network pharmacology analysis and transcriptomic analysis were employed to explore the underlying mechanism of EAESI against PCa. Activating transcription factor 3 (ATF3) and androgen receptor (AR) were confirmed to be the targets of EAESI against PCa by RT‒qPCR, western blotting, and rescue assays. In addition, the interaction between ATF3 and AR was assessed by coimmunoprecipitation, immunofluorescence, and nuclear-cytoplasmic separation assays. RESULTS: EAESI decreased cell viability, inhibited cell proliferation and migration, and induced apoptosis in AR+ and AR- PCa cells. Moreover, EAESI suppressed the growth of xenograft tumors in vivo. Network pharmacology analysis revealed that the hub targets of EAESI against PCa included AR, AKT1, TP53, and CCND1. Transcriptomic analysis indicated that activating transcription factor 3 (ATF3) was the most likely critical target of EAESI. EAESI downregulated AR expression and decreased the transcriptional activity of AR through ATF3 in AR+ PCa cells; and EAESI promoted the expression of ATF3 and exerted its antitumor effect via ATF3 in AR+ and AR- PCa cells. CONCLUSIONS: EAESI exerts good antitumor effects on PCa both in vitro and in vivo, and ATF3 and AR are the critical targets through which EAESI exerts antitumor effects on AR+ and AR- PCa cells.


Assuntos
Acetatos , Fator 3 Ativador da Transcrição , Camundongos Nus , Farmacologia em Rede , Neoplasias da Próstata , Receptores Androgênicos , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Animais , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Acetatos/química , Linhagem Celular Tumoral , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Camundongos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Transcriptoma/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos
10.
Arch Toxicol ; 98(7): 2065-2084, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38630284

RESUMO

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.


Assuntos
Fator 3 Ativador da Transcrição , Arsenicais , Células Epiteliais , Estresse Oxidativo , Espécies Reativas de Oxigênio , Bexiga Urinária , Humanos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fator 3 Ativador da Transcrição/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Linhagem Celular , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos
11.
J Med Chem ; 67(8): 6810-6821, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38613772

RESUMO

Anti-PD-L1 immunotherapy, a new lung cancer treatment, is limited to a few patients due to low PD-L1 expression and tumor immunosuppression. To address these challenges, the upregulation of PD-L1 has the potential to elevate the response rate and efficiency of anti-PD-L1 and alleviate the immunosuppression of the tumor microenvironment. Herein, we developed a novel usnic acid-derived Iridium(III) complex, Ir-UA, that boosts PD-L1 expression and converts "cold tumors" to "hot". Subsequently, we administered Ir-UA combined with anti-PD-L1 in mice, which effectively inhibited tumor growth and promoted CD4+ and CD8+ T cell infiltration. To our knowledge, Ir-UA is the first iridium-based complex to stimulate the expression of PD-L1 by explicitly regulating its transcription factors, which not only provides a promising platform for immune checkpoint blockade but, more importantly, provides an effective treatment strategy for patients with low PD-L1 expression.


Assuntos
Antígeno B7-H1 , Imunoterapia , Irídio , Animais , Irídio/química , Irídio/farmacologia , Antígeno B7-H1/metabolismo , Camundongos , Humanos , Imunoterapia/métodos , Fator 3 Ativador da Transcrição/metabolismo , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Microambiente Tumoral/efeitos dos fármacos , Feminino , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antineoplásicos/síntese química
12.
Cell Mol Biol (Noisy-le-grand) ; 70(3): 125-129, 2024 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-38650141

RESUMO

Myocardial ischemia/reperfusion injury (MIRI) is an irreversible adverse event during the management of coronary heart disease that lacks effective controls. The underlying mechanism of MIRI still requires further investigation. Recent studies have suggested that overexpression of ATF3 protects against MIRI by regulating inflammatory responses, ferroptosis, and autophagy. The downstream target of ATF3, EGR1, also showed cardioprotective properties against MIRI by promoting autophagy. Therefore, further investigating the effect of ATF3/EGR1 pathway on MIRI-induced inflammation and autophagy is needed. Cardiomyocyte MIRI model was established by challenging H9C2 cells with hypoxia/reoxygenation (H/R). The ATF3 overexpression-H/R cell model by transfecting ATF3 plasmid into the H9C2 cell line. The transcription levels of ATF3 and EGR1 were determined using RT-qPCR, the levels of TNF-α and IL-6 were determined using ELISA kits, the protein expression of LC3 I, LC3 II, and P62 was determined via WB, and microstructure of H9C2 cell was observed by transmission electron microscopy (TEM). Overexpression of ATF3 significantly downregulated Egr1 levels, indicating that EGR1 might be the target of ATF3. By upregulating ATF3 levels, the extracellular levels of the inflammatory cytokines TNF-α and IL-6 significantly decreased, and the protein expression of the autophagy markers LC3 I, LC3 II, and P62 significantly increased. TEM results revealed that the cell line in the H/R-ATF3 group exhibited a higher abundance of autophagosome enclosures of mitochondria. The results indicated that ATF3/EGR1 may alleviate inflammation and improve autophagy in an H/R-induced MIRI model of cardiomyocytes.


Assuntos
Fator 3 Ativador da Transcrição , Autofagia , Proteína 1 de Resposta de Crescimento Precoce , Inflamação , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Fator de Necrose Tumoral alfa , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Autofagia/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Animais , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Ratos , Linhagem Celular , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Transdução de Sinais , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética
13.
Cell Death Dis ; 15(4): 290, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38658567

RESUMO

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.


Assuntos
Fator 3 Ativador da Transcrição , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Neoplasias Ovarianas , Humanos , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Feminino , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Animais , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos
14.
Clin Transl Med ; 14(4): e1650, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38649772

RESUMO

BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.


Assuntos
Fator 3 Ativador da Transcrição , Biomarcadores , AVC Isquêmico , Neurônios , Traumatismos da Medula Espinal , Animais , Feminino , Humanos , Masculino , Camundongos , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Biomarcadores/metabolismo , Biomarcadores/sangue , Modelos Animais de Doenças , AVC Isquêmico/metabolismo , AVC Isquêmico/genética , AVC Isquêmico/sangue , Camundongos Knockout , Neurônios/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/complicações
15.
Circ Res ; 134(11): 1495-1511, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38686580

RESUMO

BACKGROUND: Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. METHODS: Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. RESULTS: In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor ß) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. CONCLUSIONS: Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.


Assuntos
Fator 3 Ativador da Transcrição , Aneurisma da Aorta Abdominal , Músculo Liso Vascular , Miócitos de Músculo Liso , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/induzido quimicamente , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Apoptose , Células Cultivadas , Angiotensina II , Proliferação de Células , Aorta Abdominal/patologia , Aorta Abdominal/metabolismo , Modelos Animais de Doenças
16.
Cell Commun Signal ; 22(1): 240, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38664711

RESUMO

BACKGROUND: The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear. METHODS: We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs). RESULTS: The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites. CONCLUSION: These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration.


Assuntos
Fator 3 Ativador da Transcrição , Axônios , Quebras de DNA de Cadeia Dupla , Gânglios Espinais , Células-Tronco Mesenquimais , Mitocôndrias , Regeneração Nervosa , Espécies Reativas de Oxigênio , Nervo Isquiático , Regulação para Cima , Animais , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Espécies Reativas de Oxigênio/metabolismo , Axônios/metabolismo , Regeneração Nervosa/genética , Regulação para Cima/genética , Camundongos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Gânglios Espinais/metabolismo , Camundongos Endogâmicos C57BL , Masculino
17.
Redox Biol ; 71: 103118, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38490069

RESUMO

The induction of ferroptosis is promising for cancer therapy. However, the mechanisms enabling cancer cells to evade ferroptosis, particularly in low-cystine environments, remain elusive. Our study delves into the intricate regulatory mechanisms of Activating transcription factor 3 (ATF3) on Cystathionine ß-synthase (CBS) under cystine deprivation stress, conferring resistance to ferroptosis in colorectal cancer (CRC) cells. Additionally, our findings establish a positively correlation between this signaling axis and CRC progression, suggesting its potential as a therapeutic target. Mechanistically, ATF3 positively regulates CBS to resist ferroptosis under cystine deprivation stress. In contrast, the suppression of CBS sensitizes CRC cells to ferroptosis through targeting the mitochondrial tricarboxylic acid (TCA) cycle. Notably, our study highlights that the ATF3-CBS signaling axis enhances ferroptosis-based CRC cancer therapy. Collectively, the findings reveal that the ATF3-CBS signaling axis is the primary feedback pathway in ferroptosis, and blocking this axis could be a potential therapeutic approach for colorectal cancer.


Assuntos
Neoplasias Colorretais , Ferroptose , Humanos , Cistationina beta-Sintase/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Ferroptose/genética , Cistina , Carcinogênese/genética , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo
18.
Cancer Lett ; 588: 216812, 2024 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-38490327

RESUMO

The efficacy of temozolomide (TMZ) treatment in glioblastoma (GBM) is influenced by various mechanisms, mainly including the level of O6-methylguanine-DNA methyltransferase (MGMT) and the activity of DNA damage repair (DDR) pathways. In our previous study, we had proved that long non-coding RNA HOTAIR regulated the GBM progression and mediated DDR by interacting with EZH2, the catalytic subunit of PRC2. In this study, we developed a small-molecule inhibitor called EPIC-0628 that selectively disrupted the HOTAIR-EZH2 interaction and promoted ATF3 expression. The upregulation of ATF3 inhibited the recruitment of p300, p-p65, p-Stat3 and SP1 to the MGMT promoter. Hence, EPIC-0628 silenced MGMT expression. Besides, EPIC-0628 induced cell cycle arrest by increasing the expression of CDKN1A and impaired DNA double-strand break repair via suppressing the ATF3-p38-E2F1 pathway. Lastly, EPIC-0628 enhanced TMZ efficacy in GBM in vitro and vivo. Hence, this study provided evidence for the combination of epigenetic drugs EPIC-0628 with TMZ for GBM treatment through the above mechanisms.


Assuntos
Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Dacarbazina/farmacologia , Linhagem Celular Tumoral , Enzimas Reparadoras do DNA/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Quebras de DNA de Cadeia Dupla , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Fator 3 Ativador da Transcrição/genética
19.
Hum Genet ; 143(3): 343-355, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38480539

RESUMO

Colorectal cancer (CRC) is the third most prevalent diagnosed cancer in men and second most prevalent cancer in women. H3K27ac alterations are more commonly than gene mutations in colorectal cancer. Most colorectal cancer genes have significant H3K27ac changes, which leads to an over-expression disorder in gene transcription. Over-expression of STEAP3 is involved in a variety of tumors, participating in the regulation of cancer cell proliferation and migration. The purpose of this work is to investigate the role of STEAP3 in the regulation of histone modification (H3K27ac) expression in colon cancer. Bioinformatic ChIP-seq, ChIP-qPCR and ATAC-seq were used to analyze the histone modification properties and gene accessibility of STEAP3. Western blot and qRT-PCR were used to evaluate relative protein and gene expression, respectively. CRISPR/Cas9 technology was used to knockout STEAP3 on colon cancer cells to analyze the effect of ATF3 on STEAP3. STEAP3 was over-expressed in colon cancer and associated with higher metastases and more invasive and worse stage of colon cancer. ChIP-seq and ChIP-qPCR analyses revealed significant enrichment of H3K27ac in the STEAP3 gene. In addition, knocking down STEAP3 significantly inhibits colon cancer cell proliferation and migration and down-regulates H3K27ac expression. ChIP-seq found that ATF3 is enriched in the STEAP3 gene and CRISPR/Cas9 technology used for the deletion of the ATF3 binding site suppresses the expression of STEAP3. Over-expression of STEAP3 promotes colon cancer cell proliferation and migration. Mechanical studies have indicated that H3K27ac and ATF3 are significantly enriched in the STEAP3 gene and regulate the over-expression of STEAP3.


Assuntos
Movimento Celular , Proliferação de Células , Neoplasias do Colo , Regulação Neoplásica da Expressão Gênica , Histonas , Humanos , Proliferação de Células/genética , Movimento Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Histonas/metabolismo , Histonas/genética , Acetilação , Feminino , Linhagem Celular Tumoral , Masculino , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo
20.
ACS Chem Biol ; 19(3): 753-762, 2024 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-38412264

RESUMO

Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C-C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five a positions to identify those that provided improved binding, with all e/g positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten e/g positions with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto e/g options provided. The combined a/e/g library represents ∼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined a/e/g exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.


Assuntos
Fator 3 Ativador da Transcrição , Biblioteca de Peptídeos , Fator 3 Ativador da Transcrição/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Peptídeos/metabolismo
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