RESUMO
Mammalian barrier surfaces are constitutively colonized by numerous microorganisms. We explored how the microbiota was sensed by the immune system and the defining properties of such responses. Here, we show that a skin commensal can induce T cell responses in a manner that is restricted to non-classical MHC class I molecules. These responses are uncoupled from inflammation and highly distinct from pathogen-induced cells. Commensal-specific T cells express a defined gene signature that is characterized by expression of effector genes together with immunoregulatory and tissue-repair signatures. As such, non-classical MHCI-restricted commensal-specific immune responses not only promoted protection to pathogens, but also accelerated skin wound closure. Thus, the microbiota can induce a highly physiological and pleiotropic form of adaptive immunity that couples antimicrobial function with tissue repair. Our work also reveals that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota.
Assuntos
Imunidade Adaptativa , Bactérias/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Microbiota/imunologia , Pele/imunologia , Linfócitos T/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Camundongos TransgênicosRESUMO
Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/química , Metiltransferases/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Fatores de Transcrição/química , Iniciação da Transcrição Genética , Sequência de Aminoácidos , Bacteriófago T7/enzimologia , Bacteriófago T7/metabolismo , DNA Mitocondrial/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Regiões Promotoras Genéticas , Alinhamento de Sequência , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream "sliding clamp," providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA.
Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Quadruplex G , Proteínas Mitocondriais/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , DNA Mitocondrial/química , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Modelos Moleculares , Elongação da Transcrição Genética , Fatores de Transcrição/química , Terminação da Transcrição GenéticaRESUMO
Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Edição de Genes/métodos , Inativação Gênica , Marcação de Genes/métodos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Repressoras/metabolismo , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Metiltransferase 3A , Células-Tronco Embrionárias/metabolismo , Engenharia Genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas Repressoras/genética , Linfócitos T/metabolismoRESUMO
A majority of T cells bearing the αß T cell receptor (TCR) are specific for peptides bound to polymorphic classical major histocompatibility complex (MHC) molecules. Smaller subsets of T cells are reactive toward various nonpeptidic ligands associated with nonpolymorphic MHC class-Ib (MHC-Ib) molecules. These cells have been termed unconventional for decades, even though only the composite antigen is different from the one seen by classical T cells. Herein, we discuss the identity of these particular T cells in light of the coevolution of their TCR and MHC-Ib restricting elements. We examine their original thymic development: selection on hematopoietic cells leading to the acquisition of an original differentiation program. Most of these cells acquire memory cell features during thymic maturation and exhibit unique patterns of migration into peripheral nonlymphoid tissues to become tissue resident. Thus, these cells are termed preset T cells, as they also display a variety of effector functions. They may act as microbial or danger sentinels, fight microbes, or regulate tissue homeostasis.
Assuntos
Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismo , Animais , Antígenos de Histocompatibilidade/metabolismo , Humanos , Ligantes , Timo/citologiaRESUMO
The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in â¼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer.
RESUMO
Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.
Assuntos
DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA Mitocondrial/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Sítios de Ligação , Microscopia Crioeletrônica , DNA Mitocondrial/química , DNA Mitocondrial/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Elongação da Transcrição Genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Iniciação da Transcrição GenéticaRESUMO
A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.
Assuntos
Proteínas de Protozoários , Toxoplasma , Toxoplasma/metabolismo , Toxoplasma/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Fase G2/genética , Centrossomo/metabolismo , Divisão Celular , Ciclinas/metabolismo , Ciclinas/genéticaRESUMO
Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
Assuntos
Caspase 8/metabolismo , Núcleo Celular/enzimologia , Proliferação de Células , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias/enzimologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Antineoplásicos/farmacologia , Apoptose , Caspase 8/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , Estabilidade Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genéticaRESUMO
The stem-loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3'-UTR. Although s2m has been reported to display characteristics of a mobile genomic element that might lead to an evolutionary advantage, its function has remained unknown. The secondary structure of the original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020, delineating the base-pairing pattern and revealing substantial differences in secondary structure compared to SARS-CoV-1 (SCoV-1). The existence of a single G29742-A29756 mismatch in the upper stem of s2m leads to its destabilization and impedes a complete NMR analysis. With Delta, a variant of concern has evolved with one mutation compared to the original sequence that replaces G29742 by U29742. We show here that this mutation results in a more defined structure at ambient temperature accompanied by a rise in melting temperature. Consequently, we were able to identify >90% of the relevant NMR resonances using a combination of selective RNA labeling and filtered 2D NOESY as well as 4D NMR experiments. We present a comprehensive NMR analysis of the secondary structure, (sub)nanosecond dynamics, and ribose conformation of s2m Delta based on heteronuclear 13C NOE and T 1 measurements and ribose carbon chemical shift-derived canonical coordinates. We further show that the G29742U mutation in Delta has no influence on the druggability of s2m compared to the Wuhan-Hu-1 sequence. With the assignment at hand, we identify the flexible regions of s2m as the primary site for small molecule binding.
Assuntos
Conformação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/química , SARS-CoV-2/metabolismo , RNA Viral/genética , RNA Viral/química , RNA Viral/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância Magnética/métodos , Regiões 3' não Traduzidas , Ligantes , Humanos , Mutação , COVID-19/virologia , Pareamento de Bases , Motivos de NucleotídeosRESUMO
UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.
Assuntos
Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Ciclopentanos/farmacologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pirimidinas/farmacologia , Estresse Fisiológico/fisiologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/biossíntese , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
The s2m, a highly conserved 41-nt hairpin structure in the SARS-CoV-2 genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and the subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a >99% correlation of a single nucleotide mutation at the 15th position (G15U) in Delta SARS-CoV-2. Based on 1H NMR spectroscopy assignments comparing the imino proton resonance region of s2m and the s2m G15U at 19°C, we show that the U15-A29 base pair closes, resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we show that the s2m G15U mutation drastically impacts the binding of host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on immune responses.
Assuntos
COVID-19 , MicroRNAs , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , COVID-19/genética , Dimerização , Mutação , MicroRNAs/metabolismoRESUMO
Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.
Assuntos
Meiose , Fuso Acromático , Feminino , Camundongos , Animais , Fuso Acromático/metabolismo , Metáfase , Oócitos/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas Repressoras/metabolismo , Cinesinas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Neddylation, a ubiquitylation-like post-translational modification, is catalyzed by a cascade composed of three enzymes: E1 activating enzyme, E2 conjugating enzyme, and E3 ligase with cullins as physiological substrates. Specifically, neddylation E2 UBE2M couples with E3 RBX1 to neddylate cullins 1-4, whereas neddylation E2 UBE2F couples with E3 RBX2/SAG to neddylate cullin 5, leading to activation of CRL1-4 (Cullin-RING ligases 1-4) and CRL5, respectively. While over-activation of the neddylation-CRLs axis occurs frequently in many human cancers, how neddylation-CRLs regulate the function of immune cells, particularly Treg cells was previously unknown. To this end, we recently performed Treg selective knockout of two neddylation E2s and two E3s, individually, driven by Foxp3-Cre, and found that while the Ube2f-Sag E2/E3 pair plays a minimal role, if any, the Ube2m-Rbx1 pair is essential for the maintenance of Treg functionality, since their deletion triggers robust inflammatory response with autoimmune phenotypes. Milder phenotype severity upon Treg KO of upstream Ube2m than that of downstream Rbx1 strongly suggested that Rbx1 regulates Treg function in a manner dependent and independent of neddylation.
Assuntos
Proteínas Culina , Linfócitos T Reguladores , Humanos , Proteínas Culina/genética , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Processamento de Proteína Pós-Traducional , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
COVID-19 is an acute respiratory disorder that is caused by SARS-CoV-2, in which excessive systemic inflammation is associated with adverse patient clinical outcomes. Here, we observed elevated expression levels of NLRP12 (nucleotide-binding leucine-rich repeat-containing receptor 12) in human peripheral monocytes and lung tissue during infection with SARS-CoV-2. Co-immunoprecipitation analysis revealed that NLRP12 directly interacted with the M protein through its leucine-rich repeat domain. Moreover, in vitro studies demonstrated that NLRP12 interacted with TRAF3 and promoted its ubiquitination and degradation, which counteracted the inhibitory effect of TRAF3 on the NF-κB/MAPK signaling pathway and promoted the production of inflammatory cytokines. Furthermore, an in vivo study revealed that NLRP12 knockout mice displayed attenuated tissue injury and ameliorated inflammatory responses in the lungs when infected with a SARS-CoV-2 M protein-reconstituted pseudovirus and mouse coronavirus. Taken together, these findings suggest that NLRP12 mediates the inflammatory responses during coronavirus infection.
Assuntos
COVID-19 , Fator 3 Associado a Receptor de TNF , Humanos , Animais , Camundongos , Fator 3 Associado a Receptor de TNF/metabolismo , SARS-CoV-2/metabolismo , Leucina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismoRESUMO
BACKGROUND: ZNF468 is a relatively unexplored gene that has been implicated in potential oncogenic properties in various cancer types. However, the exact role of ZNF468 in radiotherapy resistance of esophageal squamous cell carcinomas (ESCCs) is not well understood. METHODS: Bioinformatic analysis was performed using the TCGA database to assess ZNF468 expression and prognostic significance in pan-cancer and ESCC. Functional experiments were conducted using ZNF468 overexpressing and knockdown cell lines to assess its impact on cell survival, DNA damage response, cell cycle, and apoptosis upon radiation. A luciferase reporter assay was utilized to validate ZNF468 binding to the AURKA promoter. RESULTS: ZNF468 was significantly upregulated in diverse cancer types, including ESCC, and its high expression correlated with adverse prognosis in specific tumors. In the ESCC cohort, ZNF468 exhibited substantial upregulation in post-radiotherapy tissues, indicating its potential role in conferring radiotherapy resistance. Functional experiments revealed that ZNF468 enhances cell viability and facilitates DNA damage repair in radiotherapy-treated ESCC cells, while dampening the G2/M cell cycle arrest and apoptosis induced by radiation. Moreover, ZNF468 facilitated AURKA transcription, resulting in upregulated Aurora A expression, and subsequently inhibited P53 expression, unveiling key molecular mechanisms underlying radiotherapy resistance in ESCC. CONCLUSION: ZNF468 plays an oncogenic role in ESCC and contributes to radiotherapy resistance. It enhances cell survival while dampening radiation-induced G2/M cell cycle arrest and apoptosis. By modulating AURKA and P53 expression, ZNF468 represents a promising therapeutic target for enhancing radiotherapy efficacy in ESCC.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Apoptose/genética , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/radioterapia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Tolerância a Radiação/genética , Proteína Supressora de Tumor p53RESUMO
Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.
Assuntos
Vírus da Bronquite Infecciosa , Mamastrovirus , Mutagênese Insercional , Animais , Humanos , Regiões 3' não Traduzidas/genética , Galinhas/virologia , Vírus da Bronquite Infecciosa/genética , Mamastrovirus/genética , Mutagênese Insercional/genética , Doenças das Aves Domésticas/virologia , RNA Viral/genética , Replicação Viral/genética , Estabilidade de RNA/genética , Deleção de Sequência/genéticaRESUMO
The stem-loop II motif (s2m) is an RNA structural element that is found in the 3' untranslated region (UTR) of many RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Though the motif was discovered over 25 years ago, its functional significance is unknown. In order to understand the importance of s2m, we created viruses with deletions or mutations of the s2m by reverse genetics and also evaluated a clinical isolate harboring a unique s2m deletion. Deletion or mutation of the s2m had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also compared the secondary structure of the 3' UTR of wild-type and s2m deletion viruses using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) and dimethyl sulfate mutational profiling and sequencing (DMS-MaPseq). These experiments demonstrate that the s2m forms an independent structure and that its deletion does not alter the overall remaining 3'-UTR RNA structure. Together, these findings suggest that s2m is dispensable for SARS-CoV-2. IMPORTANCE RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), contain functional structures to support virus replication, translation, and evasion of the host antiviral immune response. The 3' untranslated region of early isolates of SARS-CoV-2 contained a stem-loop II motif (s2m), which is an RNA structural element that is found in many RNA viruses. This motif was discovered over 25 years ago, but its functional significance is unknown. We created SARS-CoV-2 with deletions or mutations of the s2m and determined the effect of these changes on viral growth in tissue culture and in rodent models of infection. Deletion or mutation of the s2m element had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also observed no impact of the deletion on other known RNA structures in the same region of the genome. These experiments demonstrate that s2m is dispensable for SARS-CoV-2.
Assuntos
Motivos de Nucleotídeos , SARS-CoV-2 , Animais , Cricetinae , Regiões 3' não Traduzidas/genética , COVID-19/virologia , Mesocricetus , Mutação , SARS-CoV-2/genética , Motivos de Nucleotídeos/genética , RNA Viral/química , RNA Viral/genéticaRESUMO
BACKGROUND: Our previous study demonstrated that ß2-microglobulin (ß2M) promoted ER+/HER2- breast cancer survival via the SGK1/Bcl-2 signaling pathway. However, the role of ß2M has not been investigated in ER-/HER2+ breast cancer. Here, we aimed to determine the role of ß2M in ER-/HER2+ breast cancer. METHODS: The interaction between ß2M and HFE was confirmed by co-immunoprecipitation, mass spectrometry, yeast two-hybrid screening, and His pull-down. The knockdown and overexpression of ß2M or HFE were performed in MDA-MB-453 cells, and ERK signaling pathway was subsequently analyzed via western blotting. Apoptotic cells were detected using flow cytometer. ß2M, HFE, and p-ERK1/2 were examined in tumor and paired adjacent tissues via immunohistochemistry. RESULTS: HFE was found to be an interacting protein of ß2M in ER-/HER2+ breast cancer cells MDA-MB-453 by co-immunoprecipitation and mass spectrometry. A yeast two-hybrid system and His-pull down experiments verified that ß2M directly interacted with HFE. ß2M and HFE as a complex were mainly located in the cytoplasm, with some on the cytomembrane of MDA-MB-453 cells. In addition to breast cancer cells BT474, endogenous ß2M directly interacted with HFE in breast cancer cells MDA-MB-453, MDA-MB-231, and MCF-7. ß2M activated the ERK signaling pathway by interacting with HFE and induced apoptosis of MDA-MB-453 cells. The expression of HFE and p-ERK1/2 showed significantly high levels in HER2-overexpressing breast cancer tumor tissue compared with adjacent normal tissue, consistent with the results obtained from the cell experiments. CONCLUSIONS: ß2M induced apoptosis of tumor cells via activation of the ERK signal pathway by directly interacting with HFE in HER2-overexpressing breast cancer.
Assuntos
Apoptose , Neoplasias da Mama , Proteína da Hemocromatose , Sistema de Sinalização das MAP Quinases , Receptor ErbB-2 , Microglobulina beta-2 , Humanos , Microglobulina beta-2/metabolismo , Microglobulina beta-2/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Linhagem Celular Tumoral , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Ligação Proteica , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant brain tumor, with a poor prognosis and life expectancy of 14-16 months after diagnosis. The standard treatment for GBM consists of surgery, radiotherapy, and chemotherapy with temozolomide. Most patients become resistant to treatment after some time, and the tumor recurs. Therefore, there is a need for new drugs to manage GBM. Eslicarbazepine (ESL) is a well-known antiepileptic drug belonging to the dibenzazepine group with anticancer potentials. In this study, for the first time, we evaluated the potential effects of ESL on C6 cell growth, both in vitro and in vivo, and examined its molecular effects. METHODS: To determine the effect of ESL on the c6 cell line, cell viability, proliferation, and migration were evaluated by MTT assay, colony formation, and wound healing assay. Also, apoptosis and cell cycle were examined by flow cytometry, qRT-PCR, and western blotting. In addition, an intracranial model in Wistar rats was used to investigate the effect of ESL in vivo, and the tumor size was measured using both Caliper and MRI. RESULTS: The obtained results are extremely consistent and highly encouraging. C6 cell viability, proliferation, and migration were significantly suppressed in ESL-treated C6 cells (p < 0.001), as determined by cell-based assays. ESL treatment led to significant enhancement of apoptosis (p < 0.01), as determined by flow cytometry, and upregulation of genes involved in cell apoptosis, such as the Bax/Bcl2 ratio at RNA (p < 0.05) and protein levels (5.37-fold). Flow cytometric analysis of ESL-treated cells revealed G2/M phase cell cycle arrest. ESL-treated cells demonstrated 2.49-fold upregulation of p21 alongside, 0.22-fold downregulation of cyclin B1, and 0.34-fold downregulation of cyclin-dependent kinase-1 at the protein level. Administration of ESL (30 mg/kg) to male rats bearing C6 intracranial tumors also suppressed the tumor volume and weight (p < 0.01). CONCLUSIONS: Based on these novel findings, ESL has the potential for further experimental and clinical studies in glioblastoma.