RESUMEN
Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.
Asunto(s)
Retrovirus Endógenos/genética , Histona Demetilasas/metabolismo , Complejo Silenciador Inducido por ARN/genética , Animales , Línea Celular Tumoral , Cromatina , Terapia Combinada , Regulación de la Expresión Génica/genética , Histona Demetilasas/genética , Humanos , Inmunidad Celular , Inmunoterapia , Interferón Tipo I , Células MCF-7 , Ratones , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , ARN Bicatenario/genética , Linfocitos TRESUMEN
Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino- or carboxyl-termini are eliminated by the N- or C-degron pathways, respectively. We aimed to elucidate the function of C-degron pathways and to unveil how normal proteomes are exempt from C-degron pathway-mediated destruction. Our data reveal that C-degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C-degron and N-degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C-degron pathways, but not of defective proteins, suggesting proteolysis-based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.
Asunto(s)
Proteolisis , Ubiquitinación , Secuencias de Aminoácidos , Línea Celular Tumoral , Células HEK293 , Humanos , Transporte de Proteínas , Proteoma/química , Proteoma/metabolismo , Receptores de Citocinas/metabolismoRESUMEN
Metastasis is the major cause of cancer death. An increased level of circulating tumor cells (CTCs), metastatic cancer cells that have intravasated into the circulatory system, is particularly associated with colonization of distant organs and poor prognosis. However, the key factors required for tumor cell dissemination and colonization remain elusive. We found that high expression of desmoglein2 (DSG2), a component of desmosome-mediated intercellular adhesion complexes, promoted tumor growth, increased the prevalence of CTC clusters, and facilitated distant organ colonization. The dynamic regulation of DSG2 by hypoxia was key to this process, as down-regulation of DSG2 in hypoxic regions of primary tumors led to elevated epithelial-mesenchymal transition (EMT) gene expression, allowing cells to detach from the primary tumor and undergo intravasation. Subsequent derepression of DSG2 after intravasation and release of hypoxic stress was associated with an increased ability to colonize distant organs. This dynamic regulation of DSG2 was mediated by Hypoxia-Induced Factor1α (HIF1α). In contrast to its more widely observed function to promote expression of hypoxia-inducible genes, HIF1α repressed DSG2 by recruitment of the polycomb repressive complex 2 components, EZH2 and SUZ12, to the DSG2 promoter in hypoxic cells. Consistent with our experimental data, DSG2 expression level correlated with poor prognosis and recurrence risk in breast cancer patients. Together, these results demonstrated the importance of DSG2 expression in metastasis and revealed a mechanism by which hypoxia drives metastasis.
Asunto(s)
Neoplasias de la Mama/genética , Desmogleína 2/genética , Transición Epitelial-Mesenquimal/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/genética , Recurrencia Local de Neoplasia/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Desmogleína 2/antagonistas & inhibidores , Desmogleína 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/metabolismo , Hipoxia/mortalidad , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Metástasis Linfática , Ratones , Ratones SCID , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Negative Pressure Wound Therapy (NPWT) is a commonly employed clinical strategy for wound healing, yet its early-stage mechanisms remain poorly understood. To address this knowledge gap and overcome the limitations of human trials, we establish an NPWT C57BL/6JNarl mouse model to investigate the molecular mechanisms involved in NPWT. In this study, we investigate the intricate molecular mechanisms through which NPWT expedites wound healing. Our focus is on NPWT's modulation of inflammatory immune responses and the concurrent orchestration of multiple signal transduction pathways, resulting in shortened coagulation time and reduced inflammation. Notably, we observe a significant rise in dickkopf-related protein 1 (DKK-1) concentration during NPWT, promoting the differentiation of Hair Follicle Stem Cells (HFSCs) into epidermal cells, expediting wound closure. Under negative pressure, macrophages express and release DKK-1 cytokines, crucial for stimulating HFSC differentiation, as validated in animal experiments and in vitro studies. Our findings illuminate the inflammatory dynamics under NPWT, revealing potential signal transduction pathways. The proposed framework, involving early hemostasis, balanced inflammation, and macrophage-mediated DKK-1 induction, provides a novel perspective on enhancing wound healing during NPWT. Furthermore, these insights lay the groundwork for future pharmacological advancements in managing extensive wounds, opening avenues for targeted therapeutic interventions in wound care.
Asunto(s)
Terapia de Presión Negativa para Heridas , Humanos , Ratones , Animales , Terapia de Presión Negativa para Heridas/métodos , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Cicatrización de Heridas , Inflamación/terapiaRESUMEN
BACKGROUND: Emerging evidence suggests that DNA methylation can be affected by physical activities and is associated with cardiac fibrosis. This translational research examined the implications of DNA methylation associated with the high-intensity interval training (HIIT) effects on cardiac fibrosis in patients with heart failure (HF). METHODS: Twelve HF patients were included and received cardiovascular magnetic resonance imaging with late gadolinium enhancement for cardiac fibrosis severity and a cardiopulmonary exercise test for peak oxygen consumption ([Formula: see text]O2peak). Afterwards, they underwent 36 sessions of HIIT at alternating 80% and 40% of [Formula: see text]O2peak for 30 min per session in 3-4 months. Human serum from 11 participants, as a means to link cell biology to clinical presentations, was used to investigate the exercise effects on cardiac fibrosis. Primary human cardiac fibroblasts (HCFs) were incubated in patient serum, and analyses of cell behaviour, proteomics (n = 6) and DNA methylation profiling (n = 3) were performed. All measurements were conducted after completing HIIT. RESULTS: A significant increase (p = 0.009) in [Formula: see text]O2peak (pre- vs. post-HIIT = 19.0 ± 1.1 O2 ml/kg/min vs. 21.8 ± 1.1 O2 ml/kg/min) was observed after HIIT. The exercise strategy resulted in a significant decrease in left ventricle (LV) volume by 15% to 40% (p < 0.05) and a significant increase in LV ejection fraction by approximately 30% (p = 0.010). LV myocardial fibrosis significantly decreased from 30.9 ± 1.2% to 27.2 ± 0.8% (p = 0.013) and from 33.4 ± 1.6% to 30.1 ± 1.6% (p = 0.021) in the middle and apical LV myocardium after HIIT, respectively. The mean single-cell migration speed was significantly (p = 0.044) greater for HCFs treated with patient serum before (2.15 ± 0.17 µm/min) than after (1.11 ± 0.12 µm/min) HIIT. Forty-three of 1222 identified proteins were significantly involved in HIIT-induced altered HCF activities. There was significant (p = 0.044) hypermethylation of the acyl-CoA dehydrogenase very long chain (ACADVL) gene with a 4.474-fold increase after HIIT, which could activate downstream caspase-mediated actin disassembly and the cell death pathway. CONCLUSIONS: Human investigation has shown that HIIT is associated with reduced cardiac fibrosis in HF patients. Hypermethylation of ACADVL after HIIT may contribute to impeding HCF activities. This exercise-associated epigenetic reprogramming may contribute to reduce cardiac fibrosis and promote cardiorespiratory fitness in HF patients. TRIAL REGISTRATION: NCT04038723. Registered 31 July 2019, https://clinicaltrials.gov/ct2/show/NCT04038723 .
Asunto(s)
Insuficiencia Cardíaca , Entrenamiento de Intervalos de Alta Intensidad , Humanos , Entrenamiento de Intervalos de Alta Intensidad/métodos , Metilación de ADN/genética , Medios de Contraste , Gadolinio , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Consumo de OxígenoRESUMEN
BACKGROUND: Human polyomavirus BK (BKPyV) causes associated nephropathy and contributes to urinary tract cancer development in renal transplant recipients. Large tumor antigen (LT) is an early protein essential in the polyomavirus life cycle. Protein acetylation plays a critical role in regulating protein stability, so this study investigated the acetylation of the BKPyV LT protein. METHODS: The BKPyV LT nucleotide was synthesized, and the protein was expressed by transfection into permissive cells. The BKPyV LT protein was immunoprecipitated and subjected to LC-MS/MS analysis to determine the acetylation residues. The relative lysine was then mutated to arginine in the LT nucleotide and BKPyV genome to analyze the role of LT lysine acetylation in the BKPyV life cycle. RESULTS: BKPyV LT acetylation sites were identified at Lys3 and Lys230 by mass spectrometry. HDAC3 and HDAC8 and their deacetylation activity are required for BKPyV LT expression. In addition, mutations of Lys3 and Lys230 to arginine increased LT expression, and the interaction of HDAC3 and LT was confirmed by coimmunoprecipitation. CONCLUSIONS: HDAC3 is a newly identified protein that interacts with BKPyV LT, and LT acetylation plays a vital role in the BKPyV life cycle.
Asunto(s)
Virus BK , Trasplante de Riñón , Infecciones por Polyomavirus , Poliomavirus , Infecciones Tumorales por Virus , Humanos , Virus BK/genética , Trasplante de Riñón/efectos adversos , Lisina , Cromatografía Liquida , Espectrometría de Masas en Tándem , Antígenos de Neoplasias , Estabilidad Proteica , Histona Desacetilasas/genética , Proteínas RepresorasRESUMEN
This corrects the article DOI: 10.1038/nature21671.
RESUMEN
Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.
Asunto(s)
Daño del ADN/efectos de la radiación , Metilación , ARN/química , ARN/metabolismo , Rayos Ultravioleta , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Biocatálisis/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Metilación/efectos de la radiación , Metiltransferasas/deficiencia , Metiltransferasas/metabolismo , Ratones , Poli A/metabolismo , ARN/efectos de la radiación , Fase S/efectos de la radiaciónRESUMEN
How ACE2 functions as the major host receptor of SARS-CoV-2 despite having low expression in the lungs is still unknown. To facilitate the development of therapeutic strategies against coronaviruses, gaining a deeper comprehension of the molecular mechanism of SARS-CoV-2 infection is imperative. In our previous study, we identified several potential host factors of SARS-CoV-2 using an shRNA arrayed screen, one of which was Wnt3a. Here, we validated the significance of Wnt3a, a potent activator of the Wnt/ß-catenin signaling pathway, for SARS-CoV-2 entry into cells by evaluating the effects of its knockdown and overexpression on SARS-CoV-2 pseudotyped virus entry. Further analysis revealed that SARS-CoV-2 pseudotyped virus infection activates the canonical Wnt/ß-catenin signaling pathway, which we found could subsequently stimulate ACE2 transcription. Collectively, our study identified Wnt3a as an important host factor that facilitates ACE2-mediated virus infection. Insight into the virus entry mechanism is impactful as it will aid in developing novel therapeutic strategies against current and future coronavirus pandemics.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2/genética , Pandemias , ARN Interferente PequeñoRESUMEN
Vibrio vulnificus is a pathogen that accounts for one of the highest mortality rates and is responsible for most reported seafood-related illnesses and deaths worldwide. Owing to the threats of pathogens with ß-lactamase activity, it is important to identify and characterize ß-lactamases with clinical significance. In this study, the protein sequence of the metallo-ß-lactamase (MBL) fold metallohydrolase from V. vulnificus (designated Vmh) was analyzed, and its oligomeric state, ß-lactamase activity, and metal binding ability were determined. BLASTp analysis indicated that the V. vulnificus Vmh protein showed no significant sequence identity with any experimentally identified Ambler class B MBLs or enzymes containing the MBL protein fold; it was also predicted to have a signal peptide of 19 amino acids at its N terminus and an MBL protein fold from amino acid residues 23 to 216. Recombinant V. vulnificus Vmh protein was overexpressed and purified. Analytical ultracentrifugation and electrospray ionization-mass spectrometry (MS) data demonstrated its monomeric state in an aqueous solution. Recombinant V. vulnificus Vmh protein showed broad degrading activities against ß-lactam antibiotics, such as penicillins, cephalosporins, and imipenems, with kcat/Km values ranging from 6.23 × 102 to 1.02 × 104 M-1 s-1. The kinetic reactions of this enzyme exhibited sigmoidal behavior, suggesting the possibility of cooperativity. Zinc ions were required for the enzyme activity, which was abolished by adding the metal chelator EDTA. Inductively coupled plasma-MS indicated that this enzyme might bind two zinc ions per molecule as a cofactor.
Asunto(s)
Vibrio vulnificus , beta-Lactamasas , Antibacterianos/farmacología , Cefalosporinas , Monobactamas , Vibrio vulnificus/genética , Inhibidores de beta-Lactamasas , beta-Lactamasas/genéticaRESUMEN
NPGPx is a member of the glutathione peroxidase (GPx) family; however, it lacks GPx enzymatic activity due to the absence of a critical selenocysteine residue, rendering its function an enigma. Here, we show that NPGPx is a newly identified stress sensor that transmits oxidative stress signals by forming the disulfide bond between its Cys57 and Cys86 residues. This oxidized form of NPGPx binds to glucose-regulated protein (GRP)78 and forms covalent bonding intermediates between Cys86 of NPGPx and Cys41/Cys420 of GRP78. Subsequently, the formation of the disulfide bond between Cys41 and Cys420 of GRP78 enhances its chaperone activity. NPGPx-deficient cells display increased reactive oxygen species, accumulated misfolded proteins, and impaired GRP78 chaperone activity. Complete loss of NPGPx in animals causes systemic oxidative stress, increases carcinogenesis, and shortens life span. These results suggest that NPGPx is essential for releasing excessive ER stress by enhancing GRP78 chaperone activity to maintain physiological homeostasis.
Asunto(s)
Proteínas Portadoras/metabolismo , Estrés del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Estrés Oxidativo , Peroxidasas/metabolismo , Deficiencias en la Proteostasis/enzimología , Transducción de Señal , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Cisteína , Daño del ADN , Disulfuros/metabolismo , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Fibroblastos/enzimología , Fibroblastos/patología , Glutatión Peroxidasa , Proteínas de Choque Térmico/genética , Homeostasis , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación , Oxidantes/farmacología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Peroxidasas/genética , Unión Proteica , Pliegue de Proteína , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , TransfecciónRESUMEN
To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.
Asunto(s)
Ciclo Celular/genética , Diferenciación Celular/genética , Proteínas de Drosophila/genética , Proteínas del Grupo Polycomb/genética , Factores de Transcripción/genética , Animales , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Chaperonas de Histonas/biosíntesis , Chaperonas de Histonas/genética , Histonas/genética , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Mutación , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Proteínas del Grupo Polycomb/biosíntesis , Factores de Transcripción/biosíntesisRESUMEN
While numerous small ubiquitin-like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid-dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ-K-X-E/D consensus motif, such as CBP and Elk-1, but not substrates with core ψ-K-X-E/D motif alone or SUMO-interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia-elicited modulation of sumoylation and target gene expression of CBP and Elk-1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.
Asunto(s)
Acetiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína SUMO-1/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/fisiología , Acetilación , Acetiltransferasas/fisiología , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Células Cultivadas , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/fisiología , ARN Interferente Pequeño/farmacología , Sirtuina 1/metabolismo , Sirtuina 1/fisiología , Sumoilación/efectos de los fármacos , Sumoilación/genética , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Enzimas Ubiquitina-Conjugadoras/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Non-selenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx or GPx7) is an oxidative stress sensor that modulates the antioxidative activity of its target proteins through intermolecular disulfide bond formation. Given NPGPx's role in protecting cells from oxidative damage, identification of the oxidative stress-induced protein complexes, which forms with key stress factors, may offer novel insight into intracellular reactive oxygen species homeostasis. Here, we show that NPGPx forms a disulfide bond with the translational regulator cytoplasmic polyadenylation element-binding protein 2 (CPEB2) that results in negative regulation of hypoxia-inducible factor 1-alpha (HIF-1α) RNA translation. In NPGPx-proficient cells, high oxidative stress that disrupts this bonding compromises the association of CPEB2 with HIF-1α RNA, leading to elevated HIF-1α RNA translation. NPGPx-deficient cells, in contrast, demonstrate increased HIF-1α RNA translation under normoxia with both impaired induction of HIF-1α synthesis and blunted HIF-1α-programmed transcription following oxidative stress. Together, these results reveal a molecular mechanism for how NPGPx mediates CPEB2-controlled HIF-1α RNA translation in a redox-sensitive manner.
Asunto(s)
Proteínas Portadoras/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Estrés Oxidativo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas Portadoras/genética , Células Cultivadas , Cisteína/análisis , Disulfuros/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Ratas , Transcripción GenéticaRESUMEN
O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) is the only known enzyme that catalyzes the O-GlcNAcylation of proteins at the Ser or Thr side chain hydroxyl group. OGT participates in transcriptional and epigenetic regulation, and dysregulation of OGT has been implicated in diseases such as cancer. However, the underlying mechanism is largely unknown. Here we show that OGT is required for the trimethylation of histone 3 at K27 to form the product H3K27me3, a process catalyzed by the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the polycomb repressive complex 2 (PRC2). H3K27me3 is one of the most important histone modifications to mark the transcriptionally silenced chromatin. We found that the level of H3K27me3, but not other H3 methylation products, was greatly reduced upon OGT depletion. OGT knockdown specifically down-regulated the protein stability of EZH2, without altering the levels of H3K27 demethylases UTX and JMJD3, and disrupted the integrity of the PRC2 complex. Furthermore, the interaction of OGT and EZH2/PRC2 was detected by coimmunoprecipitation and cosedimentation experiments. Importantly, we identified that serine 75 is the site for EZH2 O-GlcNAcylation, and the EZH2 mutant S75A exhibited reduction in stability. Finally, microarray and ChIP analysis have characterized a specific subset of potential tumor suppressor genes subject to repression via the OGT-EZH2 axis. Together these results indicate that OGT-mediated O-GlcNAcylation at S75 stabilizes EZH2 and hence facilitates the formation of H3K27me3. The study not only uncovers a functional posttranslational modification of EZH2 but also reveals a unique epigenetic role of OGT in regulating histone methylation.
Asunto(s)
Acetilglucosamina/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Metilación de ADN , Regulación hacia Abajo , Proteína Potenciadora del Homólogo Zeste 2 , Técnicas de Silenciamiento del Gen , Genes Supresores de Tumor , Humanos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Estabilidad ProteicaRESUMEN
The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.
Asunto(s)
Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Fosfotreonina/metabolismo , Secuencia de Aminoácidos , Western Blotting , Calorimetría , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masas , Datos de Secuencia Molecular , FosforilaciónRESUMEN
Iron was previously shown to induce rapid nuclear translocation of a Myb3 transcription factor in the protozoan parasite, Trichomonas vaginalis. In the present study, iron was found to induce a transient increase in cellular cAMP, followed by the nuclear influx of Myb3, whereas the latter was also induced by 8-bromo-cyclic AMP. Iron-inducible cAMP production and nuclear influx of Myb3 were inhibited by suramin and SQ22536, respective inhibitors of the Gα subunit of heterotrimeric G proteins and adenylyl cyclases. In contrast, the nuclear influx of Myb3 induced by iron or 8-bromo-cAMP was delayed or inhibited, respectively, by H89, the inhibitor of protein kinase A. Using liquid chromatography-coupled tandem mass spectrometry, Thr(156) and Lys(143) in Myb3 were found to be phosphorylated and ubiquitinated, respectively. These modifications were induced by iron and inhibited by H89, as shown by immunoprecipitation-coupled Western blotting. Iron-inducible ubiquitination and nuclear influx were aborted in T156A and K143R, but T156D was constitutively ubiquitinated and persistently localized to the nucleus. Myb3 was phosphorylated in vitro by the catalytic subunit of a T. vaginalis protein kinase A, TvPKAc. A transient interaction between TvPKAc and Myb3 and the phosphorylation of both proteins were induced in the parasite shortly after iron or 8-bromo-cAMP treatment. Together, these observations suggest that iron may induce production of cAMP and activation of TvPKAc, which then induces the phosphorylation of Myb3 and subsequent ubiquitination for accelerated nuclear influx. It is conceivable that iron probably exerts a much broader impact on the physiology of the parasite than previously thought to encounter environmental changes.
Asunto(s)
Núcleo Celular/metabolismo , Hierro/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Lisina/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/genética , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Ubiquitina/metabolismoRESUMEN
Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.
Asunto(s)
Apoptosis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Neoplasias/metabolismo , Empalme del ARN/genética , Alas de Animales/anomalías , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Larva/crecimiento & desarrollo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Fenotipo , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión , Alas de Animales/crecimiento & desarrolloRESUMEN
The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type ß-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glicósido Hidrolasas/metabolismo , Pseudomonas/enzimología , Sefarosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico , Galactósidos/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Oligosacáridos/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Pseudomonas/genéticaRESUMEN
Germ line mutations of the BRCA1 gene increase the risk of breast and ovarian cancer, but the basis of this tissue-specific tumor predisposition is not fully understood. Previously, we reported that the progesterone receptors are stabilized in Brca1-deficient mammary epithelial cells, and treating with anti-progesterone delays mammary tumorigenesis in Brca1/p53 conditional knock-out mice, suggesting that the progesterone has a critical role in breast carcinogenesis. To further explore how the stability of progesterone receptor is modulated, here, we have found that glycogen synthase kinase (GSK)-3ß phosphorylation of progesterone receptor-A (PR-A) facilitates its ubiquitination. GSK-3ß-mediated phosphorylation of serine 390 in PR-A regulates its subsequent ubiquitination and protein stability. Expression of PR-A(S390A) mutant in the human breast epithelial cells, MCF-10A, results in enhanced proliferation and formation of aberrant acini structure in the three-dimensional culture. Consistently, reduction of phosphorylation of serine 390 of PR-A and GSK-3ß activity is observed in the Brca1-deficient mammary gland. Taken together, these results provide important aspects of tissue specificity of BRCA1-mediated suppression of breast carcinogenesis.