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The 21st Association for Cancer Immunotherapy (CIMT) Annual Meeting took place from May 15th to May 17th in Mainz, Germany, and was attended by a total of 855 academic and clinical professionals hailing from 33 different countries. The conference served as a platform for these experts to convene and discuss the latest breakthroughs in cancer immunology and immunotherapy research. Dedicated sessions covering advancements in artificial intelligence tools for cancer immunotherapy research, as well as the landscape of cancer care and cancer immunotherapy trials on the African continent, prompted lively and informative discussions among the attendees. This report aims to provide an overview of the most noteworthy highlights and key takeaways from CIMT2024.
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Imunoterapia , Neoplasias , Humanos , Neoplasias/terapia , Neoplasias/imunologia , Imunoterapia/métodos , Inteligência Artificial , AlemanhaRESUMO
Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors.
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Neoplasias Pancreáticas , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Neoplasias Pancreáticas/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Neoplasias PancreáticasRESUMO
The conserved Mre11-Rad50 complex is crucial for the detection, signaling, end tethering and processing of DNA double-strand breaks. While it is known that Mre11-Rad50 foci formation at DNA lesions accompanies repair, the underlying molecular assembly mechanisms and functional implications remained unclear. Combining pathway reconstitution in electron microscopy, biochemical assays and genetic studies, we show that S. cerevisiae Mre11-Rad50 with or without Xrs2 forms higher-order assemblies in solution and on DNA. Rad50 mediates such oligomerization, and mutations in a conserved Rad50 beta-sheet enhance or disrupt oligomerization. We demonstrate that Mre11-Rad50-Xrs2 oligomerization facilitates foci formation, DNA damage signaling, repair, and telomere maintenance in vivo. Mre11-Rad50 oligomerization does not affect its exonuclease activity but drives endonucleolytic cleavage at multiple sites on the 5'-DNA strand near double-strand breaks. Interestingly, mutations in the human RAD50 beta-sheet are linked to hereditary cancer predisposition and our findings might provide insights into their potential role in chemoresistance.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Hidrolases Anidrido Ácido/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.
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Amiloide/química , Proteínas de Ciclo Celular/química , Grânulos Citoplasmáticos/química , Piruvato Quinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Frutosedifosfatos/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Piruvato Quinase/química , Piruvato Quinase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The oxidative coupling of methane to ethylene using gaseous disulfur (2CH4 + S2 â C2H4 + 2H2S) as an oxidant (SOCM) proceeds with promising selectivity. Here, we report detailed experimental and theoretical studies that examine the mechanism for the conversion of CH4 to C2H4 over an Fe3O4-derived FeS2 catalyst achieving a promising ethylene selectivity of 33%. We compare and contrast these results with those for the highly exothermic oxidative coupling of methane (OCM) using O2 (2CH4 + O2 â C2H4 + 2H2O). SOCM kinetic/mechanistic analysis, along with density functional theory results, indicate that ethylene is produced as a primary product of methane activation, proceeding predominantly via CH2 coupling over dimeric S-S moieties that bridge Fe surface sites, and to a lesser degree, on heavily sulfided mononuclear sites. In contrast to and unlike OCM, the overoxidized CS2 by-product forms predominantly via CH4 oxidation, rather than from C2 products, through a series of C-H activation and S-addition steps at adsorbed sulfur sites on the FeS2 surface. The experimental rates for methane conversion are first order in both CH4 and S2, consistent with the involvement of two S sites in the rate-determining methane C-H activation step, with a CD4/CH4 kinetic isotope effect of 1.78. The experimental apparent activation energy for methane conversion is 66 ± 8 kJ/mol, significantly lower than for CH4 oxidative coupling with O2 The computed methane activation barrier, rate orders, and kinetic isotope values are consistent with experiment. All evidence indicates that SOCM proceeds via a very different pathway than that of OCM.
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Enhanced growth and proliferation of cancer cells are accompanied by profound changes in cellular metabolism. These metabolic changes are also common under physiological conditions, and include increased glucose fermentation accompanied by elevated cytosolic pH (pHc)1,2. However, how these changes contribute to enhanced cell growth and proliferation is unclear. Here, we show that elevated pHc specifically orchestrates an E2F-dependent transcriptional programme to drive cell proliferation by promoting cyclin D1 expression. pHc-dependent transcription of cyclin D1 requires the transcription factors CREB1, ATF1 and ETS1, and the histone acetyltransferases p300 and CBP. Biochemical characterization revealed that the CREB1-p300/CBP interaction acts as a pH sensor and coincidence detector, integrating different mitotic signals to regulate cyclin D1 transcription. We also show that elevated pHc contributes to increased cyclin D1 expression in malignant pleural mesotheliomas (MPMs), and renders these cells hypersensitive to pharmacological reduction of pHc. Taken together, these data demonstrate that elevated pHc is a critical cellular signal regulating G1 progression, and provide a mechanism linking elevated pHc to oncogenic activation of cyclin D1 in MPMs, and possibly other cyclin D1~dependent tumours. Thus, an increase of pHc may represent a functionally important, early event in the aetiology of cancer that is amenable to therapeutic intervention.
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Proliferação de Células , Ciclina D1/biossíntese , Citosol/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Ciclina D1/genética , Citosol/patologia , Citosol/fisiologia , Fatores de Transcrição E2F/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/genética , Mesotelioma/patologia , Metabolômica , Mitose/fisiologia , Frações Subcelulares/metabolismo , Fatores de TranscriçãoRESUMO
Parkinson's disease (PD) is a neurological disorder characterized by the progressive accumulation of neuronal α-synuclein (αSyn) inclusions called Lewy bodies. It is believed that Lewy bodies spread throughout the nervous system due to the cell-to-cell propagation of αSyn via cycles of secretion and uptake. Here, we investigated the internalization and intracellular accumulation of exogenous αSyn, two key steps of Lewy body pathogenesis, amplification and spreading. We found that stable αSyn fibrils substantially accumulate in different cell lines upon internalization, whereas αSyn monomers, oligomers, and dissociable fibrils do not. Our data indicate that the uptake-mediated accumulation of αSyn in a human-derived neuroblastoma cell line triggered an adaptive response that involved proteins linked to ubiquitin ligases of the S-phase kinase-associated protein 1 (SKP1), cullin-1 (Cul1), and F-box domain-containing protein (SCF) family. We found that SKP1, Cul1, and the F-box/LRR repeat protein 5 (FBXL5) colocalized and physically interacted with internalized αSyn in cultured cells. Moreover, the SCF containing the F-box protein FBXL5 (SCFFBXL5) catalyzed αSyn ubiquitination in reconstitution experiments in vitro using recombinant proteins and in cultured cells. In the human brain, SKP1 and Cul1 were recruited into Lewy bodies from brainstem and neocortex of patients with PD and related neurological disorders. In both transgenic and nontransgenic mice, intracerebral administration of exogenous αSyn fibrils triggered a Lewy body-like pathology, which was amplified by SKP1 or FBXL5 loss of function. Our data thus indicate that SCFFXBL5 regulates αSyn in vivo and that SCF ligases may constitute targets for the treatment of PD and other α-synucleinopathies.
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Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/metabolismo , Animais , Benzotiazóis/metabolismo , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Proteoma/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitina/metabolismoRESUMO
OBJECTIVES: We aimed to assess the safety and immunogenicity of a diphtheria/tetanus vaccine booster dose in three different patient groups with rheumatic diseases on a variety of immunosuppressive/immunomodulatory medications compared with healthy controls (HCs). METHODS: We conducted a multi-centre prospective cohort study in Switzerland. We enrolled patients with RA, axial SpA/PsA, vasculitis (Behçet's disease, ANCA-associated vasculitis) and HCs. Diphtheria/tetanus vaccination was administered according to the Swiss vaccination recommendations. Blood samples were drawn before vaccination, and 1 month and 3 months afterwards. Antibody concentrations against vaccine antigens were measured by ELISA. Immunogenicity was compared between patient and medication groups. A mixed model was applied for multivariate analysis. Missing data were dealt with using multiple imputation. RESULTS: Between January 2014 and December 2015, we enrolled 284 patients with rheumatic diseases (131 RA, 114 SpA/PsA, 39 vasculitis) and 253 HCs. Of the patients, 89% were on immunosuppressive/immunomodulatory medication. Three months post-vaccination 100% of HCs vs 98% of patients were protected against tetanus and 84% vs 73% against diphtheria. HCs and SpA/PsA patients had significantly higher responses than RA and vasculitis patients. Assessing underlying diseases and medications in a multivariate model, rituximab was the only factor negatively influencing tetanus immunogenicity, whereas only MTX treatment had a negative influence on diphtheria antibody responses. No vaccine-related serious adverse events were recorded. CONCLUSION: Diphtheria/tetanus booster vaccination was safe. Tetanus vaccination was immunogenic; the diphtheria component was less immunogenic. Vaccine responses were blunted by rituximab and MTX. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov, Identifier: NCT01947465.
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Anticorpos Antibacterianos/biossíntese , Vacina contra Difteria e Tétano/efeitos adversos , Imunogenicidade da Vacina/efeitos dos fármacos , Doenças Reumáticas/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Clostridium tetani/imunologia , Corynebacterium diphtheriae/imunologia , Difteria/prevenção & controle , Vacina contra Difteria e Tétano/imunologia , Feminino , Humanos , Imunização Secundária , Imunogenicidade da Vacina/imunologia , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Doenças Reumáticas/tratamento farmacológico , Tétano/prevenção & controle , Vacinação , Adulto JovemRESUMO
Protein aggregates, and in particular amyloids, are generally considered to be inherently irreversible aberrant clumps, and are often associated with pathologies, such as Alzheimer's disease, Parkinson's disease, or systemic amyloidosis. However, recent evidence demonstrates that some aggregates are not only fully reversible, but also perform essential physiological functions. Despite these new findings, very little is known about how these functional protein aggregates are regulated in a physiological context. Here, we take the yeast pyruvate kinase Cdc19 as an example of a protein forming functional, reversible, solid, amyloid-like aggregates in response to stress conditions. Cdc19 aggregation is regulated via an aggregation-prone low complexity region (LCR). In favorable growth conditions, this LCR is prevented from aggregating by phosphorylation or oligomerization, while upon glucose starvation it becomes exposed and allows aggregation. We suggest that LCR phosphorylation, oligomerization or partner-binding may be general and widespread mechanisms regulating LCR-mediated reversible protein aggregation. Moreover, we show that, as predicted by computational tools, Cdc19 forms amyloid-like aggregates in vitro. Interestingly, we also observe striking similarities between Cdc19 and its mammalian counterpart, PKM2. Indeed, also PKM2 harbors a LCR and contains several peptides with high amyloidogenic propensity, which coincide with known phosphorylation sites. Thus, we speculate that the formation of reversible, amyloid-like aggregates may be a general physiological mechanism for cells to adapt to stress conditions, and that the underlying regulatory mechanisms may be conserved from yeast to humans.
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Amiloide/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Humanos , Mamíferos/metabolismo , Agregados ProteicosRESUMO
In yeast, the glucose-induced degradation-deficient (GID) E3 ligase selectively degrades superfluous gluconeogenic enzymes. Here, we identified all subunits of the mammalian GID/CTLH complex and provide a comprehensive map of its hierarchical organization and step-wise assembly. Biochemical reconstitution demonstrates that the mammalian complex possesses inherent E3 ubiquitin ligase activity, using Ube2H as its cognate E2. Deletions of multiple GID subunits compromise cell proliferation, and this defect is accompanied by deregulation of critical cell cycle markers such as the retinoblastoma (Rb) tumor suppressor, phospho-Histone H3 and Cyclin A. We identify the negative regulator of pro-proliferative genes Hbp1 as a bonafide GID/CTLH proteolytic substrate. Indeed, Hbp1 accumulates in cells lacking GID/CTLH activity, and Hbp1 physically interacts and is ubiquitinated in vitro by reconstituted GID/CTLH complexes. Our biochemical and cellular analysis thus demonstrates that the GID/CTLH complex prevents cell cycle exit in G1, at least in part by degrading Hbp1.
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Proliferação de Células , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Fase G1 , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Camundongos Endogâmicos C57BL , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation.
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Basófilos/imunologia , Hipersensibilidade/genética , Imunoglobulina E/genética , Interleucina-3/genética , Receptores de IgE/genética , Animais , Basófilos/efeitos dos fármacos , Basófilos/patologia , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/fisiopatologia , Imunoglobulina E/imunologia , Interleucina-3/imunologia , Interleucina-3/farmacologia , Interleucina-5/genética , Interleucina-5/imunologia , Interleucina-5/farmacologia , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Receptores de IgE/deficiência , Receptores de IgE/imunologia , Transdução de Sinais , Transcrição GênicaRESUMO
It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.
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Adenocarcinoma de Células Claras/genética , Carcinoma Endometrioide/genética , Carcinossarcoma/genética , Neoplasias do Endométrio/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Acetanilidas/farmacologia , Adenocarcinoma de Células Claras/metabolismo , Animais , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Carcinoma Endometrioide/metabolismo , Carcinossarcoma/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Proteínas Culina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias do Endométrio/metabolismo , Epigênese Genética , Feminino , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Masculino , Espectrometria de Massas , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Transplante de Neoplasias , Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , UbiquitinaçãoRESUMO
Homologous recombination (HR) is a key pathway that repairs DNA double-strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L-TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L-TONSL associates with replication protein A (RPA)-coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR-mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L-TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51-ssDNA nucleoprotein filament formation and RAD51-dependent strand exchange activity in vitro Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L-TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR-mediated restart in vivo.
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Proteínas de Ligação a DNA/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Recombinação Genética , Dano ao DNA , Reparo do DNA , Replicação do DNA , Células HeLa , Humanos , Mapeamento de Interação de Proteínas , Multimerização ProteicaRESUMO
To maintain genome integrity and epigenetic information, mammalian cells must carefully coordinate the supply and deposition of histones during DNA replication. Here we report that the CUL4 E3 ubiquitin ligase complex CRL4(WDR23) directly regulates the stem-loop binding protein (SLBP), which orchestrates the life cycle of histone transcripts including their stability, maturation, and translation. Lack of CRL4(WDR23) activity is characterized by depletion of histones resulting in inhibited DNA replication and a severe slowdown of growth in human cells. Detailed analysis revealed that CRL4(WDR23) is required for efficient histone mRNA 3' end processing to produce mature histone mRNAs for translation. CRL4(WDR23) binds and ubiquitylates SLBP in vitro and in vivo, and this modification activates SLBP function in histone mRNA 3' end processing without affecting its protein levels. Together, these results establish a mechanism by which CUL4 regulates DNA replication and possible additional chromatin transactions by controlling the concerted expression of core histones.
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Proteínas de Transporte/metabolismo , Replicação do DNA , DNA/biossíntese , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fase S , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Proteínas de Transporte/genética , Montagem e Desmontagem da Cromatina , DNA/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Proteínas Nucleares/genética , Ligação Proteica , Processamento de Terminações 3' de RNA , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Complexos Ubiquitina-Proteína Ligase , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
DNA repair by homologous recombination is highly suppressed in G1 cells to ensure that mitotic recombination occurs solely between sister chromatids. Although many homologous recombination factors are cell-cycle regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 to constrain BRCA2 function to the S/G2 phases in human cells. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein, in complex with cullin-3 (CUL3)-RBX1 (ref. 6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA-end resection is sufficient to induce homologous recombination in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR-Cas9-based gene-targeting assay. We conclude that the mechanism prohibiting homologous recombination in G1 minimally consists of the suppression of DNA-end resection coupled with a multi-step block of the recruitment of BRCA2 to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce homologous recombination in G1 cells with defined factors could spur the development of gene-targeting applications in non-dividing cells.
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Fase G1 , Recombinação Homóloga , Sequência de Aminoácidos , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Culina/metabolismo , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi , Fase G2 , Marcação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo , Fase S , Tioléster Hidrolases/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Using abundant soft oxidants, a high methane-to-ethylene conversion might be achievable due to the low thermodynamic driving force for over-oxidation. Here we report on the oxidative coupling of methane by gaseous S2 (SOCM). The catalytic properties of Pd/Fe3O4 are compared with those of Fe3O4, and it is found that high ethylene selectivities can be achieved without noble metals; conversion and selectivity on Fe3O4 are stable for at least 48 h at SOCM conditions. SOCM data for 10 oxides are compared, and ethylene selectivities as high as 33% are found; the C2H4/C2H6 ratios of 9-12 observed at the highest S2 conversions are significantly higher than the C2H4/C2H6 ratios usually found in the CH4 coupling with O2. Complementary in-detail analytical studies show that, on Mg, Zr, Sm, W, and La catalysts, which strongly coke during the reaction, lower ethylene selectivities are observed than on Fe, Ti, and Cr catalysts, which only coke to a minor extent. Further catalyst-dependent changes during SOCM in surface area, surface composition, and partial conversion to oxysulfides and sulfides are discussed. Evidence concerning the reaction mechanism is obtained taking into account the selectivity for the different reaction products versus the contact time. CH4 coupling proceeds non-oxidatively with the evolution of H2 on some catalysts, and evidence is presented that C2H4 and C2H2 formation occur via C2H6 and C2H4 dehydrogenation, respectively.
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Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.
Assuntos
Diferenciação Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Biologia de Sistemas/métodos , Animais , Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
PROBLEM: Given the important role of regulatory T cells (Treg) for successful pregnancy, the ability of soluble maternal and fetal pregnancy factors to induce human Treg was investigated. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) or isolated CD4+CD25â cells were cultured in the presence of pooled second or third trimester pregnancy sera, steroid hormones or supernatants from placental explants, and the numbers and function of induced CD4+CD25+FOXP3+ Treg were analysed. RESULTS: Third trimester pregnancy sera and supernatants of early placental explants, but not sex steroid hormones, induced an increase of Tregs from PBMCs. Early placental supernatant containing high levels of tumour necrosis factor-α, interferon-γ, interleukins -1, -6 and -17, soluble human leucocyte antigen-G, and transforming growth factor-ß1, increased the proportion of Treg most effectively and was able to induce interleukin-10-secreting-Treg from CD4+CD25âcells. CONCLUSIONS: Compared with circulating maternal factors, placental- and fetal-derived factors appear to exert a more powerful effect on numerical changes of Treg, thereby supporting fetomaternal tolerance during human pregnancy.
Assuntos
Vilosidades Coriônicas/metabolismo , Citocinas/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Linfócitos T CD4-Positivos , Células Cultivadas , Estudos Transversais , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2 , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Gravidez , Segundo Trimestre da Gravidez , Terceiro Trimestre da GravidezRESUMO
Although chromosome partitioning during mitosis is well studied, the molecular mechanisms that allow proper segregation of cytoplasmic organelles in human cells are poorly understood. Here we show that mitochondria interact with growing microtubule tips and are transported towards the daughter cell periphery at the end of mitosis. This phenomenon is promoted by the direct and cell cycle-dependent interaction of the mitochondrial protein Miro and the cytoskeletal-associated protein Cenp-F. Cenp-F is recruited to mitochondria by Miro at the time of cytokinesis and associates with microtubule growing tips. Cells devoid of Cenp-F or Miro show decreased spreading of the mitochondrial network as well as cytokinesis-specific defects in mitochondrial transport towards the cell periphery. Thus, Miro and Cenp-F promote anterograde mitochondrial movement and proper mitochondrial distribution in daughter cells.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitose/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas dos Microfilamentos/genética , Microtúbulos/fisiologia , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Plasmídeos , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The mitotic spindle drives chromosome movement during mitosis and attaches to chromosomes at dedicated genomic loci named centromeres. Centromeres are epigenetically specified by their histone composition, namely the presence of the histone H3 variant CENP-A, which is regulated during the cell cycle by its dynamic expression and localization. Here, we combined biochemical methods and quantitative imaging approaches to investigate a new function of CUL4-RING E3 ubiquitin ligases (CRL4) in regulating CENP-A dynamics. We found that the core components CUL4 and DDB1 are required for centromeric loading of CENP-A, but do not influence CENP-A maintenance or pre-nucleosomal CENP-A levels. Interestingly, we identified RBBP7 as a substrate-specific CRL4 adaptor required for this process, in addition to its role in binding and stabilizing soluble CENP-A. Our data thus suggest that the CRL4 complex containing RBBP7 (CRL4(RBBP7)) might regulate mitosis by promoting ubiquitin-dependent loading of newly synthesized CENP-A during the G1 phase of the cell cycle.