RESUMO
DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Animais , Coenzimas/química , Coenzimas/metabolismo , Ilhas de CpG , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Embrião de Mamíferos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Oócitos/citologia , Oócitos/enzimologia , Oócitos/crescimento & desenvolvimento , Transdução de Sinais , Espermatozoides/citologia , Espermatozoides/enzimologia , Espermatozoides/crescimento & desenvolvimentoRESUMO
Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Artefatos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Transferência de Genes , Genoma Humano , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , SoftwareRESUMO
Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.
Assuntos
Ritmo Circadiano/genética , Regulação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Peroxirredoxinas/metabolismo , Animais , Domínio Catalítico , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocôndrias/ultraestrutura , Mitose , Oxirredução , Peroxirredoxinas/genética , Fosforilação , Transdução de SinaisRESUMO
Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics.
Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Membranas Mitocondriais/ultraestrutura , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Biogênese de Organelas , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Precursores de Proteínas/química , Precursores de Proteínas/genética , Transporte ProteicoRESUMO
The identification of heterozygous mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) in subsets of cancers, including secondary glioblastoma, acute myeloid leukemia, intrahepatic cholangiocarcinoma, and chondrosarcomas, led to intense discovery efforts to delineate the mutations' involvement in carcinogenesis and to develop therapeutics, which we review here. The three IDH isoforms (nicotinamide adenine dinucleotide phosphate-dependent IDH1 and IDH2, and nicotinamide adenine dinucleotide-dependent IDH3) contribute to regulating the circuitry of central metabolism. Several biochemical and genetic observations led to the discovery of the neomorphic production of the oncometabolite (R)-2-hydroxyglutarate (2-HG) by mutant IDH1 and IDH2 (mIDH). Heterozygous mutation of IDH1/2 and accumulation of 2-HG cause profound metabolic and epigenetic dysregulation, including inhibition of normal cellular differentiation, leading to disease. Crystallographic structural studies during the development of compounds targeting mIDH demonstrated common allosteric inhibition by distinct chemotypes. Ongoing clinical trials in patients with mIDH advanced hematologic malignancies have demonstrated compelling clinical proof-of-concept, validating the biology and drug discovery approach.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Glutaratos/metabolismo , Isocitrato Desidrogenase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Acetamidas/síntese química , Acetamidas/uso terapêutico , Antineoplásicos/síntese química , Benzenoacetamidas/síntese química , Benzenoacetamidas/uso terapêutico , Benzimidazóis/síntese química , Benzimidazóis/uso terapêutico , Biomarcadores Tumorais/análise , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Expressão Gênica , Glutaratos/análise , Humanos , Imidazóis/síntese química , Imidazóis/uso terapêutico , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Modelos Moleculares , Mutação , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Pesquisa Translacional BiomédicaRESUMO
Recent developments indicate that macrodomains, an ancient and diverse protein domain family, are key players in the recognition, interpretation, and turnover of ADP-ribose (ADPr) signaling. Crucial to this is the ability of macrodomains to recognize ADPr either directly, in the form of a metabolic derivative, or as a modification covalently bound to proteins. Thus, macrodomains regulate a wide variety of cellular and organismal processes, including DNA damage repair, signal transduction, and immune response. Their importance is further indicated by the fact that dysregulation or mutation of a macrodomain is associated with several diseases, including cancer, developmental defects, and neurodegeneration. In this review, we summarize the current insights into macrodomain evolution and how this evolution influenced their structural and functional diversification. We highlight some aspects of macrodomain roles in pathobiology as well as their emerging potential as therapeutic targets.
Assuntos
Reparo do DNA , Proteínas de Escherichia coli/química , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/química , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/química , Viroses/enzimologia , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Animais , Dano ao DNA , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Família Multigênica , Neoplasias/química , Neoplasias/genética , Neoplasias/patologia , Filogenia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Viroses/genética , Viroses/patologia , Viroses/virologiaRESUMO
Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCß mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCß is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.
Assuntos
Proteína Quinase C/química , Proteína Quinase C/genética , Animais , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Genes Supressores de Tumor , Xenoenxertos , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos Nus , Modelos Moleculares , Mutação , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Quinase C/metabolismo , Estrutura Terciária de ProteínaRESUMO
Cancer cells must integrate multiple biosynthetic demands to drive indefinite proliferation. How these key cellular processes, such as metabolism and protein synthesis, crosstalk to fuel cancer cell growth is unknown. Here, we uncover the mechanism by which the Myc oncogene coordinates the production of the two most abundant classes of cellular macromolecules, proteins, and nucleic acids in cancer cells. We find that a single rate-limiting enzyme, phosphoribosyl-pyrophosphate synthetase 2 (PRPS2), promotes increased nucleotide biosynthesis in Myc-transformed cells. Remarkably, Prps2 couples protein and nucleotide biosynthesis through a specialized cis-regulatory element within the Prps2 5' UTR, which is controlled by the oncogene and translation initiation factor eIF4E downstream Myc activation. We demonstrate with a Prps2 knockout mouse that the nexus between protein and nucleotide biosynthesis controlled by PRPS2 is crucial for Myc-driven tumorigenesis. Together, these studies identify a translationally anchored anabolic circuit critical for cancer cell survival and an unexpected vulnerability for "undruggable" oncogenes, such as Myc. PAPERFLICK:
Assuntos
Carcinogênese , Nucleotídeos/biossíntese , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribose-Fosfato Pirofosfoquinase/genética , Regiões 5' não Traduzidas , Animais , Linfócitos B/metabolismo , Sequência de Bases , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células-Tronco Embrionárias , Fator de Iniciação 4E em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células NIH 3T3 , Ribose-Fosfato Pirofosfoquinase/metabolismoRESUMO
Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mitocôndrias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Treonina-tRNA Ligase/genética , Treonina/metabolismo , Regulação da Expressão Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais , Treonina-tRNA Ligase/antagonistas & inibidores , Treonina-tRNA Ligase/metabolismoRESUMO
Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.
Assuntos
Adenosina Desaminase/genética , Adenosina/metabolismo , Genoma , Inosina/metabolismo , Edição de RNA , RNA Mensageiro/genética , Adenosina Desaminase/metabolismo , Elementos Alu , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de SinaisRESUMO
The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.
Assuntos
Neoplasias da Mama/metabolismo , Deleção de Genes , Neoplasias Mamárias Experimentais/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Éxons , Feminino , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação , Metástase Neoplásica , Transplante de Neoplasias , Splicing de RNARESUMO
Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isoenzimas/genética , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Xenoenxertos , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Neuroglia/metabolismo , Neuroglia/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Quinases DyrkRESUMO
Sumoylation regulates many cellular processes, but its role in signaling via the T cell antigen receptor (TCR) remains unknown. We found that the kinase PKC-θ was sumoylated upon costimulation with antigen or via the TCR plus the coreceptor CD28, with Lys325 and Lys506 being the main sumoylation sites. We identified the SUMO E3 ligase PIASxß as a ligase for PKC-θ. Analysis of primary mouse and human T cells revealed that sumoylation of PKC-θ was essential for T cell activation. Desumoylation did not affect the catalytic activity of PKC-θ but inhibited the association of CD28 with PKC-θ and filamin A and impaired the assembly of a mature immunological synapse and central co-accumulation of PKC-θ and CD28. Our findings demonstrate that sumoylation controls TCR-proximal signaling and that sumoylation of PKC-θ is essential for the formation of a mature immunological synapse and T cell activation.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Linfócitos T/imunologia , Animais , Sítios de Ligação , Antígenos CD28/metabolismo , Diferenciação Celular , Células Cultivadas , Filaminas/metabolismo , Células HEK293 , Humanos , Sinapses Imunológicas/metabolismo , Isoenzimas/química , Isoenzimas/deficiência , Isoenzimas/genética , Células Jurkat , Ativação Linfocitária , Lisina/química , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteína Quinase C/química , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Proteína Quinase C-theta , Transdução de Sinais , Sumoilação , Linfócitos T/citologia , Células Th2/citologia , Células Th2/enzimologia , Células Th2/imunologiaRESUMO
Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.
Assuntos
Mucosa Intestinal/imunologia , Neoplasias Intestinais/imunologia , Isoenzimas/imunologia , Proteína Quinase C/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Vigilância Imunológica/genética , Vigilância Imunológica/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
Protein kinase C (PKC) isozymes function as tumor suppressors in increasing contexts. In contrast to oncogenic kinases, whose function is acutely regulated by transient phosphorylation, PKC is constitutively phosphorylated following biosynthesis to yield a stable, autoinhibited enzyme that is reversibly activated by second messengers. Here, we report that the phosphatase PHLPP1 opposes PKC phosphorylation during maturation, leading to the degradation of aberrantly active species that do not become autoinhibited. Cancer-associated hotspot mutations in the pseudosubstrate of PKCß that impair autoinhibition result in dephosphorylated and unstable enzymes. Protein-level analysis reveals that PKCα is fully phosphorylated at the PHLPP site in over 5,000 patient tumors, with higher PKC levels correlating (1) inversely with PHLPP1 levels and (2) positively with improved survival in pancreatic adenocarcinoma. Thus, PHLPP1 provides a proofreading step that maintains the fidelity of PKC autoinhibition and reveals a prominent loss-of-function mechanism in cancer by suppressing the steady-state levels of PKC.
Assuntos
Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Proteína Quinase C beta/genética , Proteína Quinase C-alfa/genética , Humanos , Isoenzimas/genética , Mutação com Perda de Função/genética , Neoplasias/patologia , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Controle de Qualidade , Transdução de Sinais/genéticaRESUMO
Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Replicação do DNA , Exodesoxirribonucleases/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sinalização do Cálcio/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/química , Cromatina/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains â¼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA â¼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.
Assuntos
Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA Intergênico/química , DNA/química , Streptococcus thermophilus/genética , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , DNA Intergênico/genética , DNA Intergênico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus thermophilus/metabolismo , Especificidade por SubstratoRESUMO
PKC is a multifunctional family of Ser-Thr kinases widely implicated in the regulation of fundamental cellular functions, including proliferation, polarity, motility, and differentiation. Notwithstanding their primary cytoplasmic localization and stringent activation by cell surface receptors, PKC isozymes impel prominent nuclear signaling ultimately impacting gene expression. While transcriptional regulation may be wielded by nuclear PKCs, it most often relies on cytoplasmic phosphorylation events that result in nuclear shuttling of PKC downstream effectors, including transcription factors. As expected from the unique coupling of PKC isozymes to signaling effector pathways, glaring disparities in gene activation/repression are observed upon targeting individual PKC family members. Notably, specific PKCs control the expression and activation of transcription factors implicated in cell cycle/mitogenesis, epithelial-to-mesenchymal transition and immune function. Additionally, PKCs isozymes tightly regulate transcription factors involved in stepwise differentiation of pluripotent stem cells toward specific epithelial, mesenchymal, and hematopoietic cell lineages. Aberrant PKC expression and/or activation in pathological conditions, such as in cancer, leads to profound alterations in gene expression, leading to an extensive rewiring of transcriptional networks associated with mitogenesis, invasiveness, stemness, and tumor microenvironment dysregulation. In this review, we outline the current understanding of PKC signaling "in" and "to" the nucleus, with significant focus on established paradigms of PKC-mediated transcriptional control. Dissecting these complexities would allow the identification of relevant molecular targets implicated in a wide spectrum of diseases.
Assuntos
Regulação da Expressão Gênica , Proteína Quinase C , Transdução de Sinais , Regulação da Expressão Gênica/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Fatores de Transcrição/metabolismo , Humanos , Animais , Núcleo Celular/enzimologia , Núcleo Celular/genéticaRESUMO
Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.
Assuntos
Glucuronosiltransferase , Isoenzimas , Microssomos Hepáticos , Proteínas Recombinantes , Animais , Humanos , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Glucuronosiltransferase/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/química , Isoenzimas/metabolismo , Isoenzimas/genética , Cinética , Microssomos Hepáticos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Magnetismo , Microssomos/química , Microssomos/metabolismoRESUMO
Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.