RESUMO
Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor kappaB (NFkappaB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks, but we have found that genetic as well as acute deficiency of RIP140 leads to the inhibition of the proinflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFkappaB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína de Ligação a CREB/metabolismo , Citocinas/genética , Regulação da Expressão Gênica/imunologia , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Humanos , Inflamação/genética , Camundongos , Camundongos Knockout , NF-kappa B/imunologia , Proteína 1 de Interação com Receptor Nuclear , Ligação Proteica/imunologia , Fator de Transcrição RelA/imunologiaRESUMO
Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin-proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin-proteasome pathway constitutes the underlying regulatory system.
Assuntos
Ciclo Celular , DNA Glicosilases/metabolismo , Reparo do DNA , Timina DNA Glicosilase/metabolismo , Uracila-DNA Glicosidase/metabolismo , Linhagem Celular , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fase S , Ubiquitina/metabolismoRESUMO
Genetic screens are powerful tools to identify components that make up biological systems. Perturbations introduced by methods such as RNA interference (RNAi) or CRISPR/Cas9-mediated genome editing lead to biological phenotypes that can be examined to understand the molecular function of genes in the cell. Over the years, many of such experiments have been conducted providing a wealth of knowledge about genotype-to-phenotype relationships. These data are a rich source of information and it is in a common interest to make them available in a simplified and integrated format. Thus, an important challenge is that genetic screening data can be stored in databases in standardized ways, allowing users to gain new biological insights through data mining and integrated analyses. Here, we provide an overview of available phenotype databases for human cells. We review in detail two databases for high-throughput screens, GenomeRNAi and GenomeCRISPR, and describe how these resources are integrated into the German Network for Bioinformatics Infrastructure de.NBI as part of the European infrastructure for life-science information ELIXIR.
Assuntos
Biologia Computacional , Bases de Dados Genéticas , Testes Genéticos , Técnicas Citológicas , Humanos , FenótipoRESUMO
Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G.T and G.U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species-specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4- ethenocytosine opposite guanine and 5-fluorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5'-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual species.
Assuntos
Drosophila melanogaster/enzimologia , Endodesoxirribonucleases/metabolismo , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Animais , Pareamento Incorreto de Bases/genética , DNA/genética , DNA/metabolismo , Reparo do DNA , DNA Complementar/genética , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/genética , Escherichia coli/genética , Humanos , Cinética , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Filogenia , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Uracila/metabolismoRESUMO
Exocyclic adducts of DNA bases, such as etheno- and hydroxyalkano- ones, are generated by a variety of bifunctional agents, including endogenously formed products of lipid peroxidation. In this work we selectively modified cytosines in the 5'-d(TTT TTT CTT TTT CTT TTT CTT TTT T)-3' oligonucleotide using: chloroacetaldehyde to obtain 3,N(4)-alpha-hydroxyethano- (HEC) and 3,N(4)-etheno- (epsilonC), acrolein to obtain 3,N(4)-alpha-hydroxypropano- (HPC) and crotonaldehyde to obtain 3,N(4)-alpha-hydroxy-gamma-methylpropano- (mHPC) adducts of cytosine. The studied adducts are alkali-labile which results in oligonucleotide strain breaks at the sites of modification upon strong base treatment. The oligonucleotides carrying adducted cytosines were studied as substrates of Escherichia coli Mug, human TDG and fission yeast Thp1p glycosylases. All the adducts studied are excised by bacterial Mug although with various efficiency: epsilonC >HEC >HPC >mHPC. The yeast enzyme excises efficiently epsilonC>HEC>HPC, whereas the human enzyme excises only epsilonC. The pH-dependence curves of excision of eC, HEC and HPC by Mug are bell shaped and the most efficient excision of adducts occurs within the pH range of 8.6-9.6. The observed increase of excision of HEC and HPC above pH 7.2 can be explained by deprotonation of these adducts, which are high pK(a) compounds and exist in a protonated form at neutrality. On the other hand, since epsilonC is in a neutral form in the pH range studied, we postulate an involvement of an additional catalytic factor. We hypothesize that the enzyme structure undergoes a pH-induced rearrangement allowing the participation of Lys68 of Mug in catalysis via a hydrogen bond interaction of its epsilon-amino group with N(4) of the cytosine exocyclic adducts.
Assuntos
Pareamento Incorreto de Bases , Citosina/metabolismo , DNA Glicosilases/metabolismo , Timina DNA Glicosilase/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Citosina/química , Concentração de Íons de Hidrogênio , Uracila-DNA GlicosidaseRESUMO
OBJECTIVE: In mammals, proper storage and distribution of lipids in and between tissues is essential for the maintenance of energy homeostasis. In contrast, aberrantly high levels of triglycerides in the blood ("hypertriglyceridemia") represent a hallmark of the metabolic syndrome and type 2 diabetes. As hypertriglyceridemia has been identified as an important risk factor for cardiovascular complications, in this study we aimed to identify molecular mechanisms in aberrant triglyceride elevation under these conditions. RESEARCH DESIGN AND METHODS: To determine the importance of hepatic lipid handling for systemic dyslipidemia, we profiled the expression patterns of various hepatic lipid transporters and receptors under healthy and type 2 diabetic conditions. A differentially expressed lipoprotein receptor was functionally characterized by generating acute, liver-specific loss- and gain-of-function animal models. RESULTS: We show that the hepatic expression of lipid transporter lipolysis-stimulated lipoprotein receptor (LSR) is specifically impaired in mouse models of obesity and type 2 diabetes and can be restored by leptin replacement. Experimental imitation of this pathophysiological situation by liver-specific knockdown of LSR promotes hypertriglyceridemia and elevated apolipoprotein (Apo)B and E serum levels in lean wild-type and ApoE knockout mice. In contrast, genetic restoration of LSR expression in obese animals to wild-type levels improves serum triglyceride levels and serum profiles in these mice. CONCLUSIONS: The dysregulation of hepatic LSR under obese and diabetic conditions may provide a molecular rationale for systemic dyslipidemia in type 2 diabetes and the metabolic syndrome and represent a novel target for alternative treatment strategies in these patients.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Hiperlipidemias/fisiopatologia , Fígado/fisiologia , Receptores de LDL/genética , Receptores de Lipoproteínas/fisiologia , Animais , Apolipoproteínas E/deficiência , Glicemia/metabolismo , Colesterol/sangue , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/sangue , Corpos Cetônicos/sangue , Lipólise , Lipoproteínas VLDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Interferência de RNA , Receptores de LDL/deficiência , Receptores de LDL/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/sangueRESUMO
Eukaryotic RNA polymerases are multisubunit assemblies, whose enzymatic function in the nucleus is intensively studied. However, little is known about the biogenesis of the three RNA polymerases and coupling to nucleo-cytoplasmic transport. Here, we show that Rpc128, the second largest subunit of RNA polymerase III, was mislocalized to the cytoplasm, when a short sequence in the N-terminal domain was deleted. Importantly, nuclear import of other, but not all, RNA polymerase III subunits was impaired in this RPC128DeltaN mutant. These data suggest that RNA polymerase III subunits are not imported independently into the nucleus but may require preassembly into cytoplasmic subcomplexes for coordinated nuclear uptake. We expect these studies to be a starting point to dissect the complex biogenesis pathway of eukaryotic RNA polymerases.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Subunidades Proteicas/metabolismo , RNA Polimerase III/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos , Sinais Direcionadores de Proteínas , Subunidades Proteicas/genética , RNA Polimerase III/genética , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
DNA glycosylases initiate base excision repair (BER) through the generation of potentially harmful abasic sites (AP sites) in DNA. Human thymine-DNA glycosylase (TDG) is a mismatch-specific uracil/thymine-DNA glycosylase with an implicated function in the restoration of G*C base pairs at sites of cytosine or 5-methylcytosine deamination. The rate-limiting step in the action of TDG in vitro is its dissociation from the product AP site, suggesting the existence of a specific enzyme release mechanism in vivo. We show here that TDG interacts with and is covalently modified by the ubiquitin-like proteins SUMO-1 and SUMO-2/3. SUMO conjugation dramatically reduces the DNA substrate and AP site binding affinity of TDG, and this is associated with a significant increase in enzymatic turnover in reactions with a G*U substrate and the loss of G*T processing activity. Sumoylation also potentiates the stimulatory effect of APE1 on TDG. These observations implicate a function of sumoylation in the controlled dissociation of TDG from the AP site and open up novel perspectives for the understanding of the molecular mechanisms coordinating the early steps of BER.
Assuntos
Reparo do DNA , Endodesoxirribonucleases/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Ubiquitinas/metabolismo , Sítios de Ligação , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/genética , Células HeLa , Humanos , Lisina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Ubiquitinas/genéticaRESUMO
The ubiquitin-related protein SUMO-1 is covalently attached to proteins by SUMO-1 ligases. We have performed a proteome-wide analysis of sumoylated substrate proteins in yeast. Employing the powerful affinity purification of Protein A-Smt3 (Smt3 is the yeast homologue of SUMO-1) from yeast lysates in combination with tandem liquid chromatography mass spectrometry, we have isolated potential Smt3-carrying substrate proteins involved in DNA replication and repair, chromatin remodeling, transcription activation, Pol-I, Pol-II, and Pol-III transcription, 5' pre-mRNA capping, 3' pre-mRNA processing, proteasome function, and tubulin folding. Employing tandem affinity purifications or a rapid biochemical assay referred to as "SUMO fingerprint," we showed that several subunits of RNA polymerases I, II, and III, members of the transcription repression and chromatin remodeling machineries previously not known to be sumoylated, are modified by SUMO-1. Thus, the identification of a broad range of SUMO-1 substrate proteins is expected to lead to further insight into the regulatory aspects of sumoylation.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/fisiologia , Proteômica/métodos , Proteína SUMO-1/análise , Proteínas de Saccharomyces cerevisiae , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Proteínas Fúngicas/análise , Fenômenos Genéticos , Espectrometria de Massas , Proteínas Repressoras , Proteína SUMO-1/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Proteína Estafilocócica ARESUMO
We describe a novel regulatory mechanism for DNA polymerase beta (Polbeta), a protein involved in DNA base excision repair (BER). Polbeta colocalized in vivo and formed a complex with the transcriptional coactivator p300. p300 interacted with Polbeta through distinct domains and acetylated Polbeta in vitro. Polbeta acetylation was furthermore observed in vivo. Lysine 72 of Polbeta was identified as the main target for acetylation by p300. Interestingly, acetylated Polbeta showed a severely reduced ability to participate in a reconstituted BER assay. This was due to an impairment of the dRP-lyase activity of Polbeta. Acetylation of Polbeta thus acts as an intranuclear regulatory mechanism and implies that p300 plays a critical regulatory role in BER.