RESUMO
Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.
Assuntos
Expressão Gênica , Proteínas Recombinantes de Fusão , Proteína X Associada a bcl-2 , Cristalografia por Raios X , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/isolamento & purificaçãoRESUMO
Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.
Assuntos
Proteínas de Bactérias/metabolismo , Listeria monocytogenes/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Motivos de Aminoácidos , Cristalografia por Raios X , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Glutationa/química , Glicina/química , Ligação Proteica , Multimerização Proteica , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitinação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/química , Genes Reporter , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Lisina/metabolismo , Fatores de Transcrição HES-1 , Fatores de Transcrição/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Earlier studies demonstrated the involvement of the p300 histone acetyltransferase in Notch signaling but the precise mechanisms by which p300 might modulate Notch function remains to be investigated. In this study, we show that p300 acetylates Notch1 ICD in cell culture assay and in vitro, and conserved lysines located within the Notch C-terminal nuclear localization signal are essential for Notch acetylation. MAML1 and CSL, which are components of the Notch transcription complex, enhance Notch acetylation and we suggest that MAML1 increases Notch acetylation by potentiating p300 autoacetylation. Furthermore, MAML1-dependent acetylation of Notch1 ICD by p300 decreases the ubiquitination of Notch1 ICD in cellular assays. CDK8 has been shown to target Notch1 for ubiquitination and proteosomal degradation. We show that CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. Therefore, we speculate that acetylation of Notch1 might be a mechanism to regulate Notch activity by interfering with ubiquitin-dependent pathways.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinação , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Quinase 8 Dependente de Ciclina/metabolismo , Células HEK293 , Humanos , Lisina/química , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
The Mastermindlike (MAML) family, comprising human MAML1, MAML2, and MAML3, are transcriptional regulators in Notch signaling. MAML proteins contain two consensus sites for SUMOylation at Lysine217 and Lysine299 that are conserved in humans, mice, and Xenopus. In this report, we show that MAML1 is SUMOylated at both sites. The E2-conjugating enzyme UBC9 is essential for MAML1 SUMOylation, and the E3 ligase PIAS1 stimulates this activity. Mutation of the lysines abolishes SUMOylation of MAML1 and strongly increases MAML1-activated transcription in cell culture assays. The protease SENP1 reverses SUMOylation of MAML1 and potentiates the transcription factor activity of MAML1. Furthermore, SUMOylation enhances MAML1 interaction with HDAC7, which decreases MAML1 transcriptional activity. Taken together, our data indicate that SUMOylation of MAML1 is a mechanism for repressing MAML1 activity by influencing its interaction with HDAC7.
Assuntos
Proteínas de Ligação a DNA/genética , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Proteínas Nucleares , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Glycogen synthase kinase 3beta (GSK3beta) is involved in several cellular signaling systems through regulation of the activity of diverse transcription factors such as Notch, p53 and beta-catenin. Mastermind-like 1 (MAML1) was originally identified as a Notch coactivator, but has also been reported to function as a transcriptional coregulator of p53, beta-catenin and MEF2C. In this report, we show that active GSK3beta directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity, suggesting that GSK3beta might target a coactivator in its regulation of gene expression. We have previously shown that MAML1 increases global acetylation of histones, and here we show that the GSK3 inhibitor SB41, further enhances MAML1-dependent histone acetylation in cells. Finally, MAML1 translocates GSK3beta to nuclear bodies; this function requires full-length MAML1 protein.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Glicogênio Sintase Quinase 3 beta , Histonas/metabolismo , Humanos , Estrutura Terciária de Proteína , Receptor Notch1/metabolismo , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
MAML1 is a transcriptional coregulator originally identified as a Notch coactivator. MAML1 is also reported to interact with other coregulator proteins, such as CDK8 and p300, to modulate the activity of Notch. We, and others, previously showed that MAML1 recruits p300 to Notch-regulated genes through direct interactions with the DNA-CSL-Notch complex and p300. MAML1 interacts with the C/H3 domain of p300, and the p300-MAML1 complex specifically acetylates lysines of histone H3 and H4 tails in chromatin in vitro. In this report, we show that MAML1 potentiates p300 autoacetylation and p300 transcriptional activation. MAML1 directly enhances p300 HAT activity, and this coincides with the translocation of MAML1, p300 and acetylated histones to nuclear bodies.
Assuntos
Transativadores/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Linhagem Celular , Histonas/metabolismo , Estrutura Terciária de Proteína , Deleção de Sequência , Transativadores/química , Transativadores/genética , Fatores de Transcrição de p300-CBP/químicaRESUMO
Amyotrophic lateral sclerosis is a neurodegenerative syndrome associated with 114 mutations in the gene encoding the cytosolic homodimeric enzyme Cu/Zn superoxide dismutase (SOD). In this article, we report that amyotrophic lateral sclerosis-associated SOD mutations with distinctly different disease progression can be rationalized in terms of their folding patterns. The mutations are found to perturb the protein in multiple ways; they destabilize the precursor monomers (class 1), weaken the dimer interface (class 2), or both at the same time (class 1 + 2). A shared feature of the mutational perturbations is a shift of the folding equilibrium toward poorly structured SOD monomers. We observed a link, coupled to the altered folding patterns, between protein stability, net charge, and survival time for the patients carrying the mutations.
Assuntos
Esclerose Lateral Amiotrófica/genética , Variação Genética , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Dobramento de Proteína , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/classificação , Cromatografia em Gel , Dimerização , Progressão da Doença , Humanos , Cinética , Prognóstico , Superóxido Dismutase-1RESUMO
The molecular mechanism by which the homodimeric enzyme Cu/Zn superoxide dismutase (SOD) causes neural damage in amytrophic lateral sclerosis is yet poorly understood. A striking, as well as an unusual, feature of SOD is that it maintains intrasubunit disulfide bonds in the reducing environment of the cytosol. Here, we investigate the role of these disulfide bonds in folding and assembly of the SOD apo protein (apoSOD) homodimer through extensive protein engineering. The results show that apoSOD folds in a simple three-state process by means of two kinetic barriers: 2D<==>2M<==>M(2). The early predominant barrier represents folding of the monomers (M), and the late barrier the assembly of the dimer (M(2)). Unique for this mechanism is a dependence of protein concentration on the unfolding rate constant under physiological conditions, which disappears above 6 M Urea where the transition state for unfolding shifts to first-order dissociation of the dimer in accordance with Hammond-postulate behavior. Although reduction of the intrasubunit disulfide bond C57-C146 is not critical for folding of the apoSOD monomer, it has a pronounced effect on its stability and abolishes subsequent dimerization. Thus, impaired ability to form, or retain, the C57-C146 bond in vivo is predicted to increase the cellular load of marginally stable apoSOD monomers, which may have implications for the amytrophic lateral sclerosis neuropathology.
Assuntos
Superóxido Dismutase/química , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/etiologia , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Cisteína/química , Dimerização , Dissulfetos/química , Estabilidade Enzimática , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Engenharia de Proteínas , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents. We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents. This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu(2+) and Zn(2+). The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein. The enzymes were purified to homogeneity and active enzymes were obtained in high yield. The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy.
Assuntos
Cobre/química , Escherichia coli/enzimologia , Superóxido Dismutase/química , Superóxido Dismutase/fisiologia , Western Blotting , Citosol/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Imunoquímica , Espectrometria de Massas , Plasmídeos/metabolismo , Proteínas/química , Proteínas Recombinantes/química , Saccharomyces cerevisiae/enzimologia , Espectrofotometria Atômica , Superóxido Dismutase/metabolismo , Zinco/químicaRESUMO
More than 100 point mutations of the superoxide scavenger Cu/Zn superoxide dismutase (SOD; EC ) have been associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, these mutations are scattered throughout the protein and provide no clear functional or structural clues to the underlying disease mechanism. Therefore, we undertook to look for folding-related defects by comparing the unfolding behavior of five ALS-associated mutants with distinct structural characteristics: A4V at the interface between the N and C termini, C6F in the hydrophobic core, D90A at the protein surface, and G93A and G93C, which decrease backbone flexibility. With the exception of the disruptive replacements A4V and C6F, the mutations only marginally affect the stability of the native protein, yet all mutants share a pronounced destabilization of the metal-free apo state: the higher the stability loss, the lower the mean survival time for ALS patients carrying the mutation. Thus organism-level pathology may be directly related to the properties of the immature state of a protein rather than to those of the native species.
Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação , Superóxido Dismutase/química , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/etiologia , Dicroísmo Circular , Humanos , Fosfinas , Desnaturação Proteica , Dobramento de ProteínaRESUMO
An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.