RESUMO
BACKGROUND: Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. RESULTS: At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10 mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. CONCLUSION: Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.
Assuntos
Complexo Mycobacterium avium , Infecção por Mycobacterium avium-intracellulare , Humanos , Idoso , Complexo Mycobacterium avium/genética , Transcriptoma , Infecção por Mycobacterium avium-intracellulare/microbiologia , Perfilação da Expressão Gênica , Estresse Oxidativo , Nitrogênio , EnxofreRESUMO
Helicobacter pylori is a bacterium that causes gastritis, peptic ulcer disease and adenocarcinoma while infecting human stomach. In the stomach H. pylori is under stresses caused by reactive oxygen and nitrogen species from host immune response, which causes oxidative DNA damage. The DNA damage in single base is repaired by base excision repair (BER) and/or nucleotide incision repair (NIR) pathways. H. pylori retains a minimal set of enzymes involved in the BER and NIR pathways. The HP1526 protein is a single apurinic/apyrimidinic (AP) endonuclease homologous to E. coli Xth protein but little is known for its structure up to now. In this study, the structure of the recombinant HP1526 protein from H. pylori (HpXthA) has been determined at a high resolution of 1.84 Å. From the structural analysis the HpXthA was found to belong to the Xth-like AP endonuclease family carrying the common fold of a central bilayer ß-sheet flanked by α-helices with a divalent metal ion bound. A Mn2+ ion and a 1,3-butanediol were unusually found and modeled around the active site. Structural and sequence comparisons among the AP endonucleases show well-conserved residues for metal and DNA binding and for catalysis. Interestingly, the presence of a small polar residue Ser201 of the HpXthA commonly found in NIR-proficient AP endonucleases instead of an aspartate residue in NIR-deficient enzymes suggests that the HpXthA retain a nucleotide incision repair activity.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Helicobacter pylori , Humanos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Helicobacter pylori/metabolismo , Escherichia coli/metabolismo , Dano ao DNA , Reparo do DNA , Nucleotídeos , Endonucleases/metabolismoRESUMO
BACKGROUND AND AIMS: Nuclear targeting of bacterial proteins has a significant impact on host cell pathology. Helicobacter pylori have many nuclear targeting proteins that translocate into the nucleus of host cells. H. pylori HP0425, annotated as hypothetical, has a nuclear localization signal (NLS) sequence, but its function has not been demonstrated. The aim of this experiment was to address the nuclear translocation of HP0425 and determine the effect of HP0425 pathology on host cells. MATERIALS AND METHODS: To investigate the nuclear localization of HP0425, it was expressed in AGS and MKN-1 cells as a GFP fusion protein (pEGFP-HP0425), and its localization was analyzed by confocal microscopy. Recombinant HP0425 (rHP0425) protein was overproduced as a GST fusion protein in Escherichia coli and purified by glutathione-affinity column chromatography. Purified rHP0425 was examined for cytotoxicity and DNase activity. RESULTS: The pEGFP-HP0425 fluorescence was expressed in the nucleus and cytosol fraction of cells, while it was localized in the cytoplasm in the negative control. This protein exhibited DNase activity under various conditions, with the highest DNase activity in the presence of manganese. In addition, the rHP0425 protein efficiently decreased cell viability in a concentration-dependent manner. CONCLUSIONS: These results suggest that HP0425 carrying a nuclear localization signal sequence translocates into the nucleus of host cells and degrades genomic DNA by DNase I-like enzymatic activity, which is a new pathogenic strategy of H. pylori in the host.
Assuntos
Núcleo Celular/microbiologia , Desoxirribonuclease I/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Sinais de Localização Nuclear , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Núcleo Celular/enzimologia , Citoplasma/metabolismo , Desoxirribonuclease I/genética , Proteínas de Fluorescência Verde , Helicobacter pylori/enzimologia , Humanos , Microscopia Confocal , Transporte Proteico , Proteínas Recombinantes de FusãoRESUMO
Methionine aminopeptidases (MetAPs), ubiquitous enzymes that play an important role in nascent protein maturation, have been recognized as attractive targets for the development of drugs against pathogenic protozoa including Plasmodium spp. Here, we characterized partial biochemical properties of a type I MetAP of Plasmodium vivax (PvMetAP1). PvMetAP1 had the typical amino acid residues essential for metal binding and substrate binding sites, which are well conserved in the type I MetAP family enzymes. Recombinant PvMetAP1 showed activity in a broad range of neutral pHs, with optimum activity at pH 7.5. PvMetAP1 was stable under neutral and alkaline pHs, but was relatively unstable under acidic conditions. PvMetAP1 activity was highly increased in the presence of Mn(2+), and was effectively inhibited by a metal chelator, EDTA. Fumagillin and aminopeptidase inhibitors, amastatin and bestatin, also showed an inhibitory effect on PvMetAP1. The enzyme had a highly specific hydrolytic activity for N-terminal methionine. These results collectively suggest that PvMetAP1 belongs to the family of type I MetAPs and may play a pivotal role for the maintenance of P. vivax physiology by mediating protein maturation and processing of the parasite.
Assuntos
Expressão Gênica , Metionil Aminopeptidases/biossíntese , Metionil Aminopeptidases/química , Plasmodium vivax/enzimologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Leucina/análogos & derivados , Leucina/química , Manganês/química , Metionil Aminopeptidases/genética , Peptídeos/química , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro compared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn(2+) binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.
Assuntos
Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mamíferos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fósforo-Oxigênio Liases/genética , Filogenia , Conformação Proteica , Pterinas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria. Subsequent bioactivity-guided fractionation of this extract led to six cinnamic amides (1-6) and ferulic acid (7). Compound 6 emerged as new compound possessing the very rare carbinolamide motif. These compounds (1-7) were evaluated for severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro inhibitory activity to identify their potencies and kinetic behavior. Compounds (1-6) displayed significant inhibitory activity with IC50 values in the range 15.8-70.1 µM. The new cinnamic amide 6 was found to be most potent inhibitor with an IC50 of 15.8 µM. In kinetic studies, all inhibitors exhibited mixed type inhibition. Furthermore, the most active PLpro inhibitors (1-6) were proven to be present in the native fruits in high quantities by HPLC chromatogram and liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS).
Assuntos
Cinamatos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Extratos Vegetais/química , Tribulus/química , Proteínas Virais/antagonistas & inibidores , Amidas , Cinamatos/isolamento & purificação , Cinamatos/uso terapêutico , Proteases 3C de Coronavírus , Cisteína Endopeptidases/genética , Inibidores de Cisteína Proteinase/isolamento & purificação , Inibidores de Cisteína Proteinase/uso terapêutico , Relação Dose-Resposta a Droga , Escherichia coli/genética , Frutas/química , Humanos , Concentração Inibidora 50 , Cinética , Estrutura Molecular , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/virologia , Relação Estrutura-Atividade , Proteínas Virais/genéticaRESUMO
Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro) is a key enzyme that plays an important role in SARS virus replication. The ethanol extract of the seeds of Psoralea corylifolia showed high activity against the SARS-CoV PLpro with an IC50 of value of 15 µg/ml. Due to its potency, subsequent bioactivity-guided fractionation of the ethanol extract led to six aromatic compounds (1-6), which were identified as bavachinin (1), neobavaisoflavone (2), isobavachalcone (3), 4'-O-methylbavachalcone (4), psoralidin (5) and corylifol A (6). All isolated flavonoids (1-6) inhibited PLpro in a dose-dependent manner with IC50 ranging between 4.2 and 38.4 µM. Lineweaver-Burk and Dixon plots and their secondary replots indicated that inhibitors (1-6) were mixed inhibitors of PLpro. The analysis of KI and KIS values proved that the two most promising compounds (3 and 5) had reversible mixed type I mechanisms.
Assuntos
Fenóis/farmacologia , Psoralea/embriologia , Sementes/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacosRESUMO
CsStefin-2, the second cysteine protease inhibitor of Clonorchis sinensis, was identified and characterized. CsStefin-2 is a cysteine protease inhibitor that belongs to family 1 stefins based on its phylogenetic and structural properties. However, CsStefin-2 had a QIVSG cystatin motif distinct from the common QVVAG cystatin motif that is well conserved in family 1 stefins. Mutagenesis analysis revealed that the two amino acid substitutions in the QIVSG cystatin motif of CsStefin-2 did not affect its inhibitory activity. Molecular modeling also indicated that no critical change was induced in the interaction between CsStefin-2 and its target enzyme. CsStefin-2 showed broad inhibitory activities against several cysteine proteases, including human cathepsins B and L, papain, and cathepsin Fs of C. sinensis (CsCFs), and effectively inhibited the autocatalytic maturation of CsCF-6. Native CsStefin-2 was assembled into a homo-tetramer, in which intermolecular disulfide bonds are not involved in the assembly of the tetramer. CsStefin-2 was expressed throughout the various developmental stages of the parasite and was localized in the intestinal epithelium, where CsCFs are actively synthesized. These results suggest that CsStefin-2 is the second active cysteine protease inhibitor of C. sinensis that shares functional redundancy with CsStefin-1 to modulate the activity and processing of CsCFs.
Assuntos
Clonorchis sinensis/genética , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Proteínas de Helminto/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Clonagem Molecular , Clonorchis sinensis/metabolismo , Cistatinas/genética , Inibidores de Cisteína Proteinase/genética , Proteínas de Helminto/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Papaína/antagonistas & inibidores , Filogenia , Estrutura Terciária de ProteínaRESUMO
Given the intricate etiology and pathogenesis of atopic dermatitis (AD), the complete cure of AD remains challenging. This study aimed to investigate if topically applying N-benzyl-N-methyldecan-1-amine (BMDA), derived from garlic, and its derivative [decyl-(4-methoxy-benzyl)-methyl-1-amine] (DMMA) could effectively alleviate AD-like skin lesions in 2,4-dinitrochlorobenzene (DNCB)-treated mice. Administering these compounds to the irritated skin of DNCB-treated mice significantly reduced swelling, rash, and excoriation severity, alongside a corresponding decrease in inflamed epidermis and dermis. Moreover, they inhibited spleen and lymph node enlargement and showed fewer infiltrated mast cells in the epidermis and dermis through toluidine-blue staining. Additionally, they led to a lower IgE titer in mouse sera as determined by ELISA, compared to vehicle treatment. Analyzing skin tissue from the mice revealed decreased transcript levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6), IL-4, iNOS, and COX-2, compared to control mice. Simultaneously, the compounds impeded the activation of inflammation-related signaling molecules such as JNK, p38 MAPK, and NF-κB in the mouse skin. In summary, these findings suggest that BMDA and DMMA hold the potential to be developed as a novel treatment for healing inflammatory AD.
Assuntos
Dermatite Atópica , Alho , Anidridos Maleicos , Animais , Camundongos , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Dinitroclorobenzeno/toxicidade , Pele/patologia , Citocinas , Aminas/farmacologia , NF-kappa B/farmacologia , Camundongos Endogâmicos BALB CRESUMO
SARS-CoV papain-like protease (PLpro) is an important antiviral target due to its key roles in SARS virus replication. The MeOH extracts of the fruits of the Paulownia tree yielded many small molecules capable of targeting PLpro. Five of these compounds were new geranylated flavonoids, tomentin A, tomentin B, tomentin C, tomentin D, tomentin E (1-5). Structure analysis of new compounds (1-5) by NMR showed that they all contain a 3,4-dihydro-2H-pyran moiety. This chemotype is very rare and is derived from cyclization of a geranyl group with a phenol functionality. Most compounds (1-12) inhibited PLpro in a dose dependent manner with IC50's raging between 5.0 and 14.4 µM. All new compounds having the dihydro-2H-pyran group showed better inhibition than their parent compounds (1 vs 11, 2 vs 9, 4 vs 12, 5 vs 6). In kinetic studies, 1-12 emerged to be reversible, mixed inhibitors.
Assuntos
Antivirais/química , Flavonoides/química , Magnoliopsida/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas Virais/antagonistas & inibidores , Antivirais/isolamento & purificação , Proteases 3C de Coronavírus , Cisteína Endopeptidases/química , Escherichia coli/genética , Flavonoides/isolamento & purificação , Frutas/química , Cinética , Extratos Vegetais/química , Proteínas Recombinantes/química , Proteínas Virais/químicaRESUMO
Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32â M MgCl2, 0.08â M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a=97.11, b=135.92, c=46.03â Å. Diffraction data were collected to 2.08â Å resolution.
Assuntos
Methanosarcina/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/metabolismo , Catálise , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trometamina/análogos & derivados , Difração de Raios XRESUMO
A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.22 M NaCl, 0.1 M bis-tris pH 6.5, 21% polyethylene glycol 3350. AtDJ-1D crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.78, b = 75.21, c = 141.68 Å, ß = 96.87°, and contained a trimer in the asymmetric unit. Diffraction data were collected to 2.05 Å resolution. The structure of AtDJ-1D has been determined using the multiple-wavelength anomalous dispersion (MAD) method.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Cristalização , Cristalografia por Raios XRESUMO
Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2-4 (MP2-4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.
RESUMO
Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.
Assuntos
Biopterinas/análogos & derivados , Cianobactérias/enzimologia , Microbiologia Ambiental , Glucose/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Xilose/metabolismo , Sequência de Aminoácidos , Biopterinas/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Análise por Conglomerados , Cianobactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Glicosiltransferases/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Dictyostelium discoideum phenylalanine hydroxylase (DicPAH; residues 1-415) was expressed in Escherichia coli and purified for structural analysis. Apo DicPAH and DicPAH complexed with dihydrobiopterin (BH(2)) and Fe(III) were crystallized using 0.06 M PIPES pH 7.0, 26%(w/v) PEG 2000 by the hanging-drop vapour-diffusion method. Crystals of apo DicPAH and the DicPAH-BH(2)-Fe(III) complex diffracted to 2.6 and 2.07 A resolution, respectively, and belonged to space group P2(1), with unit-cell parameters a = 70.02, b = 85.43, c = 74.86 A, beta = 110.12 degrees and a = 70.97, b = 85.33, c = 74.89 A, beta = 110.23 degrees , respectively. There were two molecules in the asymmetric unit. The structure of DicPAH has been solved by molecular replacement.
Assuntos
Biopterinas/análogos & derivados , Dictyostelium/enzimologia , Compostos Férricos/química , Fenilalanina Hidroxilase/química , Biopterinas/química , Biopterinas/metabolismo , Cristalização , Cristalografia por Raios X , Compostos Férricos/metabolismo , Fenilalanina Hidroxilase/isolamento & purificação , Fenilalanina Hidroxilase/metabolismo , Ligação ProteicaRESUMO
p23 is a heat shock protein 90 (Hsp90) co-chaperone and stabilizes the Hsp90 heterocomplex in mammals and yeast. In this study, we isolated a complementary DNA (cDNA) encoding p23 from orchardgrass (Dgp23) and characterized its functional roles under conditions of thermal stress. Dgp23 is a 911 bp cDNA with an open reading frame predicted to encode a 180 amino acid protein. Northern analysis showed that expression of Dgp23 transcripts was heat inducible. Dgp23 has a well-conserved p23 domain and interacted with an orchardgrass Hsp90 homolog in vivo, like mammalian and yeast p23 homologs. Recombinant Dgp23 is a small acidic protein with a molecular mass of approximately 27 kDa and pI 4.3. Dgp23 was also shown to function as a chaperone protein by suppression of malate dehydrogenase thermal aggregation. Differential scanning calorimetry thermograms indicated that Dgp23 is a heat-stable protein, capable of increasing the T (m) of lysozyme. Moreover, overexpression of Dgp23 in a yeast p23 homolog deletion strain, Deltasba1, increased cell viability. These results suggest that Dgp23 plays a role in thermal stress-tolerance and functions as a co-chaperone of Hsp90 and as a chaperone.
Assuntos
Dactylis/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Animais , Dactylis/genética , Teste de Complementação Genética , Proteínas de Choque Térmico HSP90/genética , Temperatura Alta , Humanos , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Plantas/genética , Desnaturação Proteica , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
The iron superoxide dismutase found in the pathogenic amoeba Acanthamoeba castellanii (AcFeSOD) may play essential roles in the survival of the parasite, not only by protecting it from endogenous oxidative stress but also by detoxifying oxidative killing of the parasite by host immune effector cells. The AcFeSOD protein was expressed in a stable form using an Escherichia coli expression system and was crystallized by the microbatch and hanging-drop vapour-diffusion methods. The structure was determined to 2.33â Å resolution from a single AcFeSOD crystal. The crystal belonged to the hexagonal space group P61 and contained 12 molecules forming three tetramers in the asymmetric unit, with an iron ion bound in each molecule. Structural comparisons and sequence alignment of AcFeSOD with other FeSODs showed a well conserved overall fold and conserved active-site residues with subtle differences.
Assuntos
Acanthamoeba castellanii/enzimologia , Superóxido Dismutase/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
Snail is a key regulator of epithelial-mesenchymal transition (EMT), which is a major step in tumor metastasis. Although the induction of Snail transcription precedes EMT, posttranslational regulation, especially phosphorylation of Snail, is critical for determining Snail protein levels or stability, subcellular localization, and the ability to induce EMT. To date, several kinases are known that enhance the stability of Snail by preventing its ubiquitination; however, the molecular mechanism(s) underlying this are still unclear. Here, we identified p38 MAPK as a crucial posttranslational regulator that enhances the stability of Snail. p38 directly phosphorylated Snail at Ser107, and this effectively suppressed DYRK2-mediated Ser104 phosphorylation, which is critical for GSK3ß-dependent Snail phosphorylation and ßTrCP-mediated Snail ubiquitination and degradation. Importantly, functional studies and analysis of clinical samples established a crucial role for the p38-Snail axis in regulating ovarian cancer EMT and metastasis. These results indicate the potential therapeutic value of targeting the p38-Snail axis in ovarian cancer. SIGNIFICANCE: These findings identify p38 MAPK as a novel regulator of Snail protein stability and potential therapeutic target in ovarian cancer.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Humanos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Neoplasias Ovarianas/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Serina/metabolismo , Fatores de Transcrição da Família Snail/química , Fatores de Transcrição da Família Snail/genética , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Quinases DyrkRESUMO
Maspin is a tumor suppressor protein that stimulates apoptosis and inhibits motility, invasion and cancer metastasis. We report on a previously uncharacterized Pro/Ser (C to T) polymorphism at amino acid 176 of the human maspin protein. We analyzed the maspin mutation in 17 cancer cell lines and 36 cancer tissues. Association of polymorphic variants on apoptosis, colony formation and in vivo tumor formation was evaluated. Mutant maspin was found to be frequently expressed in gastric cancer (32/36, 89%). According to predicted maspin tertiary structure, the polymorphic residue is located on the surface of the protein proximal to the reactive site loop domain, and thus may significantly affect the protein interactions of maspin. Stable expression of the Pro and Ser forms of maspin in lung cancer cells revealed that cells expressing Ser176 maspin showed significantly decreased apoptosis and increased colony formation compared with those expressing Pro176 maspin. In a mouse model, cells expressing Ser176 maspin showed a higher rate of tumor formation in vivo than those harboring Pro176 maspin. Therefore, P176S (C526T) substitution of maspin may result in a partial loss of the tumor suppressor function of this protein, contributing to decreased susceptibility to apoptosis and malignant progression.
Assuntos
Apoptose , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Polimorfismo Genético/genética , Serpinas/genética , Serpinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Suscetibilidade a Doenças , Feminino , Camundongos , Camundongos Nus , Modelos Moleculares , Mutação/genética , Estrutura Terciária de Proteína , Serpinas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 A resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 A , and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 A , and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement.