RESUMO
Predicting cellular responses to perturbations requires interpretable insights into molecular regulatory dynamics to perform reliable cell fate control, despite the confounding non-linearity of the underlying interactions. There is a growing interest in developing machine learning-based perturbation response prediction models to handle the non-linearity of perturbation data, but their interpretation in terms of molecular regulatory dynamics remains a challenge. Alternatively, for meaningful biological interpretation, logical network models such as Boolean networks are widely used in systems biology to represent intracellular molecular regulation. However, determining the appropriate regulatory logic of large-scale networks remains an obstacle due to the high-dimensional and discontinuous search space. To tackle these challenges, we present a scalable derivative-free optimizer trained by meta-reinforcement learning for Boolean network models. The logical network model optimized by the trained optimizer successfully predicts anti-cancer drug responses of cancer cell lines, while simultaneously providing insight into their underlying molecular regulatory mechanisms.
Assuntos
Aprendizado de Máquina , Humanos , Algoritmos , Linhagem Celular Tumoral , Modelos Biológicos , Simulação por Computador , Biologia de SistemasRESUMO
Tumor suppressor p53 plays a pivotal role in suppressing cancer, so various drugs has been suggested to upregulate its function. However, drug resistance is still the biggest hurdle to be overcome. To address this, we developed a deep learning model called AnoDAN (anomalous gene detection using generative adversarial networks and graph neural networks for overcoming drug resistance) that unravels the hidden resistance mechanisms and identifies a combinatorial target to overcome the resistance. Our findings reveal that the TGF-ß signaling pathway, alongside the p53 signaling pathway, mediates the resistance, with THBS1 serving as a core regulatory target in both pathways. Experimental validation in lung cancer cells confirms the effects of THBS1 on responsiveness to a p53 reactivator. We further discovered the positive feedback loop between THBS1 and the TGF-ß pathway as the main source of resistance. This study enhances our understanding of p53 regulation and offers insights into overcoming drug resistance.
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Successful embryo implantation is the first step for establishing natural pregnancy and is dependent on the crosstalk between the embryo and a receptive endometrium. However, the molecular signaling events for successful embryo implantation are not entirely understood. To identify differentially expressed transcripts [long-noncoding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs] and competing endogenous RNA (ceRNA) networks associated with endometrial receptivity, the current study analyzed gene expression profiles between proliferative and mid-secretory endometria in fertile women. A total of 247 lncRNAs, 67 miRNAs and 2,154 mRNAs were identified as differentially expressed between proliferative and mid-secretory endometria. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that these differentially expressed genes were significantly enriched for 'cell adhesion molecules.' Additionally, 98 common mRNAs were significantly involved in tryptophan metabolism, metabolic pathways and FoxO signaling. From the differentially expressed lncRNA/miRNA/mRNA ceRNA network, hub RNAs that formed three axes were identified: The DLX6-AS1/miR-141 or miR-200a/OLFM1 axis, the WDFY3-AS2/miR-135a or miR-183/STC1 axis, and the LINC00240/miR-182/NDRG1 axis. These may serve important roles in the regulation of endometrial receptivity. The hub network of the current study may be developed as a candidate marker for endometrial receptivity.
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Homeobox A9 (HOXA9) expression is associated with the aggressive growth of cancer cells and poor prognosis in lung cancer. Previously, we showed that HOXA9 can serve as a potential therapeutic target for the treatment of metastatic non-small cell lung cancer (NSCLC). In the present study, we have carried out additional studies toward the development of a peptide-based therapeutic agent. Vectors expressing partial DNA fragments of HOXA9 were used to identify a unique domain involved in the inhibition of NSCLC cell invasion. Next, we performed in vitro invasion assays and examined the expression of EMT-related genes in transfected NSCLC cells. The C-terminal fragment (HOXA9-C) of HOXA9 inhibited cell invasion and led to upregulation of CDH1 and downregulation of SNAI2 in A549 and NCI-H1299 cells. Reduced SNAI2 expression was consistent with the decreased binding of transcription factor NF-kB to the SNAI2 promoter region in HOXA9-C overexpressing cells. Based on the above results, we synthesized a cell-permeable peptide, CPP33-HADP (HOXA9 active domain peptide), for lung-specific delivery and tested its therapeutic efficiency. CPP33-HADP effectively reduced the invasion ability of NSCLC cells in both in vitro and in vivo mouse models. Our results suggest that CPP33-HADP has significant potential for therapeutic applications in metastatic NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Peptídeos Penetradores de Células/farmacologia , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/uso terapêutico , Modelos Animais de Doenças , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/metabolismo , Invasividade Neoplásica , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the importance of Lactobacillus iners and its unique characteristics for the study of vaginal adaption, its genome and genomic researches for identifying molecular backgrounds of these specific phenotypes are still limited. In this study, the first complete genome of L. iners was constructed using a cost-effective long-read sequencing platform, Flongle from Oxford Nanopore, and comparative genome analysis was conducted using a total of 1,046 strain genomes from 10 vaginal Lactobacillus species. Single-molecule sequencing using Flongle effectively resolved the limitation of the 2nd generation sequencing technologies in dealing with genomic regions of high GC contents, and comparative genome analysis identified three potential core genes (INY, ZnuA, and hsdR) of L. iners which was related to its specific adaption to the vaginal environment. In addition, we performed comparative prophage analysis for 1,046 strain genomes to further identify the species specificity. The number of prophages in L. iners genomes was significantly smaller than other vaginal Lactobacillus species, and one of the specific genes (hsdR) was suggested as the means for defense against bacteriophage. The first complete genome of L. iners and the three specific genes identified in this study will provide useful resources to further expand our knowledge of L. iners and its specific adaption to the vaginal econiche.
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BACKGROUND: Embryo implantation is essential for a successful pregnancy, and an elaborate synchronization between the receptive endometrium and trophoblast is required to achieve this implantation. To increase 'endometrial receptivity', the endometrium undergoes transformation processes including responses of adhesion molecules and cellular and molecular cell to cell communication. Many natural substances from traditional herbs have been studied to aid in the achievement of successful implantation. In this study, we investigated positive effects on embryonic implantation with decursinol that is a major compound extracted from Angelica gigas Nakai known to be associated with promotion of healthy pregnancy in the traditional Korean herbal medicine. METHODS: Expression of cell adhesion molecules after treatment of endometrial epithelial cells by decursinol (40 or 80 µM) was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analysis. The alteration of endometrial receptivity by decursinol (40 or 80 µM) was identified with the in vitro implantation model between Ishikawa cells and JAr cell spheroids (diameter, 143 ± 16 µm). Exosomes secreted from Ishikawa cells after treatment of 80 µM decursinol or dimethyl sulfoxide (DMSO) as the vehicle were investigated with invasion of JAr cells and attachment of JAr spheroids to Ishikawa cells. RESULTS: Decursinol significantly (P < 0.05) increased the expression of important endometrial adhesion molecules such as integrin ß1, ß3, ß5 and L-selectin mRNAs and integrin ß5 and L-selectin in protein. The adhesion rate of JAr spheroids to decursinol-treated Ishikawa cells also increased significantly which was 2.4-fold higher than that of the control (P < 0.05). Furthermore, decursinol induced an increase in the release of exosomes from Ishikawa cells and decursinol-induced exosomes showed autocrine (to Ishikawa cells) and paracrine (to JAr cells) positive effects on our implantation model. CONCLUSION: These results propose that decursinol could serve as a new and alternative solution for patients who are infertile.
Assuntos
Angelica/química , Benzopiranos/farmacologia , Butiratos/farmacologia , Moléculas de Adesão Celular/metabolismo , Implantação do Embrião/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , Estrutura Molecular , Esferoides Celulares/metabolismoRESUMO
Cancer cells exhibit properties of cells in a less differentiated state than the adjacent normal cells in the tissue. We explored whether cancer cells can be converted to a differentiated normal-like state by restoring the gene regulatory network (GRN) of normal cells. Here, we report that colorectal cancer cells exhibit a range of developmental states from embryonic and intestinal stem-like cells to differentiated normal-like cells. To identify the transcription factors (TF) that commit stem-like colorectal cancer cells into a differentiated normal-like state, we reconstructed GRNs of normal colon mucosa and identified core TFs (CDX2, ELF3, HNF4G, PPARG, and VDR) that govern the cellular state. We further found that SET Domain Bifurcated 1 (SETDB1), a histone H3 lysine 9-specific methyltransferase, hinders the function of the identified TFs. SETDB1 depletion effectively converts stem-like colorectal cancer cells into postmitotic cells and restores normal morphology in patient-derived colorectal cancer organoids. RNA-sequencing analyses revealed that SETDB1 depletion recapitulates global gene expression profiles of normal differentiated cells by restoring the transcriptional activity of core TFs on their target genes. IMPLICATIONS: Our study provides insights into the molecular regulatory mechanism underlying the developmental hierarchy of colorectal cancer and suggests that induction of a postmitotic state may be a therapeutic alternative to destruction of cancer cells.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Histona-Lisina N-Metiltransferase/genética , Células CACO-2 , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Células-Tronco Embrionárias/patologia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Células HCT116 , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Células-Tronco Neoplásicas/patologia , Transfecção , Células Tumorais CultivadasRESUMO
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas F-Box/química , Peptidomiméticos/química , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteínas F-Box/metabolismo , Fertilização/efeitos dos fármacos , Meiose/efeitos dos fármacos , Mesotelina , Camundongos , Modelos Moleculares , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Peptidomiméticos/administração & dosagem , Peptidomiméticos/farmacologia , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/metabolismo , Relação Estrutura-Atividade , Xenopus , Quinase 1 Polo-LikeRESUMO
Chronic ethanol consumption causes hepatic steatosis and inflammation, which are associated with liver hypoxia. Monocyte chemoattractant protein-1 (MCP-1) is a hypoxia response factor that determines recruitment and activation of monocytes to the site of tissue injury. The level of MCP-1 is elevated in the serum and liver of patients with alcoholic liver disease (ALD); however, the molecular details regarding the regulation of MCP-1 expression are not yet understood completely. Here, we show the role of liver X receptor α (LXRα) in the regulation of MCP-1 expression during the development of ethanol-induced fatty liver injury, using an antagonist, 22-S-hydroxycholesterol (22-S-HC). First, administration of 22-S-HC attenuated the signs of liver injury with decreased levels of MCP-1 and its receptor CCR2 in ethanol-fed mice. Second, hypoxic conditions or treatment with the LXRα agonist GW3965 significantly induced the expression of MCP-1, which was completely blocked by treatment with 22-S-HC or infection by shLXRα lentivirus in the primary hepatocytes. Third, over-expression of LXRα or GW3965 treatment increased MCP-1 promoter activity by increasing the binding of hypoxia-inducible factor-1α to the hypoxia response elements, together with LXRα. Finally, treatment with recombinant MCP-1 increased the level of expression of LXRα and LXRα-dependent lipid droplet accumulation in both hepatocytes and Kupffer cells. These data show that LXRα and its ligand-induced up-regulation of MCP-1 and MCP-1-induced LXRα-dependent lipogenesis play a key role in the autocrine and paracrine activation of MCP-1 in the pathogenesis of alcoholic fatty liver disease, and that this activation may provide a promising new target for ALD therapy.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Fígado Gorduroso Alcoólico/prevenção & controle , Hidroxicolesteróis/farmacologia , Fígado/efeitos dos fármacos , Receptores Nucleares Órfãos/antagonistas & inibidores , Comunicação Parácrina/efeitos dos fármacos , Animais , Sítios de Ligação , Hipóxia Celular , Células Cultivadas , Quimiocina CCL2/genética , Citoproteção , Modelos Animais de Doenças , Etanol , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células de Kupffer/efeitos dos fármacos , Células de Kupffer/metabolismo , Células de Kupffer/patologia , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Receptores X do Fígado , Masculino , Camundongos Endogâmicos C57BL , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Transfecção , Regulação para CimaRESUMO
Recent progress in the development of peptide-derived Polo-like kinase (Plk1) polo-box domain (PBD) inhibitors has led to the synthesis of multiple peptide ligands with high binding affinity and selectivity. However, few systematic analyses have been conducted to identify key Plk1 residues and characterize their interactions with potent Plk1 peptide inhibitors. We performed systematic deletion analysis using the most potent 4j peptide and studied N-terminal capping of the minimal peptide with diverse organic moieties, leading to the identification of the peptidomimetic 8 (AB-103) series with high binding affinity and selectivity. To evaluate the bioavailability of short peptidomimetic ligands, PEGylated 8 series were synthesized and incubated with HeLa cells to test for cellular uptake, antiproliferative activity, and Plk1 kinase inhibition. Finally, crystallographic studies of the Plk1 PBD in complex with peptidomimetics 8 and 22 (AB-103-5) revealed the presence of two hydrogen bond interactions responsible for their high binding affinity and selectivity.
Assuntos
Proteínas de Ciclo Celular/química , Peptídeos/química , Peptidomiméticos/química , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Ligação Competitiva , Transporte Biológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Células HeLa , Humanos , Ligação de Hidrogênio , Ligantes , Microscopia de Fluorescência , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Peptidomiméticos/metabolismo , Peptidomiméticos/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-Atividade , Quinase 1 Polo-LikeRESUMO
The bacterium Streptomyces coelicolor produces useful antibiotics from its secondary metabolites. DraK is a sensory histidine kinase involved in the differential regulation of antibiotics in S. coelicolor through the DraR/DraK two-component system. Here, the extracellular sensory domain of DraK was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal diffracted to 2.2 Å resolution and belonged to space group C2221, with unit-cell parameters a = 41.91, b = 174.50, c = 145.25 Å, α = ß = γ = 90°.
Assuntos
Proteínas de Bactérias/biossíntese , Líquido Extracelular/enzimologia , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/biossíntese , Streptomyces coelicolor/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , Histidina Quinase , Proteínas Quinases/química , Proteínas Quinases/isolamento & purificação , Estrutura Terciária de ProteínaRESUMO
The crystal structures of CsGST in two different space groups revealed that Asp26 and His79 coordinate a zinc ion. In one space group, His46 of an adjacent molecule participates in the coordination within 2.0Å. In the other space group, Asp26, His79 and a water molecule coordinate a zinc ion. The CsGST-D26H structure showed that four histidine residues - His26 and His79 from one molecule and the same residues from a symmetry-related neighboring molecule - coordinate a zinc ion. The coordinated zinc ions are located between two molecules and mediate molecular contacts within the crystal.
Assuntos
Clonorchis sinensis/enzimologia , Glutationa Transferase/química , Metais/química , Mutação , Zinco/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/metabolismo , Histidina/química , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5-10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.
Assuntos
Proteínas Quinases/química , Streptomyces coelicolor/enzimologia , Cromatografia em Gel , Citoplasma/enzimologia , Ácido Glutâmico/química , Ácido Glutâmico/genética , Histidina Quinase , Concentração de Íons de Hidrogênio , Proteínas Quinases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
NORE1 is an important tumour suppressor in human cancers that interacts with the pro-apoptotic protein kinase MST1/2 through SARAH domains. The SARAH domain (residues 366-413) of human NORE1 was expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 2.7â Å resolution and belonged to space group P6(1)22, with unit-cell parameters a = b = 73.041, c = 66.092â Å, α = ß = 90, γ = 120°.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Cristalização , Cristalografia por Raios X , Expressão Gênica , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/isolamento & purificaçãoRESUMO
Glutathione S-transferases (GSTs) are multifunctional enzymes that are used as fusion tags on recombinant proteins in mammalian and Escherichia coli expression systems. We recently found that the Schistosoma japonicum GST (SjGST) displays weak Ni(2+) ion binding affinity. Glu26 and His79 were assumed to be its Ni(2+) binding sites based on the structure of the 26-kDa Clonorchis sinensis GST. To enhance SjGST Ni(2+) binding affinity, Glu26 was mutated to His. SjGST-E26H was expressed and purified at a high concentration of imidazole to a higher purity than wild type SjGST. In addition, human biotin protein ligase fused to SjGST-E26H was purified with a immobilized Ni affinity column.
Assuntos
Cromatografia de Afinidade/métodos , Glutationa Transferase/metabolismo , Histidina/metabolismo , Níquel/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma japonicum/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Biotina/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Glutationa Transferase/química , Glutationa Transferase/genética , Histidina/química , Histidina/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Schistosoma japonicum/genética , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a beta-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (alpha/alpha)(6) fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.
Assuntos
Bacteroides/metabolismo , Heparina Liase/química , Heparina/química , Catálise , Clonagem Molecular , Análise Mutacional de DNA , Cinética , Conformação Molecular , Mutagênese Sítio-Dirigida , Polissacarídeos/química , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Especificidade por SubstratoRESUMO
The target of the RNAIII-activating protein (TRAP) is a 21 kDa protein in which phosphorylation is activated by the RNAIII-activating protein (RAP), which causes an increase in RNAII and RNAIII synthesis and the production of the virulence factors. In an attempt to examine the structural role of TRAP in the signal transduction pathway, TRAP from Staphylococcus aureus was overexpressed, purified and crystallized using PEG 8000 and 5% Jeffamine M600 (pH 7.0), as precipitants by hanging-drop vapour diffusion methods at 287 K. The crystals belong to the orthorhombic space group, P2(1)2(1)2(1), with unit cell parameters of a=39.68, b=50.41, c=85.45 A. There is one monomer of TRAP per crystallographic asymmetric unit with a crystal volume per protein mass (V(M)) of 2.06 A(3) Da(-1) and a solvent content of 40.3%. A complete data set diffracting to 1.9 A resolution was collected from a single crystal at 100 K using a synchrotron-radiation source.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Bactérias/genética , Cristalografia por Raios X , Humanos , Fosfoproteínas/genética , Transdução de Sinais/fisiologiaRESUMO
UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose, an essential metabolite in all living organisms. An X-ray crystallographic study of UGPase from Helicobacter pylori has been performed in order to elucidate its role in the regulation of this important metabolic pathway. UGPase was crystallized from 0.1 M sodium acetate trihydrate pH 4.6, 2.0 M ammonium sulfate and 0.1 M guanidine-HCl. According to diffraction data collected at a resolution of 2.9 A using a synchrotron-radiation source, the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 91.47, b = 98.61, c = 245.70 A, alpha = beta = gamma = 90.0 degrees.
Assuntos
Helicobacter pylori/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/química , Cristalização , Cristalografia por Raios XRESUMO
ClpX, a member of the HSP (heat-shock protein) 100 family, functions as a molecular chaperone and is a regulatory subunit of the ClpXP protease. To understand the chaperone and regulatory mechanisms of ClpX, Helicobacter pylori ClpX has been overexpressed in Escherichia coli and crystallized at 295 K using (NH(4))(2)HPO(4) as precipitant. X-ray diffraction data have been collected to 2.6 A resolution using a synchrotron-radiation source. The crystals belong to the hexagonal space group P6(5) or P6(1), with unit-cell parameters a = b = 78.52 (04), c = 131.51 (09) A, alpha = beta = 90, gamma = 120 degrees. The crystallographic asymmetric unit contains one molecule of ClpX, with a corresponding V(M) of 2.78 A(3) Da(-1) and a solvent content of 55.8%.