RESUMEN
This study combined the herbal pair Platycodonis Radix-Curcumae Rhizoma(PR-CR) possessing an inhibitory effect on tumor cell proliferation and metastasis with the active component of traditional Chinese medicine(TCM) silibinin-loaded nanoparticles(NPs) with a regulatory effect on tumor microenvironment based on the joint effect on tumor cells and tumor microenvironment to inhi-bit cell metastasis. The effects of PR-CR on the cellular uptake of NPs and in vitro inhibition against breast cancer proliferation and metastasis were investigated to provide an experimental basis for improving nanoparticle absorption and enhancing therapeutic effects. Silibinin-loaded lipid-polymer nanoparticles(LPNs) were prepared by the nanoprecipitation method and characterized by transmission electron microscopy. The NPs were spherical or quasi-spherical in shape with obvious core-shell structure. The mean particle size was 107.4 nm, Zeta potential was-27.53 mV. The cellular uptake assay was performed by in vitro Caco-2/E12 coculture cell model and confocal laser scanning microscopy(CLSM), and the results indicated that PR-CR could promote the uptake of NPs. Further, in situ intestinal absorption assay by the CLSM vertical scanning approach showed that PR-CR could promote the absorption of NPs in the enterocytes of mice. The inhibitory effect of NPs on the proliferation and migration of 4T1 cells was analyzed using 4T1 breast cancer cells and co-cultured 4T1/WML2 cells, respectively. The results of the CCK8 assay showed that PR-CR-containing NPs could enhance the inhibition against the proliferation of 4T1 breast cancer cells. The wound healing assay indicated that PR-CR-containing NPs enhanced the inhibition against the migration of 4T1 breast cancer cells. This study enriches the research on oral absorption of TCM NPs and also provides a new idea for utilizing the advantages of TCM to inhibit breast cancer metastasis.
Asunto(s)
Neoplasias de la Mama , Nanopartículas , Humanos , Ratones , Animales , Femenino , Silibina/uso terapéutico , Células CACO-2 , Polímeros/química , Nanopartículas/química , Línea Celular Tumoral , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Microambiente Tumoral , Melanoma Cutáneo MalignoRESUMEN
GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent 'off-target' of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.
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Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/fisiología , Multimerización de Proteína/fisiología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/fisiología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/fisiología , Animales , Apoproteínas/química , Apoproteínas/fisiología , Camelus , Dominio Catalítico/fisiología , Cristalización/métodos , Activación Enzimática/fisiología , Humanos , Conformación ProteicaRESUMEN
IceA of Helicobacter pylori (H. pylori) has been suggested as a virulence factor for the bacteria, but its pathogenic role remains unelucidated. Here, we examined the effect of iceA mutation on the secretion of IL-8 by human gastric epithelial cells. We also investigated whether the changes in IL-8 production caused by iceA mutation were associated with impaired adherence of H. pylori to the epithelial cells or with impaired apoptosis of these cells. The iceA mutant strain was constructed from wildtype H. pylori strain by insertional mutagenesis of iceA. The human gastric epithelial cells SGC7901 were infected with wildtype or mutant H. pylori for appropriate lengths of time. The adherence of the bacteria to the epithelial cells was examined by fluorescent microscopy using an anti-H. pylori antibody and flow cytometry. The apoptosis of the epithelial cells was studied by annexin-V staining and flow cytometry. The production of IL-8 by SGC 7901 cells was determined by ELISA. We found that iceA mutation was associated with significantly impaired production of IL-8 from the epithelial cells, which was not due to impaired adherence by the bacteria to the epithelial cells as wildtype and mutant H. pylori exhibited similar levels of binding to the epithelial cells. Furthermore, inactivation of iceA did not affect the apoptotic cell death of SGC7901. Our findings indicate that iceA may contribute to the pathogenicity of H. pylori by modulating the production of IL-8 by host epithelial cells.
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Proteínas de la Membrana Bacteriana Externa/genética , Células Epiteliales/inmunología , Mucosa Gástrica/inmunología , Infecciones por Helicobacter/inmunología , Helicobacter pylori/genética , Interleucina-8/metabolismo , Apoptosis , Adhesión Bacteriana , Línea Celular , Células Epiteliales/metabolismo , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Humanos , Interleucina-8/inmunología , Mutagénesis Insercional , MutaciónRESUMEN
BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is a group of rare, heterogeneous autoinflammatory disease characterized by interleukin (IL)-1ß-mediated systemic inflammation and clinical symptoms involving skin, joints, central nervous system, and eyes. It encompasses a spectrum of three clinically overlapping autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease. CAPS is associated with gain-of-function missense mutations in NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), the gene encoding NLRP3. Moreover, most mutations leading to MWS occurred in exon 3 of NLRP3 gene. Here, we reported a novel mutation occurred in exon 1 of NLRP3 gene in an MWS patient and attempted to explore the pathogenic mechanism. METHODS: Genetic sequence analysis of NLRP3 was performed in an MWS patient who presented with periodic fever, arthralgia, and multiform skin lesions. NLRP3 was also analyzed in this patient's parents and 50 healthy individuals. Clinical examinations including X-ray examination, skin biopsy, bone marrow aspiration smear, and blood test of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum levels of IL-1ß, immunoglobulin E (IgE), antineutrophil cytoplasmic antibodies, antinuclear antibodies, and extractable nuclear antigen were also analyzed. The protein structure of mutant NLRP3 inflammasome was calculated by SWISS-MODEL software. Proteins of wild type and mutant components of NLRP3 inflammasome were expressed and purified, and the interaction abilities between these proteins were tested by surface plasmon resonance (SPR) assay. RESULTS: X-ray examination showed no abnormality in the patient's knees. Laboratory tests indicated an elevation of CRP (233.24 mg/L) and ESR (67 mm/h) when the patient had fever. Serum IL-1ß increased to 24.37 pg/ml, and serum IgE was higher than 2500.00 IU/ml. Other blood tests were normal. Bone marrow aspiration smear was normal. A novel point mutation c.92A>T in exon 1 of NLRP3 gene was identified, which caused a p.D31V mutation in pyrin domain (PYD) of NLRP3. SPR assay showed that this point mutation may strengthen the interaction between the PYD of NLRP3 and the PYD of the apoptosis-associated speck-like protein. The mutation c.92A>T in exon 1 of the NLRP3 gene was not found in the patient's parents and 50 healthy individuals. CONCLUSIONS: The mutation c.92A>T in exon 1 of the NLRP3 gene is a novel mutation associated with MWS. The p.D31V mutation might promote the activation of NLRP3 inflammasome and induce MWS in this patient.
Asunto(s)
Síndromes Periódicos Asociados a Criopirina/genética , Mutación/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Adolescente , Síndromes Periódicos Asociados a Criopirina/metabolismo , Exones/genética , Humanos , Inmunoglobulina E/sangre , Interleucina-1beta/sangre , Masculino , Resonancia por Plasmón de SuperficieAsunto(s)
Proteínas/química , Cristalización , Cristalografía por Rayos X , Metilación , Conformación ProteicaRESUMEN
The Dachshund proteins are essential components of a regulatory network controlling cell fate determination. They have been implicated in eye, limb, brain, and muscle development. These proteins cannot be assigned to any recognizable structural or functional class based on amino acid sequence analysis. The 1.65 A crystal structure of the most conserved domain of human DACHSHUND is reported here. The protein forms an alpha/beta structure containing a DNA binding motif similar to that found in the winged helix/forkhead subgroup of the helix-turn-helix family. This unexpected finding alters the previously proposed molecular models for the role of Dachshund in the eye determination pathway. Furthermore, it provides a rational framework for future mechanistic analyses of the Dachshund proteins in several developmental contexts.
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ADN/metabolismo , Proteínas de Drosophila , Proteínas Nucleares/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Ojo/embriología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Homología de SecuenciaRESUMEN
Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 A resolution (R factor 22.4%, R(free) 23.7%). RpiA exhibits an alpha/beta/(alpha/beta)/beta/alpha fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 A resolution. A mechanism for acid-base catalysis is proposed.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Proteínas de Escherichia coli/química , Vía de Pentosa Fosfato , Estructura Terciaria de Proteína , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Alineación de SecuenciaRESUMEN
The surface layer of archaeobacteria protects cells from extreme environments and, in Methanosarcina, may regulate cell adhesion. We identify three domain types that account for the complete architecture of numerous Methanosarcina surface layer proteins (SLPs). We solve the crystal structure for two of these domains, which correspond to the two N-terminal domains of an M. mazei SLP. One domain displays a unique, highly symmetrical, seven-bladed beta propeller fold, and the other belongs to the polycystic kidney disease (PKD) superfamily fold. The third domain is predicted to adopt a beta helix fold. These domains have homologs in metazoan cell surface proteins, suggesting remarkable relationships between domains in archaeal SLPs and metazoan cell surface proteins.
Asunto(s)
Proteínas Arqueales/química , Proteínas de la Membrana/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Infection with Helicobacter pylori is the strongest risk factor for the development of chronic gastritis, gastric ulcer and gastric carcinoma. The majority of the H. pylori-infected population remains asymptomatic, and only 1% of individuals may progress to gastric cancer. The clinical outcomes caused by H. pylori infection are considered to be associated with bacterial virulence, genetic polymorphism of hosts as well as environmental factors. Most H. pylori strains possess a cytotoxin-associated gene (cag) pathogenicity island (cagPAI), encoding a 120-140 kDa CagA protein, which is the most important bacterial oncoprotein. CagA is translocated into host cells via T4SS system and affects the expression of signaling proteins in a phosphorylation-dependent and independent manner. Thus, this review summarizes the results of relevant studies, discusses the pathogenesis of CagA-mediated gastric cancer.
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Antígenos Bacterianos/fisiología , Proteínas Bacterianas/fisiología , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/fisiología , Neoplasias Gástricas/microbiología , Animales , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Infecciones por Helicobacter/microbiología , Interacciones Huésped-Patógeno , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMEN
Gastric cancer (GC) is one of the most common carcinoma and the second leading cause of cancer-related deaths worldwide. Helicobacter pylori (H. pylori) infection causes a series of precancerous lesions like gastritis, atrophy, intestinal metaplasia and dysplasia, and is the strongest known risk factor for GC, as supported by epidemiological, preclinical and clinical studies. However, the mechanism of H. pylori developing gastric carcinoma has not been well defined. Among infected individuals, approximately 10% develop severe gastric lesions such as peptic ulcer disease, 1%-3% progresses to GC. The outcomes of H. pylori infection are determined by bacterial virulence, genetic polymorphism of hosts as well as environmental factors. It is important to gain further understanding of the pathogenesis of H. pylori infection for developing more effective treatments for this common but deadly malignancy. The recent findings on the bacterial virulence factors, effects of H. pylori on epithelial cells, genetic polymorphism of both the bacterium and its host, and the environmental factors for GC are discussed with focus on the role of H. pylori in gastric carcinogenesis in this review.
RESUMEN
The crystal structure of the Bacillus subtilis YkoF gene product, a protein involved in the hydroxymethyl pyrimidine (HMP) salvage pathway, was solved by the multiwavelength anomalous dispersion (MAD) method and refined with data extending to 1.65 A resolution. The atomic model of the protein shows a homodimeric association of two polypeptide chains, each containing an internal repeat of a ferredoxin-like betaalphabetabetaalphabeta fold, as seen in the ACT and RAM-domains. Each repeat shows a remarkable similarity to two members of the COG0011 domain family, the MTH1187 and YBL001c proteins, the crystal structures of which were recently solved by the Northeast Structural Genomics Consortium. Two YkoF monomers form a tightly associated dimer, in which the amino acid residues forming the interface are conserved among family members. A putative small-ligand binding site was located within each repeat in a position analogous to the serine-binding site of the ACT-domain of the Escherichia coli phosphoglycerate dehydrogenase. Genetic data suggested that this could be a thiamin or HMP-binding site. Calorimetric data confirmed that YkoF binds two thiamin molecules with varying affinities and a thiamine-YkoF complex was obtained by co-crystallization. The atomic model of the complex was refined using data to 2.3 A resolution and revealed a unique H-bonding pattern that constitutes the molecular basis of specificity for the HMP moiety of thiamin.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Estructura Terciaria de Proteína , Pirimidinas/metabolismo , Tiamina/metabolismo , Secuencia de Aminoácidos , Animales , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Unión Proteica , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Pirimidinas/química , Alineación de SecuenciaRESUMEN
Ribose-5-phosphate isomerases (EC 5.3.1.6) interconvert ribose 5-phosphate and ribulose 5-phosphate. This reaction permits the synthesis of ribose from other sugars, as well as the recycling of sugars from nucleotide breakdown. Two unrelated types of enzyme can catalyze the reaction. The most common, RpiA, is present in almost all organisms (including Escherichia coli), and is highly conserved. The second type, RpiB, is present in some bacterial and eukaryotic species and is well conserved. In E.coli, RpiB is sometimes referred to as AlsB, because it can take part in the metabolism of the rare sugar, allose, as well as the much more common ribose sugars. We report here the structure of RpiB/AlsB from E.coli, solved by multi-wavelength anomalous diffraction (MAD) phasing, and refined to 2.2A resolution. RpiB is the first structure to be solved from pfam02502 (the RpiB/LacAB family). It exhibits a Rossmann-type alphabetaalpha-sandwich fold that is common to many nucleotide-binding proteins, as well as other proteins with different functions. This structure is quite distinct from that of the previously solved RpiA; although both are, to some extent, based on the Rossmann fold, their tertiary and quaternary structures are very different. The four molecules in the RpiB asymmetric unit represent a dimer of dimers. Active-site residues were identified at the interface between the subunits, such that each active site has contributions from both subunits. Kinetic studies indicate that RpiB is nearly as efficient as RpiA, despite its completely different catalytic machinery. The sequence and structural results further suggest that the two homologous components of LacAB (galactose-6-phosphate isomerase) will compose a bi-functional enzyme; the second activity is unknown.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Escherichia coli/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Bases de Datos como Asunto , Relación Dosis-Respuesta a Droga , Electrones , Escherichia coli/metabolismo , Cinética , Luz , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Vía de Pentosa Fosfato , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Rayos UltravioletaRESUMEN
The open reading frame SCO4226 of Streptomyces coelicolor A3(2) encodes an 82-residue hypothetical protein. Biochemical assays revealed that each SCO4226 dimer binds four nickel ions. To decipher the molecular function, we solved the crystal structures of SCO4226 in both apo- and nickel-bound (Ni-SCO4226) forms at 1.30 and 2.04 Å resolution, respectively. Each subunit of SCO4226 dimer adopts a canonical ferredoxin-like fold with five ß-strands flanked by two α-helices. In the structure of Ni-SCO4226, four nickel ions are coordinated at the surface of the dimer. Further biochemical assays suggested that the binding of Ni2+ triggers the self-aggregation of SCO4226 in vitro. In addition, RT-qPCR assays demonstrated that the expression of SCO4226 gene in S. coelicolor is specifically up-regulated by the addition of Ni2+, but not other divalent ions such as Cu2+, Mn2+ or Co2+. All these results suggested that SCO4226 acts as a nickel binding protein, probably required for nickel sequestration and/or detoxification.
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Proteínas Bacterianas/metabolismo , Níquel/metabolismo , Streptomyces coelicolor , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Níquel/farmacología , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca(2+) binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP ß-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.
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Proteínas Recombinantes de Fusión/química , Calcio/química , Calcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Cristalografía por Rayos X , Dimerización , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 A resolution revealed a novel two-strand antiparallel beta-sheet opposite the GSH binding groove. This extra beta-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.
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Glutarredoxinas/química , Glutarredoxinas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaAsunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Clonación Molecular , Secuencia Conservada/genética , Cristalización , Proteínas de Escherichia coli/química , Isomerasas/genética , Metales/metabolismo , Datos de Secuencia Molecular , Oxigenasas/genética , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
Bacillus anthracis elaborates a poly-gamma-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the gamma-glutamyltranspeptidase CapD with and without alpha-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-gamma-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-gamma-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro(427), Gly(428), and Gly(429) activate the catalytic residue of the enzyme, Thr(352), and stabilize an oxyanion hole via main chain amide hydrogen bonds.
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Bacillus anthracis/enzimología , Proteínas Bacterianas/química , Dipéptidos/química , Modelos Químicos , Modelos Moleculares , gamma-Glutamiltransferasa/química , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Dipéptidos/metabolismo , Estructura Cuaternaria de Proteína/fisiología , gamma-Glutamiltransferasa/metabolismoRESUMEN
AIM: To determine if disruption of the cagA gene of Helicobacter pylori (H pylori) has an effect on the expression of other proteins at proteome level. METHODS: Construction of a cagA knock out mutant Hp27_Delta cagA (cagA(-)) via homologous recombination with the wild-type strain Hp27 (cagA(+)) as a recipient was performed. The method of sonication-urea-CHAPS-DTT was employed to extract bacterial proteins from both strains. Soluble proteins were analyzed by two-dimensional electrophoresis (2-DE). Images of 2-DE gels were digitalized and analyzed. Only spots that had a statistical significance in differential expression were selected and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Biological information was used to search protein database and identify the biological function of proteins. RESULTS: The proteome expressions between wild-type strain and isogenic mutant with the cagA gene knocked-out were compared. Five protein spots with high abundance in bacteria proteins of wild-type strains, down-regulated or absently expressed in bacteria proteins of mutants, were identified and analyzed. From a quantitative point of view, the identified proteins are related to the cagA gene and important antioxidant proteins of H pylori, including alkyl hydroperoxide reductase (Ahp), superoxide dismutase (SOD) and modulator of drug activity (Mda66), respectively, suggesting that cagA is important to maintain the normal activity of antioxidative stress and ensure H pylori persistent colonization in the host. CONCLUSION: cagA gene is relevant to the expressions of antioxidant proteins of H pylori, which may be a novel mechanism involved in H pylori cagA pathogenesis.