Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
Filtros adicionais











Intervalo de ano
1.
J Mol Biol ; 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31288030

RESUMO

Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multi-protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components are unknown. Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaOC, which we show to be essential for the solubility of SpaO. We establish domain-domain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaOC/OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.

2.
Structure ; 27(9): 1416-1426.e3, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31303480

RESUMO

The type-III secretion effector YopO helps pathogenic Yersinia to outmaneuver the human immune system. Injected into host cells, it functions as a Ser/Thr kinase after activation by actin binding. This activation process is thought to involve large conformational changes. We use PELDOR spectroscopy and small-angle X-ray scattering in combination with available crystal structures to study these conformational transitions. Low-resolution hybrid models of the YopO/actin structure in solution were constructed, where the kinase domain of YopO is tilted "backward" compared with the crystal structure, thus shortening the distance between actin and the kinase active site, potentially affecting the substrate specificity of YopO. Furthermore, the GDI domain of the hybrid models resembles a conformation that was previously observed in a crystal structure of the isolated GDI domain. We investigate possible structural reasons for the inactivity of the apo state, analyze its flexibility and discuss the biological implications.

3.
EMBO Rep ; 20(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30872316

RESUMO

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

4.
Mol Cell ; 73(6): 1282-1291.e8, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792174

RESUMO

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

5.
J Mol Biol ; 2018 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-30419243

RESUMO

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central ß-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the small-angle X-ray scattering analysis.

6.
Adv Exp Med Biol ; 1009: 87-105, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29218555

RESUMO

Small angle scattering of X-rays (SAXS) and neutrons (SANS) is a structural technique to study disordered systems with chaotic orientations of scattering inhomogeneities at low resolution. An important example of such systems are solutions of biological macromolecules. Rapid development in the methodology for solution scattering data interpretation and model building during the last two decades brought the analysis far beyond the determination of just few overall structural parameters (which was the only possibility in the past) and ensured SAS a firm position in the methods palette of the modern life sciences. The advances in the methodology include ab initio approaches for shape and domain structure restoration from scattering curves without a priori structural knowledge, classification and validation of the models, evaluation of potential ambiguity associated with the reconstruction. In rigid body and hybrid modelling approaches, solution scattering is synergistically used with other structural techniques utilizing the complementary information such as atomic models of the components, intramolecular contacts, subunits orientations etc. for the reconstruction of complex systems. The usual requirement of the sample monodispersity has been loosed recently and the technique can now address such systems as weakly bound oligomers and transient complexes. These state-of-the-art methods are described together with the examples of their applications and the possible ways of post-processing of the models.


Assuntos
Modelos Moleculares , Ácidos Nucleicos/ultraestrutura , Proteínas/ultraestrutura , Espalhamento a Baixo Ângulo , Simulação por Computador , Interpretação Estatística de Dados , Humanos , Conformação Molecular , Difração de Nêutrons/instrumentação , Difração de Nêutrons/métodos , Ácidos Nucleicos/química , Proteínas/química , Síncrotrons/instrumentação , Difração de Raios X/instrumentação , Difração de Raios X/métodos
7.
IUCrJ ; 4(Pt 5): 518-528, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28989709

RESUMO

Small-angle X-ray scattering (SAXS) is an established technique that provides low-resolution structural information on macromolecular solutions. Recent decades have witnessed significant progress in both experimental facilities and in novel data-analysis approaches, making SAXS a mainstream method for structural biology. The technique is routinely applied to directly reconstruct low-resolution shapes of proteins and to generate atomistic models of macromolecular assemblies using hybrid approaches. Very importantly, SAXS is capable of yielding structural information on systems with size and conformational polydispersity, including highly flexible objects. In addition, utilizing high-flux synchrotron facilities, time-resolved SAXS allows analysis of kinetic processes over time ranges from microseconds to hours. Dedicated bioSAXS beamlines now offer fully automated data-collection and analysis pipelines, where analysis and modelling is conducted on the fly. This enables SAXS to be employed as a high-throughput method to rapidly screen various sample conditions and additives. The growing SAXS user community is supported by developments in data and model archiving and quality criteria. This review illustrates the latest developments in SAXS, in particular highlighting time-resolved applications aimed at flexible and evolving systems.

8.
IUCrJ ; 3(Pt 6): 440-447, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27840683

RESUMO

Spatial resolution is an important characteristic of structural models, and the authors of structures determined by X-ray crystallography or electron cryo-microscopy always provide the resolution upon publication and deposition. Small-angle scattering of X-rays or neutrons (SAS) has recently become a mainstream structural method providing the overall three-dimensional structures of proteins, nucleic acids and complexes in solution. However, no quantitative resolution measure is available for SAS-derived models, which significantly hampers their validation and further use. Here, a method is derived for resolution assessment for ab initio shape reconstruction from scattering data. The inherent variability of the ab initio shapes is utilized and it is demonstrated how their average Fourier shell correlation function is related to the model resolution. The method is validated against simulated data for proteins with known high-resolution structures and its efficiency is demonstrated in applications to experimental data. It is proposed that henceforth the resolution be reported in publications and depositions of ab initio SAS models.

9.
J Biol Chem ; 290(35): 21365-75, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26160175

RESUMO

Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.


Assuntos
Autoanticorpos/imunologia , Doença Celíaca/enzimologia , Doença Celíaca/imunologia , Proteínas de Ligação ao GTP/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Transglutaminases/imunologia , Autoanticorpos/química , Doença Celíaca/genética , Epitopos/química , Epitopos/imunologia , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Espalhamento a Baixo Ângulo , Ressonância de Plasmônio de Superfície , Transglutaminases/química , Transglutaminases/genética , Difração de Raios X
10.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 4): 907-17, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25849401

RESUMO

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.


Assuntos
Eubacterium/enzimologia , Liases Intramoleculares/química , Domínio Catalítico , Cristalografia por Raios X , Eubacterium/química , Flavonoides/metabolismo , Liases Intramoleculares/metabolismo , Modelos Moleculares , Conformação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
11.
Nat Struct Mol Biol ; 22(1): 50-56, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25504321

RESUMO

Long-chain bacterial polysaccharides have important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter-dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow size distribution is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Combining crystallography and small-angle X-ray scattering, we found that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions in the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/enzimologia , Antígenos O/química , Antígenos O/metabolismo , Cristalografia por Raios X , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Manosiltransferases/química , Manosiltransferases/metabolismo , Conformação Proteica , Espalhamento a Baixo Ângulo
12.
J Biol Chem ; 290(5): 2644-58, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25451923

RESUMO

Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.


Assuntos
Proteínas de Plantas/química , Receptores de Superfície Celular/química , Arabidopsis/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína , Receptores de Superfície Celular/metabolismo
13.
Biochemistry ; 53(15): 2494-504, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24689960

RESUMO

GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a ß-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure-function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.


Assuntos
Glucosiltransferases/metabolismo , Streptomyces coelicolor/enzimologia , Fosfatos Açúcares/metabolismo , Sequência de Bases , Cristalografia por Raios X , Primers do DNA , Glucosiltransferases/química , Mutagênese Sítio-Dirigida , Conformação Proteica , Espalhamento de Radiação , Especificidade por Substrato
14.
FEBS J ; 281(8): 1974-87, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24588935

RESUMO

Small-angle X-ray scattering (SAXS) is a powerful technique for studying weak interactions between proteins and their ligands (other proteins, DNA/RNA or small molecules) in solution. SAXS provides knowledge about the equilibrium state, the stoichiometry of binding and association-dissociation processes. The measurements are conducted in a solution environment that allows easy monitoring of modifications in protein-ligand association state upon environmental changes. Model-free parameters such as the molecular mass of a system and the radius of gyration can be obtained directly from the SAXS data and give indications about the association state. SAXS is also widely employed to build models of biological assemblies at a resolution of approximately 10-20 Å. Low-resolution shapes can be generated ab initio, although more detailed and biologically interpretable information can be obtained by hybrid modelling. In the latter approach, composite structures of protein-ligand complexes are constructed using atomic models of individual molecules. These may be predicted homology models or experimental structures from X-ray crystallography or NMR. This review focuses on using SAXS data to model structures of protein-ligand complexes and to study their dynamics. The combination of SAXS with other methods such as size exclusion chromatography and dynamic light scattering is discussed.


Assuntos
Proteínas/química , Proteínas/metabolismo , Espalhamento a Baixo Ângulo , Ligantes , Modelos Teóricos , Ligação Proteica
15.
J Struct Biol ; 179(3): 347-58, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22595401

RESUMO

Over the past 10years, much research has been dedicated to the understanding of protein interactions. Large-scale experiments to elucidate the global structure of protein interaction networks have been complemented by detailed studies of protein interaction interfaces. Understanding the evolution of interfaces allows one to identify convergently evolved interfaces which are evolutionary unrelated but share a few key residues and hence have common binding partners. Understanding interaction interfaces and their evolution is an important basis for pharmaceutical applications in drug discovery. Here, we review the algorithms and databases on 3D protein interactions and discuss in detail applications in interface evolution, drug discovery, and interface prediction.


Assuntos
Algoritmos , Simulação por Computador , Descoberta de Drogas , Modelos Moleculares , Proteínas/química , Bases de Dados de Proteínas , Evolução Molecular , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas/genética
16.
Proc Natl Acad Sci U S A ; 109(8): 2878-83, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22328151

RESUMO

The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.


Assuntos
Comunicação Celular , Tecido Elástico/metabolismo , Tropoelastina/química , Tropoelastina/metabolismo , Adesão Celular , Células Cultivadas , Tecido Elástico/química , Tecido Elástico/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Hidrogéis , Microscopia Confocal , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Tamanho da Partícula , Estrutura Terciária de Proteína , Proteólise , Soluções , Relação Estrutura-Atividade , Temperatura Ambiente , Tropoelastina/ultraestrutura
17.
J Cancer Res Clin Oncol ; 137(9): 1349-61, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21833720

RESUMO

BACKGROUND: Several reports describe the importance of the chaperone HSP27 (HSPB1) in cancer progression, and the demand for drugs that modulate HSPB1-activity is increasing rapidly. We reported earlier that RP101 (Bromovinyldeoxyuridine, BVDU, Brivudine) improves the efficacy of chemotherapy in pancreatic cancer. METHODS: Chemistry: Binding of RP101 and HSPB1 was discovered by affinity chromatography. Molecular and cell biology: HSPB1 in vitro transcription/translation (TNT), Pull down using RP101-coupled magnetic beads, Immuno Co-precipitations, Structural modeling of HSP27 (HSPB1), Introduction of point mutations into linear expression templates by PCR, Heat shock, Tumor Invasion. Animal experiments: Treatment of AH13r Sarcomas in SD-rats. Clinical Studies with late-stage pancreatic cancer patients: Pilot study, Dose finding study, Phase II study (NCT00550004). RESULTS: Here, we report that RP101 binds in vitro to the heat shock protein HSPB1 and inhibits interaction with its binding partners. As a result, more activated CASP9 was detected in RP101-treated cancer cells. We modeled HSPB1-structure and identified the RP101 binding site. When we tested RP101 as an anti-cancer drug in a rat model, we found that it improved chemotherapy. In clinical studies with late-stage pancreatic cancer patients, the dose of 500 mg/day was safe and efficient, but 760 mg/day turned out to be too high for lightweight patients. CONCLUSIONS: The development of RP101 as a cancer drug represents a truly novel approach for prevention of chemoresistance and enhancement of chemosensitivity.


Assuntos
Adenocarcinoma/mortalidade , Bromodesoxiuridina/análogos & derivados , Proteínas de Choque Térmico HSP27/metabolismo , Neoplasias Pancreáticas/mortalidade , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Bromodesoxiuridina/administração & dosagem , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Método Duplo-Cego , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Projetos Piloto , Placebos , Ratos , Ratos Sprague-Dawley , Sarcoma/metabolismo , Sarcoma/mortalidade , Análise de Sobrevida
18.
Proteomics ; 10(23): 4186-95, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21046623

RESUMO

Set1C is a histone methyltransferase playing an important role in yeast gene regulation. Modeling the structure of this eight-subunit protein complex is an important open problem to further elucidate its functional mechanism. Recently, there has been progress in modeling of larger complexes using constraints to restrict the combinatorial explosion in binary docking of subunits. Here, we model the subunits of Set1C and develop a constraint-based docking approach, which uses high-quality protein interaction as well as functional data to guide and constrain the combinatorial assembly procedure. We obtained 22 final models. The core complex consisting of the subunits Set1, Bre2, Sdc1 and Swd2 is conformationally conserved in over half of the models, thus, giving high confidence. We characterize these high-confidence and the lower confidence interfaces and discuss implications for the function of Set1C.


Assuntos
Histona-Lisina N-Metiltransferase/química , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , RNA Polimerase II/química
19.
BMC Bioinformatics ; 11: 204, 2010 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-20420672

RESUMO

BACKGROUND: A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. RESULTS: We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to alpha-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94%) appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1) a dimer interface motif found in voltage-gated chloride channels, (2) a proton transfer motif found in heme-copper oxidases, and (3) a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. CONCLUSIONS: Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.


Assuntos
Biologia Computacional/métodos , Proteínas de Membrana/química , Motivos de Aminoácidos , Sítios de Ligação , Análise por Conglomerados , Bases de Dados de Proteínas , Modelos Moleculares , Dobramento de Proteína , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína
20.
Nucleic Acids Res ; 38(Database issue): D181-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19910368

RESUMO

Membrane proteins are important for many processes in the cell and used as main drug targets. The increasing number of high-resolution structures available makes for the first time a characterization of local structural and functional motifs in alpha-helical transmembrane proteins possible. MeMotif (http://projects.biotec.tu-dresden.de/memotif) is a database and wiki which collects more than 2000 known and novel computationally predicted linear motifs in alpha-helical transmembrane proteins. Motifs are fully described in terms of several structural and functional features and editable. Motifs contained in MeMotif can be used in different biological applications, from the identification of biochemically important functional residues which are candidates for mutagenesis experiments to the improvement of tools for transmembrane protein modeling.


Assuntos
Motivos de Aminoácidos , Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Proteínas de Membrana/química , Proteínas de Bactérias/química , Biologia Computacional/tendências , Bases de Dados de Proteínas , Armazenamento e Recuperação da Informação/métodos , Internet , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Software
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA