RESUMO
The ancient protein TSPO (translocator protein 18kD) is found in all kingdoms and was originally identified as a binding site of benzodiazepine drugs. Its physiological function remains unclear, although porphyrins are conserved ligands. Several crystal structures of bacterial TSPO and nuclear magnetic resonance structures of a mouse form have revealed monomer and dimer configurations, but there have been no reports of structures with a physiological ligand. Here, we present the first X-ray structures of Rhodobacter sphaeroides TSPO with a physiological ligand bound. Two different variants (substituting threonine for alanine at position 139 (A139T) and phenylalanine for alanine at position 138 (A138F)) yielded well-diffracting crystals giving structures of both apo- and heme-containing forms. Both variants have wild-type micromolar affinity for heme and protoporphyrin IX, but A139T has very low ability to accelerate the breakdown of porphyrin in the presence of light and oxygen. The binding of heme to one protomer of the dimer of either mutant induces a more rigid structure, both in the heme-binding protomer and the protomer without heme bound, demonstrating an allosteric response. Ensemble refinement of the X-ray data reveals distinct regions of altered flexibility in response to single heme binding to the dimer. The A139T variant shows a more rigid structure overall, which may relate to extra hydrogen bonding of waters captured in the heme crevice. As TSPO has been suggested to have a role in heme delivery from mitochondria to the cytoplasm, the new structures provide potential clues regarding the structural basis of such activity.
Assuntos
Proteínas de Bactérias , Heme , Porfirinas , Rhodobacter sphaeroides , Alanina , Sítios de Ligação , Proteínas de Transporte/metabolismo , Heme/metabolismo , Ligantes , Porfirinas/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.
Assuntos
Evolução Molecular , Membranas Mitocondriais , Proteínas Mitocondriais , Receptores de GABA , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ratos , Receptores de GABA/química , Receptores de GABA/genética , Receptores de GABA/metabolismo , Rhodobacter/química , Rhodobacter/genética , Rhodobacter/metabolismo , Relação Estrutura-AtividadeRESUMO
Oxicams are widely used nonsteroidal anti-inflammatory drugs (NSAIDs), but little is known about the molecular basis of the interaction with their target enzymes, the cyclooxygenases (COX). Isoxicam is a nonselective inhibitor of COX-1 and COX-2 whereas meloxicam displays some selectivity for COX-2. Here we report crystal complexes of COX-2 with isoxicam and meloxicam at 2.0 and 2.45 angstroms, respectively, and a crystal complex of COX-1 with meloxicam at 2.4 angstroms. These structures reveal that the oxicams bind to the active site of COX-2 using a binding pose not seen with other NSAIDs through two highly coordinated water molecules. The 4-hydroxyl group on the thiazine ring partners with Ser-530 via hydrogen bonding, and the heteroatom of the carboxamide ring of the oxicam scaffold interacts with Tyr-385 and Ser-530 through a highly coordinated water molecule. The nitrogen atom of the thiazine and the oxygen atom of the carboxamide bind to Arg-120 and Tyr-355 via another highly ordered water molecule. The rotation of Leu-531 in the structure opens a novel binding pocket, which is not utilized for the binding of other NSAIDs. In addition, a detailed study of meloxicam·COX-2 interactions revealed that mutation of Val-434 to Ile significantly reduces inhibition by meloxicam due to subtle changes around Phe-518, giving rise to the preferential inhibition of COX-2 over COX-1.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/metabolismo , Piroxicam/análogos & derivados , Tiazinas/metabolismo , Tiazóis/metabolismo , Substituição de Aminoácidos , Animais , Anti-Inflamatórios não Esteroides/química , Arginina/química , Arginina/genética , Arginina/metabolismo , Sítios de Ligação , Domínio Catalítico , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase/química , Ligação de Hidrogênio , Leucina/química , Leucina/genética , Leucina/metabolismo , Meloxicam , Camundongos , Mutação , Piroxicam/química , Piroxicam/metabolismo , Estrutura Secundária de Proteína , Serina/química , Serina/genética , Serina/metabolismo , Tiazinas/química , Tiazóis/química , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , ÁguaRESUMO
The translocator protein 18 kDa (TSPO) has been the focus of intense research by the biomedical community and the pharmaceutical industry because of its apparent involvement in many disease-related processes. These include steroidogenesis, apoptosis, inflammation, neurological disease and cancer, resulting in the use of TSPO as a biomarker and its potential as a drug target. Despite more than 30 years of study, the precise function of TSPO remains elusive. A recent breakthrough in determining the high-resolution crystal structures of bacterial homologs of mitochondrial TSPO provides new insight into the structural and functional properties at a molecular level and new opportunities for investigating the significance of this ancient and highly conserved protein family. The availability of atomic level structural information from different species also provides a platform for structure-based drug development. Here we briefly review current knowledge regarding TSPO and the implications of the new structures with respect to hypotheses and controversies in the field.
Assuntos
Receptores de GABA/metabolismo , Biomarcadores/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismoRESUMO
Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-Å resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Glucosiltransferases/química , Uridina Difosfato Glucose/química , Catálise , Cristalografia por Raios X , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm(3)) and small pore diameters in the membrane lead to digestion in <1 s, the low membrane thickness (170 µm) affords control over residence times at the millisecond level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kDa) that increase the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion). With this approach, we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, Root Hair Defective 3 (RHD3) and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.
Assuntos
Apoproteínas/metabolismo , Mioglobina/metabolismo , Nylons/química , Peptídeo Hidrolases/metabolismo , Poliestirenos/química , Proteólise , Soroalbumina Bovina/metabolismo , Adsorção , Animais , Apoproteínas/química , Arabidopsis/enzimologia , Bovinos , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Mioglobina/química , Nylons/metabolismo , Peptídeos/análise , Poliestirenos/metabolismo , Porosidade , Soroalbumina Bovina/química , Propriedades de Superfície , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
Intestinal tumors in Apc(Min/+) mice are suppressed by over-production of HPGDS, which is a glutathione transferase that forms prostaglandin D(2) (PGD(2)). We characterized naturally occurring HPGDS isoenzymes, to see if HPGDS variation is associated with human colorectal cancer risk. We used DNA heteroduplex analysis and sequencing to identify HPGDS variants among healthy individuals. HPGDS isoenzymes were produced in bacteria, and their catalytic activities were tested. To determine in vivo effects, we conducted pooled case-control analyses to assess whether there is an association of the isoenzyme with colorectal cancer. Roughly 8% of African Americans and 2% of Caucasians had a highly stable Val187lle isoenzyme (with isoleucine instead of valine at position 187). At 37°C, the wild-type enzyme lost 15% of its activity in 1h, whereas the Val187Ile form remained >95% active. At 50°C, the half life of native HPGDS was 9min, compared to 42 min for Val187Ile. The odds ratio for colorectal cancer among African Americans with Val187Ile was 1.10 (95% CI, 0.75-1.62; 533 cases, 795 controls). Thus, the Val187Ile HPGDS isoenzyme common among African Americans is not associated with colorectal cancer risk. Other approaches will be needed to establish a role for HPGDS in occurrence of human intestinal tumors, as indicated by a mouse model.
Assuntos
Substituição de Aminoácidos , Negro ou Afro-Americano/genética , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Predisposição Genética para Doença/genética , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Lipocalinas/química , Lipocalinas/genética , Adulto , Animais , Estudos de Casos e Controles , Neoplasias Colorretais/etnologia , Estabilidade Enzimática , Técnicas de Inativação de Genes , Humanos , Oxirredutases Intramoleculares/deficiência , Isoenzimas/química , Isoenzimas/deficiência , Isoenzimas/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Transgenes/genéticaRESUMO
Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein. RIP governs diverse processes in a wide variety of organisms and is carried out by different types of intramembrane proteases (IPs), including a large family of metalloproteases. The Bacillus subtilis SpoIVFB protein is a putative metalloprotease that cleaves membrane-tethered Pro-sigma(K), releasing sigma(K) to direct transcription of genes necessary for spore formation. By attaching an extra transmembrane segment to the N terminus of SpoIVFB, expression in E. coli was improved more than 100-fold, facilitating purification and demonstration of metalloprotease activity, which accurately cleaved purified Pro-sigma(K). Uniquely for IPs examined so far, SpoIVFB activity requires ATP, which binds to the C-terminal cystathionine-beta-synthase (CBS) domain of SpoIVFB. Deleting just 10 residues from the C-terminal end of SpoIVFB nearly eliminated cleavage of coexpressed Pro-sigma(K) in E. coli. The CBS domain of SpoIVFB was shown to interact with Pro-sigma(K) and ATP changed the interaction, suggesting that ATP regulates substrate access to the active site and renders cleavage sensitive to the cellular energy level, which may be a general feature of CBS-domain-containing IPs. Incorporation of SpoIVFB into preformed liposomes stimulated its ability to cleave Pro-sigma(K). Cleavage depended on ATP and the correct peptide bond was shown to be cleaved using a rapid and sensitive mass spectrometry assay. A system for biochemical studies of RIP by a metalloprotease in a membrane environment has been established using methods that might be applicable to other IPs.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico/genética , Eletroforese em Gel de Poliacrilamida , Endopeptidases/genética , Hidrólise , Immunoblotting , Espectrometria de Massas/métodos , Proteínas de Membrana/genética , Modelos Biológicos , Mutação , Precursores de Proteínas/metabolismo , Transdução de SinaisRESUMO
Succinic semialdehyde reductase (SSAR) is an important enzyme involved in γ-aminobutyrate (GABA) metabolism. By converting succinic semialdehyde (SSA) to γ-hydroxybutyrate (GHB), the SSAR facilitates an alternative pathway for GABA degradation. In this study, we identified SSARs from Geobacter sulfurreducens and Geobacter metallireducens (GsSSAR and GmSSAR, respectively). The enzymes were over-expressed in Escherichia coli and purified to near homogeneity. Both GsSSAR and GmSSAR showed the activity of reducing SSA using nicotinamide adenine dinucleotide phosphate as a co-factor. The oligomeric sizes of GsSSAR and GmSSAR, as determined by analytical size exclusion chromatography, suggest that the enzymes presumably exist as tetramers in solution. The recombinant GsSSAR and GmSSAR crystallized in the presence of NADP(+), and the resulting crystals diffracted to 1.89 Å (GsSSAR) and 2.25 Å (GmSSAR) resolution. The GsSSAR and GmSSAR crystals belong to the space groups P2(1)22(1) (a= 99.61 Å, b= 147.49 Å, c= 182.47 Å) and P1 (a= 75.97 Å, b= 79.14 Å, c= 95.47 Å, α = 82.15°, ß = 88.80°, γ = 87.66°), respectively. Preliminary crystallographic data analysis suggests the presence of eight protein monomers in the asymmetric units for both GsSSAR and GmSSAR.
Assuntos
Cromatografia por Troca Iônica/métodos , Cristalografia por Raios X/métodos , Geobacter/enzimologia , Hidroxibutirato Desidrogenase/química , Hidroxibutirato Desidrogenase/genética , Sequência de Aminoácidos , Cristalização/métodos , Geobacter/metabolismo , Hidroxibutirato Desidrogenase/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Specific interactions between lipids and membrane proteins have been observed in recent high-resolution crystal structures of membrane proteins. A number of cytochrome oxidase structures were analyzed, along with many amino acid sequences of membrane-spanning regions aligned according to their location in the membrane. The results reveal conservation of lipid-binding sites and of the residues that form them. These studies imply that bound lipids have important roles that are crucial to the assembly, structure, or activity of the protein. Evidence for some of these roles in subunit interactions, membrane insertion, and protein-protein complex formation is reviewed.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Lipídeos de Membrana/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Evolução Molecular , Humanos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência MolecularRESUMO
A tethered bilayer lipid membrane (tBLM) was fabricated on a gold electrode using 1,2-dipalmitoyl-sn-glycero-phosphothioethanol as a tethering lipid and the membrane fractions of Saccharomyces pombe yeast cells to deposit the upper leaflet. The membrane fractions were characterized using transmission electron microscopy and dynamic light scattering and found to be similar in size to small unilamellar vesicles of synthetic lipids. The dynamics of membrane-fraction deposition and rupture on the tethering-lipid layer were measured using quartz crystal microgravimetry. The electrochemical properties of the resulting tBLM were characterized using electrical impedance spectroscopy and cyclic voltammetry. The tBLM's electrical resistance was greater than 1 MOmegacm(2), suggesting a defect-free membrane. The suitability of tBLM produced using membrane fractions for measuring ion-channel activities was shown by a decrease in membrane resistance from 1.6 to 0.43 MOmegacm(2) following addition of gramicidin. The use of membrane fractions to form high-quality tBLM on gold electrodes suggests a new approach to characterize membrane proteins, in which the upper leaflet of the tBLM is deposited, and overexpressed membrane proteins are incorporated, in a single step. This approach would be especially useful for proteins whose activity is lost or altered during extraction, purification, and reconstitution, or whose activities are strongly influenced by the lipid composition of the bilayer.
Assuntos
Canais Iônicos/análise , Bicamadas Lipídicas/química , Técnicas Biossensoriais , Membrana Celular/química , Eletroquímica , Ouro , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Schizosaccharomyces/química , Propriedades de SuperfícieRESUMO
The 18-kilodalton translocator protein (TSPO), proposed to be a key player in cholesterol transport into mitochondria, is highly expressed in steroidogenic tissues, metastatic cancer, and inflammatory and neurological diseases such as Alzheimer's and Parkinson's. TSPO ligands, including benzodiazepine drugs, are implicated in regulating apoptosis and are extensively used in diagnostic imaging. We report crystal structures (at 1.8, 2.4, and 2.5 angstrom resolution) of TSPO from Rhodobacter sphaeroides and a mutant that mimics the human Ala(147)âThr(147) polymorphism associated with psychiatric disorders and reduced pregnenolone production. Crystals obtained in the lipidic cubic phase reveal the binding site of an endogenous porphyrin ligand and conformational effects of the mutation. The three crystal structures show the same tightly interacting dimer and provide insights into the controversial physiological role of TSPO and how the mutation affects cholesterol binding.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Rhodobacter sphaeroides/química , Sequência de Aminoácidos , Sítios de Ligação , Colesterol/metabolismo , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Isoquinolinas/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Polimorfismo de Nucleotídeo Único , Porfirinas/metabolismo , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Protoporfirinas/metabolismo , Receptores de GABA/química , Receptores de GABA/genéticaRESUMO
Wang comments that the diffraction data for the structure of the A139T mutant of translocator protein TSPO from Rhodobacter sphaeroides should be used to 1.65 instead of 1.8 angstroms and that the density interpreted as porphyrin and monoolein is better fitted as polyethylene glycol. Although different practices of data processing exist, in this case they do not substantially influence the final map. Additional data are presented supporting the fit of a porphyrin and monooleins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Rhodobacter sphaeroides/química , HumanosRESUMO
d-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection. The biosynthesis of d-rhamnose proceeds through the conversion of GDP-d-mannose by GDP-d-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-d-rhamnose by a reductase. We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP. GMD belongs to the NDP-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes, all of which exhibit bidomain structures and a conserved catalytic triad (Tyr-XXX-Lys and Ser/Thr). Although most members of this enzyme subfamily display homodimeric structures, this bacterial GMD forms a tetramer in the same fashion as the plant MUR1 from Arabidopsis thaliana. The cofactor binding sites are adjoined across the tetramer interface, which brings the adenosyl phosphate moieties of the adjacent NADPH molecules to within 7 A of each other. A short peptide segment (Arg35-Arg43) stretches into the neighboring monomer, making not only protein-protein interactions but also hydrogen bonding interactions with the neighboring cofactor. The interface hydrogen bonds made by the Arg35-Arg43 segment are generally conserved in GMD and MUR1, and the interacting residues are highly conserved among the sequences of bacterial and eukaryotic GMDs. Outside of the Arg35-Arg43 segment, residues involved in tetrameric contacts are also quite conserved across different species. These observations suggest that a tetramer is the preferred, and perhaps functionally relevant, oligomeric state for most bacterial and eukaryotic GMDs.
Assuntos
Açúcares de Guanosina Difosfato/biossíntese , Açúcares de Guanosina Difosfato/metabolismo , Hidroliases/química , Pseudomonas/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Hidroliases/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Prostaglandin H synthase 2 (also known as cyclooxygenase-2) is thought to play a role in the prevention of colon cancer by aspirin, an inhibitor of the enzyme. We used DNA heteroduplex analysis to screen the prostaglandin H synthase 2 gene, to search for naturally occurring enzyme variants that may simulate the effects of aspirin. We found among African-Americans a single-nucleotide polymorphism that changes valine to alanine at residue 511 (V511A; GTT>GCT; g.5939T>C; allele frequency 0.045). The polymorphism was also seen among Asian-Indians (allele frequency, 0.03) but not among Chinese, Filipinos, Hispanics, Japanese, Koreans, Samoans, and Caucasians. The amino acid change is predicted to open a 53 cubic angstrom cavity near the active site of the enzyme, but no change in V(max), K(m), or thermal stability was observed for the variant enzyme in COS-1 cell transfection assays. Case-control analysis of African-Americans from two different study populations showed a 0.56 odds ratio for colorectal adenomas among polymorphism carriers (95% confidence interval, 0.25-1.27; 161 cases and 219 controls). A similar analysis of African-Americans nested in the Multiethnic Cohort Study showed a 0.67 odds ratio for colorectal cancer (95% confidence interval, 0.28-1.56; 138 cases and 258 controls). Consistency of the results across all three of the studies is potentially compatible with a protective effect of the polymorphism, mimicking aspirin.
Assuntos
Adenoma/enzimologia , Adenoma/genética , População Negra/genética , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/genética , Adenoma/epidemiologia , Adolescente , Adulto , Idoso , Anti-Inflamatórios não Esteroides/uso terapêutico , Aspirina/uso terapêutico , Estudos de Casos e Controles , Códon/efeitos dos fármacos , Códon/genética , Estudos de Coortes , Neoplasias Colorretais/epidemiologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/uso terapêutico , Feminino , Seguimentos , Frequência do Gene/efeitos dos fármacos , Frequência do Gene/genética , Análise Heteroduplex , Humanos , Isoenzimas/classificação , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Polimorfismo Genético/efeitos dos fármacos , Polimorfismo Genético/genética , Prostaglandina-Endoperóxido Sintases/classificação , Fatores de Risco , Análise de Sequência de DNA , Análise de Sequência de Proteína , TransfecçãoRESUMO
Despite the marked differences in their physiological roles, the structures and catalytic functions of the prostaglandin H2 endoperoxide synthases-1 and -2 (PGHS-1 and -2) are almost completely identical. These integral membrane proteins catalyze the conversion of arachidonic acid to PGG2 and finally to PGH2. The crystal structures of PGHS-1 and -2 provide new insights into the catalytic mechanism for fatty acid oxygenation. Moreover, a clearer picture emerges to explain how a handful of amino acid substitutions can give rise to subtle differences in ligand binding between the two isoforms. These "small" alterations of isozyme structure are sufficient to allow the design of new, isoform-selective drugs.
Assuntos
Isoenzimas/química , Prostaglandina-Endoperóxido Sintases/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Ácido Araquidônico/química , Ácido Araquidônico/metabolismo , Domínio Catalítico , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/metabolismo , Dimerização , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana , Modelos Moleculares , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The prostaglandin H synthase structure reveals a fold similar to those of other heme peroxidases, but within this fold a second active site has evolved which catalyzes the cyclooxygenase reaction. The protein has also acquired two additional domains: a membrane-binding motif that mediates the protein's monotopic attachment to the membrane, and an epidermal growth factor-like module which is found just before the membrane-binding motif and is located in the dinner interface.
RESUMO
Beta-hydroxyacid dehydrogenase (ß-HAD) genes have been identified in all sequenced genomes of eukaryotes and prokaryotes. Their gene products catalyze the NAD(+)- or NADP(+)-dependent oxidation of various ß-hydroxy acid substrates into their corresponding semialdehyde. In many fungal and bacterial genomes, multiple ß-HAD genes are observed leading to the hypothesis that these gene products may have unique, uncharacterized metabolic roles specific to their species. The genomes of Geobacter sulfurreducens and Geobacter metallireducens each contain two potential ß-HAD genes. The protein sequences of one pair of these genes, Gs-ßHAD (Q74DE4) and Gm-ßHAD (Q39R98), have 65% sequence identity and 77% sequence similarity with each other. Both proteins are observed to reduce succinic semialdehyde, a 4-carbon substrate instead of the typical ß-HAD 3-carbon substrate, to γ-hydroxybutyric acid. To further explore the structural and functional characteristics of these two ß-HADs with a less frequently observed substrate specificity, crystal structures for Gs-ßHAD and Gm-ßHAD in complex with NADP(+) were determined to a resolution of 1.89 Å and 2.07 Å, respectively. The structures of both proteins are similar, composed of 14 α-helices and nine ß-strands organized into two domains. Domain 1 (1-165) adopts a typical Rossmann fold composed of two α/ß units: a six-strand parallel ß-sheet surrounded by six α-helices (α1-α6) followed by a mixed three-strand ß-sheet surrounded by two α-helices (α7 and α8). Domain 2 (166-287) is composed of a bundle of seven α-helices (α9-α14). Four functional regions conserved in all ß-HADs are spatially located near each other, with a buried molecule of NADP(+), at the interdomain cleft. Comparison of these Geobacter structures to a closely related ß-HAD from Arabidopsis thaliana in the apo-NADP(+) and apo-substrate bound state suggests that NADP(+) binding effects a rigid body rotation between Domains 1 and 2. Bound near the Substrate-Binding and Catalysis Regions in two of the eight protomers in the asymmetric unit of Gm-ßHAD is a glycerol molecule that may mimic features of bound biological substrates.
Assuntos
Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/metabolismo , Geobacter/enzimologia , Hidroxibutirato Desidrogenase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , NADP/metabolismoRESUMO
Electrochemical impedance spectroscopy (EIS) was used to characterize voltage-dependent closure of PorB class II (PorBII) porin from Neisseria meningitidis incorporated in a tethered bilayer lipid membrane (tBLM). The tBLM's lower leaflet was fabricated by depositing a self assembled monolayer (SAM) of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) on a gold electrode, and the upper leaflet was formed by depositing1,2-dioleoyl-sn-glycero-3-phoshocholine (DOPC) liposomes. At 0mV bias DC potential, incorporation of PorBII decreased the membrane resistance (R(m)) from 2.5 MΩc m(2) to 0.6 MΩ cm(2), giving a ΔR(m) of 1.9 MΩ cm(2) and a normalized ΔR(m) (ΔR(m) divided by the R(m) of the tBLM without PorBII) of 76%. When the bias DC potential was increased to 200 mV, the normalized ΔR(m) value decreased to 20%. The effect of applied voltage on ΔR(m) was completely reversible, suggesting voltage-dependent closure of PorBII. The voltage dependence of PorBII was further studied in a planar bilayer lipid membrane made from 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhytPC). Following a single insertion event, PorBII exhibited multiple conductance states, with reversible, voltage-dependent closure of PorBII porin occurring at high transmembrane potentials. The trimetric porin closed in three discrete steps, each step corresponding to closure of one conducting monomer unit. The most probable single channel conductance was 4.2 nS. The agreement between results obtained with the tBLM and pBLM platforms demonstrates the utility of EIS to screen channel proteins immobilized in tBLM for voltage-gated behavior.