RESUMO
A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of â¼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galß1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.
Assuntos
Linfoma de Burkitt/metabolismo , Lectinas/química , Lectinas/toxicidade , Mytilus/metabolismo , Polissacarídeos/metabolismo , Triexosilceramidas/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/genética , Linfoma de Burkitt/fisiopatologia , Linhagem Celular Tumoral , Humanos , Células K562 , Lectinas/genética , Lectinas/metabolismo , Dados de Sequência Molecular , Mytilus/química , Mapeamento de Peptídeos , Alinhamento de Sequência , Triexosilceramidas/genéticaRESUMO
The deposition of the abundant presynaptic brain protein alpha-synuclein as fibrillary aggregates in neurons or glial cells is a hallmark lesion in a subset of neurodegenerative disorders. These disorders include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy, collectively referred to as synucleinopathies. Importantly, the identification of missense mutations in the alpha-synuclein gene in some pedigrees of familial PD has strongly implicated alpha-synuclein in the pathogenesis of PD and other synucleinopathies. However, specific post-translational modifications that underlie the aggregation of alpha-synuclein in affected brains have not, as yet, been identified. Here, we show by mass spectrometry analysis and studies with an antibody that specifically recognizes phospho-Ser 129 of alpha-synuclein, that this residue is selectively and extensively phosphorylated in synucleinopathy lesions. Furthermore, phosphorylation of alpha-synuclein at Ser 129 promoted fibril formation in vitro. These results highlight the importance of phosphorylation of filamentous proteins in the pathogenesis of neurodegenerative disorders.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Idoso , Sequência de Aminoácidos , Química Encefálica , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Humanos , Corpos de Lewy/química , Corpos de Lewy/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Sinucleínas , alfa-SinucleínaRESUMO
The high yield expression of BLT1, a G-protein coupled receptor for leukotriene B(4), was established in Pichia pastoris for structural studies. Guinea pig BLT1 was expressed in a functional form without post-translational modifications for the rapid purification and the crystallization. Among the BLT1s from four species, only guinea pig BLT1 was successfully expressed with the comparable binding affinity to BLT1 of native guinea pig tissues for several ligands. Only Asn4 of the two putative N-glycosylation sites was glycosylated, and the mutation to Ala to avoid glycosylation did not affect the ligand binding affinity. However, the N-terminal region of the mutant was digested at the carboxyl ends of Arg3 and Arg8, as detected by N-terminal amino acid sequencing, and Ser309 in the C-terminal region was partially phosphorylated, as identified in the micro-sequencing by Q-TOF-MS/MS. To avoid chemical heterogeneity, the N-terminal peptide (1-14) truncated and the C-terminal phosphorylation-site eliminated mutant was generated. The binding affinity of the mutant's membrane fraction for LTB(4) was K(d)=6.6 nM and B(max)=50.0 pmol/mg membrane protein. The yield of purified mutant was approximately 0.3-0.4 mg from 1L culture, and the protein showed a single peak at molecular weight of 100 kDa in gel-filtration and no glycosylation or phosphorylation in MALDI-TOF MS.
Assuntos
Expressão Gênica , Pichia/genética , Receptores do Leucotrieno B4/genética , Receptores do Leucotrieno B4/isolamento & purificação , Sequência de Aminoácidos , Animais , Glicosilação , Cobaias , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Receptores do Leucotrieno B4/química , Receptores do Leucotrieno B4/metabolismo , Regulação para CimaRESUMO
Autotaxin (ATX) is a tumor cell motility-stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5'-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein-coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.
Assuntos
Glucose-6-Fosfato Isomerase/metabolismo , Glicoproteínas/metabolismo , Lisofosfolipídeos/metabolismo , Complexos Multienzimáticos , Diester Fosfórico Hidrolases/metabolismo , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Glucose-6-Fosfato Isomerase/análise , Glucose-6-Fosfato Isomerase/farmacologia , Glicoproteínas/análise , Glicoproteínas/farmacologia , Neoplasias/enzimologia , Neoplasias/patologia , Fosfodiesterase I , Diester Fosfórico Hidrolases/análise , Diester Fosfórico Hidrolases/farmacologia , Pirofosfatases , Ratos , Proteínas Recombinantes/farmacologia , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Escherichia coli cytosolic glycerophosphodiester phosphodiesterase, UgpQ, functions in the absence of other proteins encoded by the ugp operon and requires Mg2+, Mn2+, or Co2+, in contrast to Ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, GlpQ. UgpQ has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. UgpQ accumulates under conditions of phosphate starvation, suggesting that it allows the utilization of glycerophosphodiesters as a source of phosphate. These results clarify how E. coli utilizes glycerophosphodiesters using two homologous enzymes, UgpQ and GlpQ.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Metais/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cobalto/metabolismo , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Immunoblotting , Magnésio/metabolismo , Manganês/metabolismo , Fosfatos/metabolismo , Diester Fosfórico Hidrolases/genética , Prótons , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.
Assuntos
Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Chaperonas Moleculares/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Deleção de Sequência , Coloração e Rotulagem , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
PH1421 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is a hypothetical protein belonging to the haloacid dehalogenase (HAD) superfamily. To gain insight into its biological function and thermostabilization mechanism, the crystal structure of PH1421 has been determined at 1.6 A resolution. The crystallographic asymmetric unit contains a homodimer. The monomeric protomer is composed of two distinct domains, a small cap domain and a large core domain, which agrees well with the typical domain organization of HAD subfamily II. Based on structure-based amino-acid sequence alignment and enzymatic analysis, PH1421 is suggested to be a magnesium-dependent phosphatase that is similar to the dimeric HAD phosphatase TA0175 from the mesothermophilic archaeon Thermoplasma acidophilum. Further comparison between the crystal structures of PH1421 and TA0175 revealed a marked structural similarity in the interprotomer dimer association. The common dimer interface with interprotomer twofold symmetry is characterized by a well conserved hydrophobic core consisting of the beta1-alpha1 loop and helices alpha1 and alpha2 of the core domain and additional contacts including the beta7-beta8 loop of the cap domain, which constitutes part of the putative active site of the enzyme. Several factors that potentially contribute to the higher thermal stability of PH1421 were identified: (i) an increase in intraprotomer hydrophobic interactions, (ii) a decrease in denaturation entropy from amino-acid composition and (iii) an increased number of intraprotomer ion pairs. These results suggest that the PH1421 protomer itself has an intrinsically higher thermal stability when compared with the mesothermophilic orthologue TA0175.
Assuntos
Biopolímeros/química , Hidrolases/química , Fosfoproteínas Fosfatases/química , Pyrococcus horikoshii/enzimologia , Adaptação Fisiológica , Sequência de Aminoácidos , Cristalização , Bases de Dados de Proteínas , Luz , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Pyrococcus horikoshii/fisiologia , Espalhamento de Radiação , Homologia de Sequência de AminoácidosRESUMO
The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.
Assuntos
Escherichia coli/genética , Proteínas Recombinantes/metabolismo , Esteroide 11-beta-Hidroxilase/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Citocromo P-450 CYP11B2/metabolismo , Eletroforese em Gel de Poliacrilamida , Ferredoxinas/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Cinética , Espectrometria de Massas , NADP/metabolismo , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Esteroide 11-beta-Hidroxilase/química , Esteroide 11-beta-Hidroxilase/genética , Especificidade por SubstratoRESUMO
The fission yeast lsd1/fas2 strain carries a temperature-sensitive mutation of the fatty-acid-synthase alpha-subunit, exhibiting an aberrant mitosis lsd phenotype, with accumulation of very-long-chain fatty-acid-containing phospholipid (VLCFA-PL). A novel 90-kDa protein, Lsd90 (SPBC16E9.16c), was found to be newly expressed in small particle-like structures in lsd1/fas2 cells under restrictive conditions. Two mismatches leading to a double frame shift were found between the sequences of the lsd90(+) gene registered in the genomic database and the sequences determined experimentally at the amino acid, cDNA and genomic DNA levels. Unexpectedly, overexpression and disruption of the lsd90(+) gene in either lsd1/fas2 or wild-type cells did not affect either cell growth or expression of the lsd phenotype. The amounts of VLCFA-PL that accumulated in lsd90-overexpressing lsd1/fas2 cells were significantly lower than those in lsd1/fas2 cells, suggesting the involvement of Lsd90 in the metabolism of VLCFA-PL.
Assuntos
Ácidos Graxos/metabolismo , Mitose , Mutação/genética , Fosfolipídeos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Peso Molecular , Fenótipo , Transporte Proteico , Proteoma/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Análise de Sequência de Proteína , Frações Subcelulares/metabolismoRESUMO
BACKGROUND: Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity. RESULTS: We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCFSkp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins in vivo. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity. CONCLUSION: SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.
Assuntos
Proteínas Culina/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Complexo do Signalossomo COP9 , Linhagem Celular , Proteínas Culina/química , Humanos , Camundongos , Poliubiquitina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/isolamento & purificação , Coelhos , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismoRESUMO
Prostaglandin E(2) is a major lipid mediator that regulates diverse biological processes. To elucidate how prostaglandin E(2) is recognized specifically by its antibody, the Fab fragment of a monoclonal anti-prostaglandin E(2) antibody was prepared and its complex with prostaglandin E(2) was crystallized. The stable Fab-prostaglandin E(2) complex was prepared by gel-filtration chromatography. Crystals were obtained by the microbatch method at 277 K using polyethylene glycol 4000 as a precipitant. A diffraction data set was collected to 2.2 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 A. The asymmetric unit was suggested to contain one molecule of the Fab-prostaglandin E(2) complex, with a corresponding crystal volume per protein weight of 2.75 A(3) Da(-1).
Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Dinoprostona , Fragmentos Fab das Imunoglobulinas/química , Animais , Anticorpos Monoclonais/imunologia , Cristalização , Cristalografia por Raios X , Dinoprostona/química , Dinoprostona/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Ligantes , Camundongos , Estrutura Molecular , Difração de Raios XRESUMO
An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.
Assuntos
Aminoácidos/química , Engenharia de Proteínas/métodos , Tirosina/análogos & derivados , Sequência de Aminoácidos , Sequência de Bases , Sistema Livre de Células , Códon , DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Variação Genética , Modelos Químicos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Fatores de Tempo , Transcrição Gênica , Tirosina/química , Tirosina/farmacologia , Proteínas ViraisRESUMO
3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme.
Assuntos
Oxirredutases do Álcool/química , Proteínas de Bactérias/química , NADP/metabolismo , Estrutura Quaternária de Proteína , Thermus thermophilus/enzimologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Sulfatos/metabolismo , Valina/metabolismoRESUMO
The stand-alone RAM (regulation of amino-acid metabolism) domain protein SraA from Thermus thermophilus HB8 (TTHA0845) was crystallized in the presence of zinc ions. The X-ray crystal structure was determined using a multiple-wavelength anomalous dispersion technique and was refined at 2.4 A resolution to a final R factor of 25.0%. The monomeric structure is a betaalphabetabetaalphabeta fold and it dimerizes mainly through interactions between the antiparallel beta-sheets. Furthermore, five SraA dimers form a ring with external and internal diameters of 70 and 20 A, respectively. This decameric structure is unique compared with the octameric and dodecameric structures found for other stand-alone RAM-domain proteins and the C-terminal RAM domains of Lrp/AsnC-family proteins.
Assuntos
Proteínas de Bactérias/química , Thermus thermophilus/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Dimerização , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Thermus thermophilus/genéticaRESUMO
A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.
Assuntos
Monoiodotirosina/metabolismo , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Animais , Western Blotting , Células CHO , Linhagem Celular , Códon/genética , Cricetinae , Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/genética , Genes Reporter/genética , Genes Supressores , Geobacillus stearothermophilus/genética , Humanos , Mamíferos , Espectrometria de Massas , Regiões Promotoras Genéticas/genética , Biossíntese de Proteínas , RNA de Transferência de Tirosina/genética , Supressão Genética/genética , Tetraciclina/farmacologia , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismoRESUMO
Mitochondrial transcription factor A (TFAM), a member of the high mobility group proteins, is essential for maintenance of mitochondrial DNA (mtDNA). Most TFAM and mtDNA (both of which are normally soluble) was recovered from the particulate fraction of human placental mitochondria when extracted with the non-ionic detergent Nonidet P-40. mtDNA and TFAM were co-immunoprecipitated by anti-TFAM antibodies. TFAM was released into the supernatant by DNase I digestion of mtDNA in the particulate fraction. Thus, TFAM and mtDNA are tightly associated with each other, and it is likely that few TFAM or mtDNA molecules exist in an unbound form in mitochondria. Based on the fact that TFAM is abundant enough to wrap mtDNA entirely, these results suggest that human mtDNA is packaged with TFAM.
Assuntos
DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA , Proteínas Mitocondriais , Proteínas Nucleares , Transativadores , Fatores de Transcrição/metabolismo , Proteínas de Xenopus , DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Desoxirribonuclease I/metabolismo , Feminino , Células HeLa , Humanos , Imuno-Histoquímica , Mitocôndrias/química , Mitocôndrias/metabolismo , Octoxinol , Polietilenoglicóis/química , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Solubilidade , Sonicação , Fatores de Transcrição/isolamento & purificação , Células U937RESUMO
Aggregates of green fluorescent protein (GFP)-fused truncated N-terminal huntingtin containing abnormally long polyglutamine tracts (150 repeats of glutamine residue) were purified from an ecdysone-inducible mutant neuro2A cell line (HD150Q-28) by using a fluorescence-activated cell sorter. To analyze the aggregate-interacting proteins, we subjected the purified aggregates to SDS-PAGE; prominent protein bands in the gel were digested with Achromobactor lysyl endopeptidase, followed by a HPLC-mass spectrometry (MS) analysis. The resulting data of tandem MS analysis revealed that, in addition to ubiquitin and widely reported chaperone proteins such as heat shock cognate 70 (HSC70), human DNA J-1 (HDJ-1), and HDJ-2, the translational elongation factor-1alpha (EF-1alpha) and heat shock protein 84 (HSP84) also were recognized as aggregate-interacting proteins. Sequestration of these proteins to aggregates was confirmed further by several immunochemical methods. We confirmed that, in addition to the other known proteins, EF-1alpha and HSP84 also colocalized with the intracellular aggregates. An assay of the transient expression of EF-1alpha and HSP84 in HD150Q-28 cells revealed that both proteins improved cell viability. Moreover, the rate of aggregate formation decreased in both transfectants. Our study suggests that both EF-1alpha and HSP84 are involved in the neurodegenerative process of polyglutamine diseases.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Doença de Huntington/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular/genética , Modelos Animais de Doenças , Citometria de Fluxo , Proteínas de Fluorescência Verde , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Humanos , Proteína Huntingtina , Doença de Huntington/etiologia , Doença de Huntington/patologia , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Corpos de Inclusão/ultraestrutura , Proteínas Luminescentes/genética , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/genética , Peptídeos/genética , Peptídeos/isolamento & purificação , Ligação Proteica/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transfecção , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate (KDGP). The genome sequence of Thermus thermophilus HB8 contains an open reading frame that has a 30% identity to Escherichia coli KDGK. The KDGK activity of T.thermophilus protein (TtKDGK) has been confirmed, and its crystal structure has been determined by the molecular replacement method and refined with two crystal forms to 2.3 angstroms and 3.2 angstroms, respectively. The enzyme is a hexamer organized as a trimer of dimers. Each subunit is composed of two domains, a larger alpha/beta domain and a smaller beta-sheet domain, similar to that of ribokinase and adenosine kinase, members of the PfkB family of carbohydrate kinases. Furthermore, the TtKDGK structure with its KDG and ATP analogue was determined and refined at 2.1 angstroms. The bound KDG was observed predominantly as an open chain structure. The positioning of ligands and the conservation of important catalytic residues suggest that the reaction mechanism is likely to be similar to that of other members of the PfkB family, including ribokinase. In particular, the Asp251 is postulated to have a role in transferring the gamma-phosphate of ATP to the 5'-hydroxyl group of KDG.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Thermus thermophilus/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.
Assuntos
Purina-Núcleosídeo Fosforilase/química , Thermus thermophilus/enzimologia , Adenosina/química , Sítios de Ligação , Calorimetria , Cristalização , Cristalografia por Raios X , Guanosina/química , Ligação de Hidrogênio , Estrutura Molecular , Conformação Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Sulfatos/químicaRESUMO
Pironetin is a potent inhibitor of tubulin assembly and arrests cell cycle progression in M phase. Analyses of its structure-activity relationships suggested that pironetin covalently binds tubulin. To determine the binding site of pironetin, we synthesized biotinylated pironetin, which inhibited tubulin assembly both in vitro and in situ. The biotinylated pironetin selectively and covalently bound with tubulin. Partial digestion of biotinylated pironetin-treated tubulin by several proteases revealed that the binding site is the C-terminal portion of alpha-tubulin. By systematic alanine scanning, the pironetin binding site was determined to be Lys352 of alpha-tubulin. Lys352 is located at the entrance of a small pocket of alpha-tubulin, and this pocket faces the beta-tubulin of the next dimer. This is the first compound that covalently binds to the alpha subunit of tubulin and Lys352 of alpha-tubulin and inhibits the interaction of tubulin heterodimers.