RESUMO
Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent haemorrhage. Linkage for some families has been established to chromosome 9q33-q34. In the present study, endoglin, a transforming growth factor beta (TGF-beta) binding protein, was analysed as a candidate gene for the disorder based on chromosomal location, expression pattern and function. We have identified mutations in three affected individuals: a C to G substitution converting a tyrosine to a termination codon, a 39 base pair deletion and a 2 basepair deletion which creates a premature termination codon. We have identified endoglin as the HHT gene mapping to 9q3 and have established HHT as the first human disease defined by a mutation in a member of the TGF-beta receptor complex.
Assuntos
Glicoproteínas de Membrana/genética , Telangiectasia Hemorrágica Hereditária/genética , Fator de Crescimento Transformador beta/metabolismo , Molécula 1 de Adesão de Célula Vascular , Sequência de Aminoácidos , Antígenos CD , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Códon , DNA Complementar , Endoglina , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Linhagem , Receptores de Superfície Celular , Regiões Terminadoras GenéticasRESUMO
Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber (ORW) syndrome, is an autosomal dominant vascular dysplasia. So far, two loci have been demonstrated for ORW. Linkage studies established an ORW locus at chromosome 9q3; endoglin was subsequently identified as the ORW1 gene. A second locus, designated ORW2, was mapped to chromosome 12. Here we report a new 4 cM interval for ORW2 that does not overlap with any previously defined. A 1.38-Mb YAC contig spans the entire interval. It includes the activin receptor like kinase 1 gene (ACVRLK1 or ALK1), a member of the serine-threonine kinase receptor family expressed in endothelium. We report three mutations in the coding sequence of the ALK1 gene in those families which show linkage of the ORW phenotype to chromosome 12. Our data suggest a critical role for ALK1 in the control of blood vessel development or repair.
Assuntos
Cromossomos Humanos Par 12 , Mutação , Proteínas Serina-Treonina Quinases/genética , Telangiectasia Hemorrágica Hereditária/genética , Receptores de Ativinas , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Telangiectasia Hemorrágica Hereditária/classificaçãoRESUMO
An approach to the generation of gas phase ions by field extraction from liquid solutions has been investigated. The method uses a polymer membrane with nano-size channels as an interface between the liquid and the atmospheric pressure gas. Ions are produced by dissociation in the polar solvent and secondary ion-molecular reactions in the solution, which fills the channels of the membrane. Field extraction of the ions from the channels is stimulated by pulses of the electric discharge between the membrane and an adjacent electrode in the gas. The gas-phase ions are removed from the extraction zone by air flow and are detected by mass spectrometry. Possibilities of the membrane interface for generation of gas phase ions have been demonstrated from mass spectral investigation curried out for angiotensin II, gramicidin S and cytochrome C solutions. The current kinetics of the membrane ion source has been investigated to elucidate the mechanism of the ion extraction.
RESUMO
The Ets gene family has a complex evolutionary history with many family members known to regulate genetic programs essential for differentiation and development, and some known for their involvement in human tumorigenesis. To understand the biological properties associated with a recently described epithelium-restricted Ets factor ESX, an 11 kb fragment from the 1q32.2 genomically localized human gene was cloned and analysed. Upstream of the ESX promoter region in this genomic fragment lies the terminal exon of a newly identified gene that encodes a ubiquitin-conjugating enzyme variant, UEV-1. Tissues expressing ESX produce a primary 2.2 kb transcript along with a 4.1 kb secondary transcript arising by alternate poly(A) site selection and uniquely recognized by a genomic probe from the 3' terminal region of the 11 kb clone. Endogenous expression of ESX results in a 42 kDa nuclear protein having fivefold greater affinity for the chromatin-nuclear matrix compartment as compared to other endogenous transcription factors like AP-2 and the homologous Ets factor, ELF-1. Exon mapping of the modular structure inferred from ESX cDNA and construction of GAL4(DBD)-ESX expression constructs were used to identify a transactivating domain encoded by exon 4 having comparable potency to the acidic transactivation domain of the viral transcription factor, VP16. This exon 4-encoded 31 amino acid domain in ESX was shown by mutation and deletion analysis to possess a 13 residue acidic transactivation core which, based on modeling and circular dichroism analysis, is predicted to form an amphipathic alpha-helical secondary structure. Using recombinant GST-ESX (exon 4) fusion proteins in an in vitro pull-down assay, this ESX transactivation domain was shown to bind specifically to one component of the general transcription machinery, TATA-binding protein (TBP). Transient transfection experiments confirmed the ability of this TBP-binding transactivation domain in ESX to squelch heterologous promoters independent of any promoter binding as efficiently as the transactivation domain from VP16.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Éxons/genética , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Células COS , Cromatina/metabolismo , Dicroísmo Circular , Clonagem Molecular , DNA Complementar/genética , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , Proteínas de Neoplasias/genética , Matriz Nuclear/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-ets , Splicing de RNA , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-Box , Fatores de Transcrição/química , TransfecçãoRESUMO
X-ray diffraction was used to study the structure of assemblies formed by synthetic peptide fragments of the prion protein (PrP) that include the hydrophobic domain implicated in the Gerstmann-Sträussler-Scheinker (GSS) mutation (P102L). The effects of hydration on polypeptide assembly and of Ala-->Val substitutions in the hydrophobic domain were characterized. Synthetic peptides included: (i) Syrian hamster (SHa) hydrophobic core, SHa106-122 (KTNMKHMAGAAAAGAVV); (ii) SHa104-122(3A-V), with A-->V mutations at 113, 115 and 118 (KPKTNMKHMVGVAAVGAVV); (iii) mouse (Mo) wild-type sequence of the N-terminal hydrophobic domain, Mo89-143WT; and (iv) the same mouse sequence with leucine substitution for proline at residue number 101, Mo89-143(P101L). Samples of SHa106-122 that formed assemblies while drying under ambient conditions showed X-ray patterns indicative of 33 A thick slab-like structures having extensive H-bonding and intersheet stacking. By contrast, lyophilized peptide that was equilibrated against 100 % relative humidity showed assemblies with only a few layers of beta-sheets. The Ala-->Val substitutions in SHa104-122 and Mo89-143(P101L) resulted in the formation of 40 A wide, cross-beta fibrils. Observation of similar size beta-sheet fibrils formed by peptides SHa104-122(3A-V) and the longer Mo89-143(P101L) supports the notion that the hydrophobic sequence forms a template or core that promotes the beta-folding of the longer peptide. The substitution of amino acids in the mutants, e.g. 3A-->V and P101L, enhances the folding of the peptide into compact structural units, significantly enhancing the formation of the extensive beta-sheet fibrils.
Assuntos
Substituição de Aminoácidos/genética , Doença de Gerstmann-Straussler-Scheinker/genética , Príons/química , Príons/metabolismo , Água/metabolismo , Sequência de Aminoácidos , Animais , Cricetinae , Humanos , Ligação de Hidrogênio , Mesocricetus , Camundongos , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Príons/genética , Conformação Proteica , Dobramento de Proteína , Difração de Raios XRESUMO
The prion protein (PrP) undergoes a profound conformational change when the cellular isoform (PrPC) is converted into the scrapie form (PrPSc). Limited proteolysis of PrPsc produces PrP 27-30 which readily polymerizes into amyloid. To study the structure of PrP amyloid, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of rod-shaped PrP amyloids; flattened ribbons with a more regular substructure were found. As the concentration of HFIP was increased, the beta-sheet content and proteinase K resistance of PrP 27-30 as well as prion infectivity diminished. HFIP reversibly decreased the binding of Congo red dye to the rods while inactivation of prion infectivity was irreversible. In contrast to 10% HFIP, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolish Congo red binding. This study separates prion infectivity from the amyloid properties of PrP 27-30 and underscores the dependence of prion infectivity on PrPSc conformation. The results also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those needed for amyloid formation as determined by Congo red binding.
Assuntos
Proteína PrP 27-30/ultraestrutura , Proteínas PrPC/ultraestrutura , Conformação Proteica , Scrapie/metabolismo , 1-Propanol/farmacologia , Acetona/análogos & derivados , Acetona/farmacologia , Álcoois/farmacologia , Animais , Vermelho Congo/metabolismo , Cricetinae , Eletroforese em Gel de Poliacrilamida , Endopeptidase K , Feminino , Fluorocarbonos/farmacologia , Glicerol/farmacologia , Microscopia Eletrônica , Proteína PrP 27-30/química , Proteína PrP 27-30/patogenicidade , Proteínas PrPC/química , Propanóis , Estrutura Secundária de Proteína , Serina Endopeptidases/metabolismo , Solubilidade , Solventes/farmacologia , Espectrofotometria , Espectroscopia de Infravermelho com Transformada de Fourier , Sacarose/farmacologiaRESUMO
Certain neurodegenerative diseases in humans and animals are caused by small proteinaceous infectious particles called prions. Limited proteolysis and detergent extraction of the prions containing PrPSc generate prion rods that are composed of a polypeptide having an apparent molecular mass of 27 to 30 kDa. This polypeptide, termed prion protein PrP 27-30, has a ragged N terminus that begins at about residue 90, but retains scrapie infectivity. Moreover, the findings in a patient having an inherited prion disease of a truncated PrP with its C terminus at residue 145 suggest that the residues 90 to 145 may be of particular importance in the pathogenesis of prion diseases. To determine the three-dimensional organization of prion rods and to identify the core region involved in amyloid formation, we recorded X-ray diffraction patterns from rods purified from scrapie-infected Syrian hamster (SHa) brains which contain PrP 27-30, and from synthetic SHaPrP peptides. Three peptides were studied corresponding to residues 113 to 120 (peptide A8A, an octamer composed of glycines and alanines), 109 to 122 (H1, the first predicted alpha-helical region of PrPC), and 90 to 145 (a 56 residue peptide containing both H1 and the second predicted alpha-helical region of PrPC, H2). Electron microscopy, carried out in parallel with the X-ray measurements, revealed that all the samples formed linear polymers which were approximately 60 to approximately 200 A wide, with fibrillar or ribbon-like morphology. Gels and dried preparations of prion rods gave X-ray patterns that indicated a beta-sheet conformation, in which the hydrogen bond distance was 4.72 A and the intersheet distance was 8.82 A. For the three PrP peptides, the intersheet spacings varied widely, owing to the side-chains of the residues involved in the formation of the beta-sheet interactions, i.e., 5.13 A for A8A, 5.91 A for lyophilized H1, 7.99 A from solubilized and dried H1 and 9.15 A for the peptide SHa 90-145. The intersheet distance of PrP 27-30 was thus within the observed range for the peptides, and suggests that the amyloidogenic core of PrP is closely modeled by the peptide SHa 90-145.
Assuntos
Proteína PrP 27-30/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Encéfalo , Cricetinae , Ligação de Hidrogênio , Mesocricetus , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Proteína PrP 27-30/isolamento & purificação , Proteína PrP 27-30/ultraestrutura , Solubilidade , Difração de Raios XRESUMO
Prions are composed largely, if not entirely, of the scrapie isoform of the prion protein (PrPSc). Conversion of the cellular isoform (PrPC) to PrPSc is accompanied by a diminution in the alpha-helical content and an increase in the beta-sheet structure. To investigate the structural basis of this transition, peptide fragments corresponding to Syrian hamster PrP residues 90 to 145 and 109 to 141, which contain the most conserved residues of the prion protein and the first two putative alpha-helical regions in a PrPC model, were studied using infrared spectroscopy and circular dichroism. The peptides could be induced to form alpha-helical structures in aqueous solutions in the presence of organic solvents, such as trifluoroethanol and hexafluoroisopropanol, or detergents, such as sodium dodecyl sulfate and dodecyl phosphocholine. NaCl at physiological concentration or acetonitrile induced the peptides to acquire substantial beta-sheet. The intermolecular nature of the beta-sheet was evident in the formation of rod-shaped polymers as detected by electron microscopy. Resistance to hydrolysis by proteinase K and epitope mapping argue that the beta-sheet structures were formed by the interaction of residues lying between 109 and 141. A similar range of residues was shown by nuclear magnetic resonance spectroscopy to be capable of forming alpha-helices. The alpha-helical structures seem to require a hydrophobic support from either intermolecular interactions or the hydrophobic environment provided by micelles, in agreement with the predicted hydrophobic nature of the packing surface among the four putative helices of PrPC and the outer surfaces of the first two helices. Our results suggest that perturbation of the packing environment of the highly conserved residues is a possible mechanism for triggering the conversion of PrPC to PrPSc where alpha-helices appear to be converted into beta-sheets.
Assuntos
Peptídeos/química , Proteínas PrPC/química , Proteínas PrPSc/química , Estrutura Secundária de Proteína , 1-Propanol/farmacologia , Acetatos/farmacologia , Ácido Acético , Acetonitrilas/farmacologia , Sequência de Aminoácidos , Dicroísmo Circular , Sequência Conservada , Detergentes/farmacologia , Endopeptidase K , Epitopos/imunologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peptídeos/síntese química , Propanóis , Serina Endopeptidases/metabolismo , Cloreto de Sódio/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Trifluoretanol/farmacologiaRESUMO
Complexes of the Syrian hamster cellular prion protein (PrPC) and synthetic Syrian hamster PrP peptides were found to mimic many of the characteristics of the scrapie PrP isoform (PrPSc). Either PrPC expressed in chinese hamster ovary (CHO) cells or a C-terminal fragment of 142 residues of recombinant PrP protein (rPrP) produced in Escherichia coli was mixed with an excess of a synthetic 56 amino acid peptide, denoted PrP(90-145). Complex formation required PrPC or rPrP to be destabilized by guanidine hydrochloride (GdnHCl) or urea and PrP(90-145) to be in a coil conformation; it was enhanced by an acidic environment, salt and detergent. If PrP(90-145) was in a beta-sheet conformation, then no complexes were formed. While complex formation was rapid, acquisition of protease resistance was a slow process. Amorphous aggregates with a PrPC/PrP(90-145) ratio of 1:1 were formed in phosphate buffer, whereas fibrils with a diameter of approximately 10 nm and a PrPC/PrP(90-145) ratio of 1:5 were formed in Tris buffer. The complexes were stable only in the presence of excess peptide in either the coil or beta-sheet conformation; they dissociated rapidly after centrifugation and resuspension in buffer without peptide. Neither a peptide having a similar hydrophobicity profile/charge distribution to PrP(90-145) nor a scrambled version, denoted hPrP(90-145) and sPrP(90-145), respectively, were able to induce complex formation. Although hPrP(90-145) could stabilize the PrPC/PrP(90-145) complexes, sPrP(90-145) could not. Studies of PrPC/peptide complexes may provide insights into how PrPC interacts with PrPSc during the formation of a nascent PrPSc molecule and into the process by which PrPC is converted into PrPSc.
Assuntos
Peptídeos/química , Príons/química , Sequência de Aminoácidos , Animais , Bioensaio , Células CHO , Cricetinae , Endopeptidases/química , Cinética , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/síntese química , Príons/administração & dosagem , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.
Assuntos
Moléculas de Adesão de Célula Nervosa/química , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Processamento Alternativo/genética , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cavéolas/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Endopeptidase K/metabolismo , Formaldeído/metabolismo , Substâncias Macromoleculares , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Moléculas de Adesão de Célula Nervosa/genética , Neuroblastoma/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Fosfatidilinositol Diacilglicerol-Liase , Proteínas PrPC/genética , Proteínas PrPSc/farmacologia , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sítios de Splice de RNA/genética , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismoRESUMO
The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the beta-form of this peptide developed signs of central nervous system dysfunction at approximately 360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Sträussler-Scheinker disease. By contrast, eight of eight mice receiving a non-beta-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.
Assuntos
Encéfalo/patologia , Doença de Gerstmann-Straussler-Scheinker/genética , Fragmentos de Peptídeos/química , Príons/genética , Sequência de Aminoácidos , Animais , Encéfalo/efeitos dos fármacos , Doença de Gerstmann-Straussler-Scheinker/patologia , Doença de Gerstmann-Straussler-Scheinker/fisiopatologia , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/toxicidade , Príons/química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Scrapie/patologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.
Assuntos
Amiloide/química , Proteínas Fúngicas/química , Príons/química , Proteínas Recombinantes de Fusão/química , Proteínas de Saccharomyces cerevisiae , Amiloide/ultraestrutura , Corantes , Vermelho Congo , Proteínas Fúngicas/genética , Glutationa Peroxidase , Glutationa Transferase/genética , Microscopia Eletrônica , Príons/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/ultraestrutura , Saccharomyces cerevisiae/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.
Assuntos
Cobre/metabolismo , Príons/química , Príons/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Dicroísmo Circular , Cobre/química , Cricetinae , Eletroquímica , Histidina/química , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Príons/genética , Conformação Proteica , Sequências Repetitivas de AminoácidosRESUMO
Insulin-like growth factor binding protein-4 (IGFBP-4), like the other five IGFBPs, is a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. However IGFBP-4 seems to be the only IGFBP with no potential to enhance the mitogenic actions of the IGFs. IGFBP-1 to -3 and -5 each contain 18 conserved cysteine residues, IGFBP-6 lacks two of the twelve N-terminal cysteines, while IGFBP-4 has two additional cysteines in the central region. A plasmid was constructed to express rat IGFBP-4 as a thioredoxin fusion protein that included a hexahistidine sequence to permit affinity purification. The fusion protein was expressed in E.coli, purified using nickel-chelate affinity chromatography and cleaved by tobacco etch virus (TEV) protease to produce mature rat IGFBP-4 with an additional glycine residue at the N-terminus. Final purification was achieved by further nickel affinity chromatography and reverse phase HPLC. The isoelectric points of the recombinant IGFBP-4 were the same as those of the non-glycosylated isoforms of IGFBP-4 in rat serum. The binding affinities of the recombinant protein and IGFBP-4 secreted by rat cells to IGF-I were compared using a newly developed binding assay. No significant difference could be detected, consistent with proper folding of the recombinant protein. This indicates that glycosylation of IGFBP-4 does not affect its binding to IGF-I. Using mass spectrometry and tandem mass spectrometry no differences between authentic and recombinant IGFBP-4 could be detected. Eight of the ten disulfide linkages have been determined, including linkages of conserved cysteine residues not previously identified in other IGFBPs. Numbering the cysteine residues sequentially from the N-terminus only the disulfide connectivity of C1, C2, C5 and C6 could not be determined. However, C1 is not linked to C1 and C5 is not linked to C6. The established linkages were C3 to C8, C4 to C7, C9 to C 11, and C10 to C12. The two cysteines in the non-conserved mid-region unique to IGFBP-4 (C13 and C14) are linked together. Linkage of the C-terminal cysteine residues is identical to that of IGFBP-2, -5 and -6 (C15 to C16, C17 to C18 and C19 to C20). The central flexible core of IGFBP-4, containing two additional cysteines may contribute to its unique biological action.
Assuntos
Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Dissulfetos/metabolismo , Eletroforese em Gel Bidimensional , Endopeptidases , Escherichia coli/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/isolamento & purificação , Fator de Crescimento Insulin-Like I/metabolismo , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant Blue. A predominant band of approximately 110-130 kDa was excised and digested in-gel with trypsin. The resulting peptides were extracted then separated by microbore reversed-phase high-performance liquid chromatography (HPLC). Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser desorption/ionization (MALDI) showed nine peptide masses, which by post source decay analysis and database searching were attributed to two proteins. Nanoflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole-quadrupole-orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the additional peptides and superior quality collision-induced dissociation spectra typical of this instrument type, two further proteins were identified. The resolution afforded by the QqTOF instrument permitted charge state determination for the fragment ions while preserving the high detection sensitivity that was essential in obtaining the composition of this mixture of proteins.
Assuntos
Proteínas/química , Animais , Calibragem , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Intestinos/química , Mapeamento de Peptídeos , Coelhos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H2N=CH-R](+), where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.
RESUMO
Full-length (67 kDa) immunoreactive estrogen receptor (ER) extracted from a third of untreated ER-positive primary breast tumors appears unable to bind to its cognate estrogen response element (ERE). We have observed partial reversibility of this ER DNA-binding defect upon treatment of these tumor extracts with excess thiol reducing agent (DTT), suggesting that ER DNA-binding is subject to redox modulation as is reported for other zinc-finger proteins and transcriptional activators. Treatment of recombinant ER DNA-binding domain (ER-DBD) or ER-enriched extracts from CHO(ER) and MCF-7 cells with thiol-reacting oxidants (diamide, iodosobenzoate, H2O2) or alkylator (iodoacetamide) produces a dose-dependent loss in ER DNA-binding capacity. Thiol-specific oxidative loss in ER DNA-binding is fully reversible by DTT reduction, unlike the defect caused by thiol-specific alkylation. Circular dichroism spectrometry shows that both forms of treatment substantially modify ER secondary structure, inducing loss of alpha-helical content within the ER-DBD that is reversible after thiol oxidation but not after thiol alkylation. Oxidant (H2O2, menadione) exposure of cultured CHO(ER) or MCF-7 cells impairs the ability of endogenous ER to bind DNA and transactivate an ER-responsive reporter gene (ERE-tk-CAT), demonstrating that extracellular redox stress can modulate intracellular ER function. Since these thiol-specific oxidant and alkylator treatments have no significant effect on either recombinant ER ligand-binding or intracellular immunoreactive ER content, our findings suggest that DNA-binding and transactivation are the most sensitive intracellular ER functions impaired by oxidant stress in some ER-positive human breast tumors.
Assuntos
Neoplasias da Mama/química , DNA/metabolismo , Estresse Oxidativo , Receptores de Estrogênio/metabolismo , Alquilantes/farmacologia , Animais , Sítios de Ligação , Células CHO , Cricetinae , Diamida/farmacologia , Humanos , Peróxido de Hidrogênio , Iodoacetamida/farmacologia , Oxidantes/farmacologia , Receptores de Estrogênio/química , Receptores de Estrogênio/genética , Proteínas Recombinantes/metabolismo , Reagentes de Sulfidrila/farmacologia , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
Liquid chromatography/electrospray ionization mass spectrometry was used to investigate the peptide composition of the venom of Conus pennaceus, a molluscivorous cone shell from the Red Sea. Based on observed M(r)s, this venom contained all known conotoxins previously isolated and identified from this species. Interestingly, the doubly protonated species of only two of these conotoxins, alpha-PnIA and alpha-PnIB, showed additional related ions at +40 m/z (+80 Da), indicating the presence of either sulfation or phosphorylation in both components. High-performance liquid chromatographic (HPLC) fractions containing these two conotoxins were examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in both positive and negative ion modes, as well as by MALDI high-energy collision-induced dissociation. These experiments established the presence of a single sulfated tyrosine residue within both alpha-PnIA and alpha-PnIB. Hence their post-translationally modified sequences are GCCSLPPCAANNPDY(S)C-NH2 (alpha-PnIA) and GCCSLPPCALSNPDY(S)C-NH2 (alpha-PnIB). This assignment was supported by comparison of their mass spectral behavior with that of known sulfated and phosphorylated peptides. This data clarified further the distinguishing features of the ionization and fragmentation of such modified peptides. Selective disulfide folding of synthetic alpha-PnIB demonstrated that both sulfated and non-sulfated toxins co-elute on reversed-phase HPLC and that alpha-PnIB possesses the same disulfide connectivity as other 'classical' alpha-conotoxins reported previously.
Assuntos
Conotoxinas , Espectrometria de Massas , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Venenos de Moluscos , Tirosina/químicaRESUMO
The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by cultured adult human fibroblasts was recently identified as pregnancy-associated plasma protein-A (PAPP-A). In this study we showed that in addition to human IGFBP-4 the IGF-dependent IGFBP-4 protease also digests recombinant rat IGFBP-4 into two fragments by specifically cleaving at the carboxyl-terminal side of methionine at position 131 for rat IGFBP-4. Thus the cleavage site is at the KMKV site, which is not represented in other IGFBPs. While kallikrein may cleave at this site, its action is not specific.
Assuntos
Metaloendopeptidases/química , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Meios de Cultivo Condicionados/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Fibroblastos/enzimologia , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Calicreínas/metabolismo , Metaloendopeptidases/metabolismo , Proteína Plasmática A Associada à Gravidez/metabolismo , Ratos , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
The medical records of 89 children followed at a multidisciplinary Meningomyelocele Clinic at the Children's Clinics for Rehabilitative Services were reviewed. Almost all children in southern Arizona with meningomyelocele are followed at this clinic. Eight children (foreign nationals) were excluded because they were not eligible for neurosurgery/neurology services at the clinic. The remaining 81 children have been followed at the clinic from 0.25 to 21 years. Seventeen children (21%; age: 1.3-17 years, mean: 9.1 +/- 4.4 years; follow-up: 1.3-16 years) manifested seizures at some time during their course. All children with seizures had shunted hydrocephalus. Neonatal seizures occurred in 2 children currently not receiving medication. An additional 3 children had an acute symptomatic seizure associated with an intraventricular hemorrhage during ventriculoperitoneal shunt revision, 2 of whom later developed epilepsy. Fourteen children (17.3%) had epilepsy; 12 were taking antiepileptic drugs. Seizures were controlled on medication in 5 children. EEG abnormalities were present in 12 children (focal slowing 4, focal spikes 8, diffuse slowing 3, generalized or bilaterally synchronous spike-wave 4). Most of these children (12/14) had evidence of additional central nervous system (CNS) pathology (i.e., areas of encephalomalacia or past stroke 7, cerebral malformations 2, CNS calcifications 1, and frequent apneic spells/cardiac arrest 2). We conclude that epilepsy occurs in approximately 17% of children with meningomyelocele, and most have other CNS pathology to account for their seizures.