RESUMO
INTRODUCTION: The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. OBJECTIVES/METHODS: We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). RESULTS: Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. CONCLUSIONS: Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.
Assuntos
Regulação da Expressão Gênica , Proteínas Hedgehog/genética , Hipopituitarismo/sangue , Transdução de Sinais , Adolescente , Animais , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Estudos de Coortes , Elementos Facilitadores Genéticos/genética , Feminino , Proteínas Ligadas por GPI/genética , Deleção de Genes , Variação Genética , Heterozigoto , Holoprosencefalia/metabolismo , Humanos , Hipopituitarismo/congênito , Hipopituitarismo/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Mutação , Células NIH 3T3 , Proteínas Nucleares/genética , Fenótipo , Análise de Sequência de DNA , Proteína Gli2 com Dedos de Zinco , Dedos de ZincoRESUMO
Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell. The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome. Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes. We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis. "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, [Fe-S] cluster formation and oxidative stress. We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16). In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.
Assuntos
Evolução Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Plastídeos/genética , Plastídeos/fisiologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estresse Oxidativo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
A homology model of the M(1) muscarinic acetylcholine receptor, based on the X-ray structure of bovine rhodopsin, has been used to interpret the results of scanning and point mutagenesis studies on the receptor's transmembrane (TM) domain. Potential intramolecular interactions that are important for the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N -methyl scopolamine, and the agonist, acetylcholine, have been mapped. The positively charged headgroups of these ligands probably bind in a charge-stabilized aromatic cage formed by amino acid side chains in TM helices TM3, TM6 and TM7, while residues in TM4 may participate as part of a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may be part of the mechanism for transducing binding energy into receptor activation, probably by disrupting a set of Van der Waals interactions between residues lying beneath the binding site that help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen-bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for achievement of the activated state.
Assuntos
Receptores Muscarínicos/química , Receptores Muscarínicos/metabolismo , Acetilcolina/química , Acetilcolina/metabolismo , Animais , Sítios de Ligação , Bovinos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Mutagênese , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptor Muscarínico M1 , Escopolamina/químicaRESUMO
Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Thermotoga maritima/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ferro/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase , Ligação Proteica/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência , Enxofre/metabolismoRESUMO
This review assembles data from three bodies of literature (bacterial genetics, plastid biogenesis and parasitology) that seldom have much direct cross-talk. After overcoming terminological complications to sort out microbial nifS from sufS genes, we connect a bacterial operon, recently found to be involved in iron metabolism, the formation of [Fe-S] clusters and oxidative stress to a potentially important gene (sufB) carried on the degenerate plastid genome of malaria and related parasites.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Genes de Protozoários , Plasmodium/genética , Plastídeos/genética , Terminologia como Assunto , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Genes de Plantas , Dados de Sequência Molecular , Óperon , Plasmodium/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Activation of the muscarinic acetylcholine receptors requires agonist binding followed by a conformational change, but the ligand binding and conformation-switching residues have not been completely identified. Systematic alanine-scanning mutagenesis has been used to assess residues 142-164 in transmembrane helix 4 and 402-421 in transmembrane helix 7 of the M(1) muscarinic acetylcholine receptor. Several inward-facing amino acid side chains in the exofacial parts of transmembrane helices 4 and 7 contribute to acetylcholine binding. Alanine substitution of the aromatic residues in this group reduced signaling efficacy, suggesting that they may form part of a charge-stabilized aromatic cage, which triggers rotation and movement of the transmembrane helices. The mutation of adjacent residues modulated receptor activation, either reducing signaling or causing constitutive activation. In the buried endofacial section of transmembrane helix 7, alanine substitution mutants of the conserved NSXXNPXXY motif displayed strongly reduced signaling efficacy, despite having increased or unchanged acetylcholine affinity. These residues may have dual functions, forming intramolecular contacts that stabilize the receptor in the inactive ground state, but that are broken, allowing them to form new intramolecular bonds in the activated state. This conformational rearrangement is critical to produce a G protein binding site and may represent a key mechanism of receptor activation.
Assuntos
Receptores Muscarínicos/química , Acetilcolina/química , Alanina/química , Animais , Sítios de Ligação , Células COS , Relação Dose-Resposta a Droga , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fenilalanina/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Receptor Muscarínico M1 , Receptores Muscarínicos/genética , Transdução de Sinais , TransfecçãoRESUMO
The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS-16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.
Assuntos
Aminopeptidases/química , Proteínas de Bactérias , Receptores da Transferrina/química , Aeromonas/enzimologia , Sequência de Aminoácidos , Sequência Conservada , Desenho de Fármacos , Dados de Sequência Molecular , Conformação Proteica , Pseudomonas/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Software , Streptomyces griseus/enzimologia , Dedos de Zinco , gama-Glutamil Hidrolase/químicaRESUMO
Humanization of rodent mAbs by CDR-grafting (also called "reshaping") is now a standard procedure for reducing immunogenicity and recruiting human effector functions. However, the design of the humanized mAb can sometimes prove circuitous. Attempts were made to humanize L-25, a mouse antibody against the human alpha-4 integrin subunit using the usual protocols. Despite reaching eight backmutations in the light chain, it was not possible to recover the binding activity to the level of the chimeric. In an effort to restore the binding activity, an analysis of the human kappa IV acceptor frameworks was undertaken. This analysis highlighted the Asp at position 9 in framework 1, which although a common amino acid in human kappa IV frameworks, was an unusual residue in mouse kappa frameworks. Backmutating this position to the mouse amino acid completely restored the binding of the humanized antibody and as a by-product also increased the secretion levels in cos cells. Mutating position 9 to the consensus residue for human kappa I also restored the binding and secretion levels although not to the same extent. The resulting humanized antibody had a light chain with only a single backmutation to the mouse sequence.
Assuntos
Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/metabolismo , Mutação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Sítios de Ligação/genética , Células COS , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Integrina alfa4 , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
With a view to obtaining a better understanding of the structural determinants of P1 glutamate specificity in glutamate-specific endopeptidases (GSEs), the active sites and specificity pockets of such enzymes from Bacillus licheniformis (gse-bl), Bacillus subtilis (mpr) and Staphylococcus aureus (v8 protease) were modelled. This approach was extended to the epidermolytic toxins (ETs), responsible for the staphylococcal scalded skin syndrome. We identify a canonical structure for the S1 subsite, composed of H213 and T190, both of which we predict to interact directly with the P1 glutamate. The possible importance of R30 (for gse-bl and mpr) and of the N-terminus (for gse-bl, mpr and v8 protease) was also examined. In the case of mpr, a G193C substitution is predicted to participate in a novel disulphide bridge which stabilizes C193 in such a way as to maintain the oxyanion hole. In v8, the loss or substitution of several important structural components around D102 of the catalytic triad probably explains its reduced catalytic efficiency in comparison with other GSEs. In the case of the epidermolytic toxins K216 may be important for the previously reported phospholipase C-like activity, since the model predicts that it may stabilize the negative charge on the phosphonyl group.
Assuntos
Simulação por Computador , Exfoliatinas/química , Modelos Moleculares , Serina Endopeptidases/química , Algoritmos , Sítios de Ligação , Bases de Dados Factuais , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.
Assuntos
Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/química , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Becaplermina , Sítios de Ligação , Cromatografia de Afinidade , Dicroísmo Circular , Espaço Extracelular/fisiologia , Humanos , Radioisótopos do Iodo , Cinética , Modelos Estruturais , Reação em Cadeia da Polimerase , Conformação Proteica , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-sis , Ensaio Radioligante , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
We present a model for the three-dimensional structure of the glutamate-specific endopeptidase from Streptomyces griseus based on the crystal structures of other bacterial proteases of the trypsin family. For the first time a structural model is described which attempts to explain the basis of P1 glutamate specificity in serine proteases. Several important changes to the S1 pocket with respect to other members of the family of different specificity are described. Of particular interest is the presence of a histidine at position 213 and the substitution of Arg-138 by lysine. Other biochemical evidence concerning substrate preferences can be rationalized on the basis of the model.
Assuntos
Conformação Proteica , Estrutura Secundária de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Streptomyces griseus/enzimologia , Sequência de Aminoácidos , Gráficos por Computador , Modelos Estruturais , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tripsina/químicaRESUMO
Present-day knowledge-based systems (or expert systems) and databases constitute 'islands of computing' with little or no connection to each other. The use of software to provide a communication channel between the two, and to integrate their separate functions, is particularly attractive in certain data-rich domains where there are already pre-existing database systems containing the data required by the relevant knowledge-based system. Our evolving program, GENPRO, provides such a communication channel. The original methodology has been extended to provide interactive Prolog clause input with syntactic and semantic verification. This enables automatic generation of clauses from the source database, together with complete management of subsequent interfacing to the specified knowledge-based system. The particular data-rich domain used in this paper is protein structure, where processes which require reasoning (modelled by knowledge-based systems), such as the inference of protein topology, protein model-building and protein structure prediction, often require large amounts of raw data (i.e., facts about particular proteins) in the form of logic programming ground clauses. These are generated in the proper format by use of the concept of metadata.
Assuntos
Inteligência Artificial , Biologia Molecular , Software , Sistemas Inteligentes , Sistemas de Informação , Design de SoftwareRESUMO
Organ specific autoimmune diseases are relatively common immunological disorders in man which include thyroid autoimmune disease, insulin-dependent diabetes mellitus and myasthenia gravis. The target autoantigens in some of these diseases have recently been characterised. In thyroid autoimmune disease this includes the key enzyme, thyroid peroxidase (TPO), which is involved in the generation of thyroid hormone. Structural knowledge about autoantigens such as thyroid peroxidase will allow a greater understanding of the interaction between autoantigens and the aberrant immune response, and facilitate the development of strategies for antigen-specific therapeutic manipulation. We report here a prediction of the secondary structure of thyroid peroxidase, together with the results of circular dichroic spectroscopy of a homologous purified enzyme. A combination of 3 secondary structure prediction programs has been used, following multiple sequence alignment, and TPO has been found to consist mainly of alpha-helical conformation, with little beta-sheet present. This structure prediction, together with knowledge of the exon-intron boundaries allows a model for the domain organisation of the TPO molecule to be proposed.