RESUMO
MOTIVATION: Combining multiple layers of information underlying biological complexity into a structured framework represent a challenge in systems biology. A key task is the formalization of such information in models describing how biological entities interact to mediate the response to external and internal signals. Several databases with signalling information, focus on capturing, organizing and displaying signalling interactions by representing them as binary, causal relationships between biological entities. The curation efforts that build these individual databases demand a concerted effort to ensure interoperability among resources. RESULTS: Aware of the enormous benefits of standardization efforts in the molecular interaction research field, representatives of the signalling network community agreed to extend the PSI-MI controlled vocabulary to include additional terms representing aspects of causal interactions. Here, we present a common standard for the representation and dissemination of signalling information: the PSI Causal Interaction tabular format (CausalTAB) which is an extension of the existing PSI-MI tab-delimited format, now designated PSI-MITAB 2.8. We define the new term 'causal interaction', and related child terms, which are children of the PSI-MI 'molecular interaction' term. The new vocabulary terms in this extended PSI-MI format will enable systems biologists to model large-scale signalling networks more precisely and with higher coverage than before. AVAILABILITY AND IMPLEMENTATION: PSI-MITAB 2.8 format and the new reference implementation of PSICQUIC are available online (https://psicquic.github.io/ and https://psicquic.github.io/MITAB28Format.html). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteômica , Biologia de Sistemas , Criança , Bases de Dados Factuais , Humanos , Transdução de Sinais , SoftwareRESUMO
BACKGROUND: Systems biologists study interaction data to understand the behaviour of whole cell systems, and their environment, at a molecular level. In order to effectively achieve this goal, it is critical that researchers have high quality interaction datasets available to them, in a standard data format, and also a suite of tools with which to analyse such data and form experimentally testable hypotheses from them. The PSI-MI XML standard interchange format was initially published in 2004, and expanded in 2007 to enable the download and interchange of molecular interaction data. PSI-XML2.5 was designed to describe experimental data and to date has fulfilled this basic requirement. However, new use cases have arisen that the format cannot properly accommodate. These include data abstracted from more than one publication such as allosteric/cooperative interactions and protein complexes, dynamic interactions and the need to link kinetic and affinity data to specific mutational changes. RESULTS: The Molecular Interaction workgroup of the HUPO-PSI has extended the existing, well-used XML interchange format for molecular interaction data to meet new use cases and enable the capture of new data types, following extensive community consultation. PSI-MI XML3.0 expands the capabilities of the format beyond simple experimental data, with a concomitant update of the tool suite which serves this format. The format has been implemented by key data producers such as the International Molecular Exchange (IMEx) Consortium of protein interaction databases and the Complex Portal. CONCLUSIONS: PSI-MI XML3.0 has been developed by the data producers, data users, tool developers and database providers who constitute the PSI-MI workgroup. This group now actively supports PSI-MI XML2.5 as the main interchange format for experimental data, PSI-MI XML3.0 which additionally handles more complex data types, and the simpler, tab-delimited MITAB2.5, 2.6 and 2.7 for rapid parsing and download.
Assuntos
Mapas de Interação de Proteínas , Proteoma/metabolismo , Proteômica , Bases de Dados de Proteínas , Humanos , Mutação/genética , Biologia de SistemasRESUMO
The current coronavirus disease of 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions can provide fine-grained resolution of the mechanisms behind the virus biology and the human organism response. We present a curated dataset of physical molecular interactions focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family that has been manually extracted by International Molecular Exchange (IMEx) Consortium curators. Currently, the dataset comprises over 4400 binarized interactions extracted from 151 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website (https://www.ebi.ac.uk/intact) and will be continuously updated as research on COVID-19 progresses.
Assuntos
Betacoronavirus , Coronaviridae , Infecções por Coronavirus , Interações Hospedeiro-Patógeno , Pandemias , Pneumonia Viral , Mapas de Interação de Proteínas , COVID-19 , Humanos , Especificidade de Órgãos , Proteômica , SARS-CoV-2 , Proteínas ViraisRESUMO
The current Coronavirus Disease 2019 (COVID-19) pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions enables studying fine-grained resolution of the mechanisms behind the virus biology and the human organism response. Here we present a curated dataset of physical molecular interactions, manually extracted by IMEx Consortium curators focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family. Currently, the dataset comprises over 2,200 binarized interactions extracted from 86 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website ( www.ebi.ac.uk/intact ), and will be continuously updated as research on COVID-19 progresses.
RESUMO
Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.
Assuntos
Diferenciação Celular , Dano ao DNA , Células-Tronco Multipotentes/patologia , Músculo Esquelético/patologia , Osteogênese , Animais , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Idoxuridina/farmacologia , Células-Tronco Multipotentes/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Ossificação Heterotópica , Proteínas Smad/metabolismoRESUMO
Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions.
Assuntos
Sistemas de Liberação de Medicamentos , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína/fisiologia , Humanos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Transdução de SinaisRESUMO
One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.
Assuntos
Plasmídeos de Bacteriocinas/genética , Replicação do DNA , RNA Antissenso/fisiologia , Proteínas de Ligação a RNA , Proteínas de Bactérias/genética , DNA Bacteriano/biossíntese , Amplificação de GenesRESUMO
The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides. By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA. The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription. The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scR1). ScR1 is neither 3'-polyadenylated nor 5'-trimethylguanosine capped. We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region. Haploid strains carrying the scr1-delta lesion grew very slowly, segregated cytoplasmic petites [( rho-]) at high frequency, and showed signs of aberrant cell division. A secondary structure model for scR1 shows some of the conserved features of the signal recognition particle 7SL RNAs.
Assuntos
RNA Fúngico/fisiologia , RNA/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sequência de Bases , Clonagem Molecular , Mitocôndrias/fisiologia , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Fenótipo , RNA/genética , RNA/isolamento & purificação , RNA de Cadeia Dupla/genética , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA Citoplasmático Pequeno , Mapeamento por Restrição , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Conventional wisdom has it that two proteins sharing 98.4% sequence identity have nearly identical three-dimensional structures. Here we provide a counter-example to this statement by showing that a single amino acid substitution can change the topology of a homodimeric 4-alpha-helical bundle protein. RESULTS: We have determined the high-resolution crystal structure of a 4-alpha-helical protein with a single alanine to proline mutation in the turn region, and show that this single amino acid substitution leads to a complete reorganisation of the whole molecule. The protein is converted from the canonical left-handed all-antiparallel form, to a right-handed mixed parallel and antiparallel bundle, which to the best of our knowledge and belief represents a novel topological motif for this class of proteins. CONCLUSIONS: The results suggest a possible new mechanism for the creation and evolution of topological motifs, show the importance of loop regions in determining the allowable folding pathways, and illustrate the malleability of protein structures.
Assuntos
Proteínas de Bactérias/química , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Eletricidade Estática , TermodinâmicaRESUMO
bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.
Assuntos
Zíper de Leucina/genética , Fragmentos de Peptídeos/química , Conformação Proteica , Proteínas Proto-Oncogênicas c-myc/química , Fatores de Transcrição , Células 3T3 , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Sequência Consenso , Proteínas de Ligação a DNA/química , Dimerização , Genes myc , Inibidores do Crescimento/farmacologia , Sequências Hélice-Alça-Hélice , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Engenharia de Proteínas , Multimerização Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Shc proteins are targets of activated tyrosine kinases and have been implicated in the transmission of activation signals to Ras. Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated proteins and with the Grb2 adaptor protein. Two Shc isoforms of 52 and 46 kDa have been characterized. They share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal phospho-tyrosine binding domain (PTB). We report her ethe initial characterization of two Shc related human cDNAs: ShcB and ShcC. The ShcB and ShcC cDNAs code for proteins that are highly similar and share the same modular organization as Shc. PTB and SH2 domains of ShcB and ShcC have similar binding specificities in vitro and bind to activated EGFR in a phosphotyrosine-dependent manner. Based on these findings we propose to rename Shc as ShcA. Anti-ShcB and anti-ShcC antibodies recognize specific polypeptides of 52, 47 kDa (ShcB) and 54 kDa (ShcC) in mammalian cells. Since these two genes are predominantly expressed in specific brain tissues, these Shc family members may be involved in cell type-specific signaling, in the nervous system.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Quitinases/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Plantas , Proteínas/genética , Proteínas de Ligação a RNA/genética , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Sequência de Bases , Quitinases/metabolismo , Clonagem Molecular , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/metabolismo , Humanos , Dados de Sequência Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/fisiologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Células Tumorais CultivadasRESUMO
Rop is the simplest and most regular member of a family of proteins characterized by a bundle of four antiparallel helices. Rop is dimeric, each monomer being formed by two helices connected by a sharp bend. In this work we have extensively mutagenized three residues that form the connection between the two alpha-helices to ask whether the bend region contains any important folding information. The characterization of a collection of random mutants indicated that this structure is rather insensitive to amino acid substitutions and that most amino acids are tolerated in these positions by the Rop native structure. In order to identify the rare amino acid sequences that would prevent Rop from folding and/or dimerizing, we exploited the observation that Rop can functionally substitute the dimerization domain of the lambda repressor. In fact plasmids expressing a hybrid protein formed by the amino-terminal domain of the lambda repressor covalently linked to Rop, confer immunity to lambda infection on their hosts. We have shown that this property depends on the ability of the Rop moiety to fold and dimerize. The analysis of 380 Rop mutants containing random amino acid sequences at positions 30, 31 and 32 allowed us to identify three mutant Rop proteins that are defective in dimerization, probably as a consequence of their inability to fold. In these mutants the tripeptides VED, VPD and YPD substitute the wild-type DAD at positions 30, 31 and 32. Other combinations of amino acids are found resulting in levels of immunity that are lower than the wild-type but still sufficient to prevent single plaque formation. This result suggests that a smaller proportion of the corresponding Rop protein reaches a thermodynamic and proteolytically stable dimeric state.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , Plasmídeos , Mutação Puntual , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Mapeamento por Restrição , Fatores de Transcrição/metabolismo , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
The analysis of a large number of independent mutants in the target of one of the inhibitors of pMB1 replication suggests that RNA1 regulates primer formation by base-pairing with the complementary sequence in the primer precursor. We conclude that the number of bases that are involved in the hydrogen bonding responsible for the specificity of the mechanism that controls plasmid replication and incompatibility properties is not much larger than seven. Five of these bases are located in the central loop and two in loop I of the RNA primer cloverleaf structure. Twenty-two single, double or triple mutants, with different nucleotide sequences in these seven bases, maintain an active mechanism of control, though with altered specificity. The efficiency of the inhibition mechanism correlates with the delta G value of the hydrogen bonds between the nucleotides of the two heptamers postulated to be involved in the interaction. The implications of these findings are discussed, and a molecular model of the interaction between RNA1 and the primer precursor is presented.
Assuntos
Replicação do DNA , Regulação da Expressão Gênica , Precursores de Ácido Nucleico/genética , Plasmídeos , RNA/genética , Bacteriófago lambda/genética , Sequência de Bases , DNA Viral , Ligação de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Precursores de Ácido Nucleico/metabolismo , RNA/biossíntese , RNA/metabolismo , Precursores de RNARESUMO
The hydrophobic core packing in four-alpha-helical bundles appears to be crucial for stabilizing the protein structure. To examine the structural basis of hydrophobic stabilization, the crystal structures of the Leu-->Val (L41V) and Leu-->Ala (L41A) substitutions of the core residue Leu41 of the ROP protein have been determined. Both substitutions are destabilizing and lead to formation of cavities. The main responses to mutations are the collapse of the central part of the alpha-helix containing the site of mutation, shifts of internal water molecules, and in L41A, the trapping of a water molecule in a cavity engineered by the mutation. For both mutants, these effects limit the increase in cavity size to less than 10 A3, while an increase of 37 A3 and 100 A3 is expected for L41V and L41A, respectively, in the absence of any cavity size reducing effects. The mobility of internal side-chains is increased and in L41A, it reaches values typical for exposed residues. A parameter (Deltanh) is introduced as a measure of the number of van der Waals contacts lost. For ROP, barnase and T4 lysozyme mutants, there is a good correlation between Deltanh and the free energy of unfolding DeltaDeltaG relative to wild-type protein. The Deltanh value turns out to be more suitable for analysing structural and energetic responses to mutation than other parameter, such as cavity volumes and packing densities. Possible evolutionary implications of the DeltaDeltaG versus Deltanh relationship are discussed.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a RNA/química , Proteínas de Bactérias/genética , Leucina/química , Mutagênese , Conformação Proteica , Proteínas de Ligação a RNA/genéticaRESUMO
Four closely related species of yeast possess multicopy nuclear plasmids whose shared molecular architecture demonstrates a common ancestor, despite their lack of discernible DNA sequence homology. Each plasmid encodes three proteins which have equivalent essential functions in plasmid maintenance. These three groups of proteins show markedly different degrees of conservation, so that although we have successfully aligned sequences for two groups, members of the third group have diverged to such an extent that they cannot be aligned. All the proteins are sufficiently different that they function only in conjunction with their encoding plasmid. These proteins have therefore conserved their functional interactions with the relevant DNA sequences of their particular plasmids, despite lack of amino acid sequence conservation. The maintenance of function in the face of DNA sequence divergence is analogous to the coevolution of ribosomal DNA promoters and RNA polymerase I, and suggests that molecular drive may be an important force in the evolution of these plasmids. This view is reinforced by the inconsistent phylogenetic relationships determined from the two alignment sets, and by the contradiction that the two plasmids known to be the closest related taxonomically and by their host interchangeability are suggested to be the most distant by their sequences.
Assuntos
Evolução Biológica , Plasmídeos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Composição de Bases , DNA Fúngico , Proteínas Fúngicas , Genes Fúngicos , Dados de Sequência MolecularRESUMO
Ff filamentous phages are long thin cylindrical structures that infect bacteria displaying the F pilus and replicate without lysing the host. These structures are exploited to display peptides by fusing them to the amino terminus of either the bacterial receptor protein (pIII) or the major coat protein (pVIII). We have analysed a vast collection of phage mutants containing substitutions and insertions in the amino terminus of pVIII to ask whether any chemical group of this solvent exposed region of the phage capsid has any key function in the phage life cycle. Any of the five amino-terminal residues can be substituted by most amino acids without affecting phage assembly suggesting that this region does not play any essential role in morphogenesis. However, a deletion of three residues delta (Gly3Asp4Asp5) results in a phage clone with an decreased ability to produce infective particles. By engineering phages designed to display peptides by fusion to the amino terminus of the major coat protein we have found that phage viability is affected by peptide length while peptide sequence plays a minor "tuning" role. Most peptides of six residues are tolerated irrespective of their sequence while only 40% of the phages carrying an amino-terminal extension of eight residues can form infective particles. This fraction drops to 20% and 1% when we attempt to insert peptides 10 and 16 amino acids long. We have used this information to build phage libraries where each phage displays approximately 2700 copies of a different octapeptide all over the phage surface.
Assuntos
Capsídeo/genética , Vetores Genéticos/genética , Inovirus/genética , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/química , Genes Virais/genética , Inovirus/crescimento & desenvolvimento , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Proteínas Recombinantes de Fusão , Deleção de Sequência/fisiologia , Proteínas Estruturais Virais/genéticaRESUMO
Preliminary crystallographic data are given for Rop, a protein involved in the control of replication of plasmids of the ColE1 family.
Assuntos
Proteínas de Bactérias , Escherichia coli/análise , Plasmídeos de Bacteriocinas , Cristalização , Replicação do DNA , DNA BacterianoRESUMO
Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.
Assuntos
Anticorpos/metabolismo , Colífagos/genética , Epitopos , Oligopeptídeos/imunologia , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/genética , Biblioteca Gênica , Vetores Genéticos , Ligantes , Dados de Sequência Molecular , Oligopeptídeos/química , Plasmídeos , Proteínas Recombinantes/imunologia , Mapeamento por RestriçãoRESUMO
One of the goals of functional genomics is the description of reliable and complete protein interaction networks. To facilitate ligand discovery from complex protein mixtures, we have developed an improved approach that is affected by a negligible fraction of false positives. We have combined a novel technique based on the display of cDNA libraries on the capsid of bacteriophage lambda and an efficient plaque assay to reveal phage displaying ligands that are enriched after only a couple of affinity purification steps. We show that the lambda display system has a unique ability to display, at high density, proteins ranging in size from a few to at least 300 amino acid residues. This characteristic permits attenuation of the size bias in the selection procedure and, at the same time, offers a sensitive plaque assay that permits us to do away with the ligand background without unduly increasing the number of selection cycles. By using a proline-rich fragment of the synaptojanin 1 protein as a bait, we have identified, in a brain cDNA display library, seven ligands all containing either SH3 or WW domains. Four of these correspond to proteins that have already been validated as physiological partners, while the remaining three are new partners, whose physiological relevance remains to be established. Two different proline-rich regions of the p21-activated protein kinase 1 (Pak1) and WAVE/SCAR2 protein retrieve from the library different proteins containing SH3 or WW domains.
Assuntos
Bacteriófago lambda/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Biblioteca de Peptídeos , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Bacteriófago T7/genética , Sítios de Ligação , Encéfalo/metabolismo , Biblioteca Gênica , Humanos , Ligantes , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Mutação/genética , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos , Monoéster Fosfórico Hidrolases/genética , Prolina/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Ensaio de Placa Viral , Família de Proteínas da Síndrome de Wiskott-Aldrich , Quinases Ativadas por p21 , Domínios de Homologia de srcRESUMO
Although some protein folding theories sustain that the peptides (loops) that connect elements of more compact secondary structure may be important in the folding process, most of the data accumulated until now seems to contradict this notion. To approach this problem we have isolated and characterized a number of mutants in which the amino acid sequence of the peptide that connects helix D and helix E in the H-chain of human ferritin has been randomized. Our results indicate that, though no single loop residue is absolutely required for ferritin to attain the native conformation, most of the mutants that we have obtained by random regional mutagenesis, affect its folding/assembly process. This conclusion was reached utilizing a sensitive test that associates the color formed by a colony synthesizing a hybrid ferritin-beta-galactosidase protein to the ability of the ferritin domain to fold and assemble as the native protein. The characterization of the folding/assembly properties of our collection of mutants and the comparison of the mutant loop sequences, have allowed us to draw the following conclusions. Mutants that have positively charged residues at position 159, 160 or 161 fail to assemble into the native protein shell and form an insoluble aggregate. Interestingly some loop amino acid sequences cause the E-helix to reverse direction and to expose its COOH group, normally hidden inside the protein cavity, to the solvent. The propensity of a given ferritin mutant to fold into this "non-native" conformation can be attenuated by the introduction of Gly at position 159 and 164, as in the natural ferritin.