Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA , Escherichia coli/genética , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Serina Endopeptidases/genética , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Two-hybrid searches with the tumor suppressor MMAC1/PTEN isolated the proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibited both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and > 200,000 proteins, but promoted coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions.
Assuntos
Proteínas de Transporte/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/metabolismo , Treonina/metabolismo , Proteínas Supressoras de Tumor , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteína 1 Homóloga a Discs-Large , Guanilato Quinases , Humanos , Proteínas de Membrana , Camundongos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Células Tumorais CultivadasRESUMO
There are several lines of evidence indicating that the carboxy-terminal region of the tumor suppressor protein BRCA1 is a functionally significant domain. Using the yeast two-hybrid and in vitro biochemical assays, we show that a protein, CtIP, interacts specifically with the carboxy-terminal segment of human BRCA1 from residues 1602-1863. A germ line truncation mutation, Y1853ter, that removes the last 11 amino acids from the carboxy-terminus of BRCA1, abolishes not only its transcriptional activation function, but also binding to CtIP. The function of CtIP is unknown, but its reported association with a transcriptional repressor CtBP lends further support that it may have a role in transcription. A sequence based screen of a panel of 89 tumor cell line cDNAs for mutations in the CtIP coding region identified five missense variants. In the pancreatic carcinoma cell line, BxPC3, the non-conservative lysine to glutamic acid change at codon 337 is accompanied with apparent loss of heterozygosity or non-expression of the wild type allele. Thus it is plausible that CtIP may itself be a tumor suppressor acting in the same pathway as BRCA1.
Assuntos
Proteína BRCA1/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sequência de Bases , Genes Reporter , Mutação em Linhagem Germinativa , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Recent work has shown that the murine BRCA2 tumor suppressor protein interacts with the murine RAD51 protein. This interaction suggests that BRCA2 participates in DNA repair. Residues 3196-3232 of the murine BRCA2 protein were shown to be involved in this interaction. Here, we report the detailed mapping of additional domains that are involved in interactions between the human homologs of these two proteins. Through yeast two-hybrid and biochemical assays, we demonstrate that the RAD51 protein interacts specifically with the eight evolutionarily conserved BRC motifs encoded in exon 11 of brca2 and with a similar motif found in a Caenorhabditis elegans hypothetical protein. Deletion analysis demonstrates that residues 98-339 of human RAD51 interact with the 59-residue minimal region that is conserved in all BRC motifs. These data suggest that the BRC repeats function to bind RAD51.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Proteína BRCA2 , Predisposição Genética para Doença , Humanos , Ligação Proteica , Rad51 Recombinase , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMO
Genome sequencing projects are predicting large numbers of novel proteins, whose interactions with other proteins must mediate the function of cellular processes. To analyse these networks, we used the yeast two-hybrid system on a genome-wide scale to identify 25 interactions among the proteins of Escherichia coli bacteriophage T7. Among these is a set of six interactions connecting proteins that function in DNA replication and DNA packaging. Remarkably, two genes, arranged such that one entirely overlaps the other and uses a different reading frame, encode interacting proteins. Several of the interactions reflect intramolecular associations of different domains of the same polypeptide, suggesting that the two-hybrid assay may be useful in the analysis of protein folding. This global approach to protein-protein interactions may be applicable to the analysis of more complex genomes whose sequences are becoming available.
Assuntos
Bacteriófago T7/ultraestrutura , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacteriófago T7/química , Bacteriófago T7/genética , Sequência de Bases , Primers do DNA/química , Replicação do DNA , Homologia de Genes , Vetores Genéticos , Substâncias Macromoleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/metabolismo , Proteínas Virais/genética , Replicação ViralAssuntos
Proteínas de Ligação a DNA/metabolismo , Plasmídeos , Multimerização Proteica , Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular/métodos , Primers do DNA , Escherichia coli/genética , Biblioteca Gênica , Marcadores Genéticos , Vetores Genéticos , Genótipo , Humanos , Indicadores e Reagentes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Transfecção/métodos , Técnicas do Sistema de Duplo-HíbridoRESUMO
p53 is a tumor-suppressor protein that can activate and repress transcription. Using the yeast two-hybrid system, we identified two previously uncharacterized human proteins, designated 53BP1 and 53BP2, that bind to p53. 53BP1 shows no significant homology to proteins in available databases, whereas 53BP2 contains two adjacent ankyrin repeats and a Src homology 3 domain. In vitro binding analyses indicate that both of these proteins bind to the central domain of p53 (residues 80-320) required for site-specific DNA binding. Consistent with this finding, p53 cannot bind simultaneously to 53BP1 or 53BP2 and to a DNA fragment containing a consensus p53 binding site. Unlike other cellular proteins whose binding to p53 has been characterized, both 53BP1 and 53BP2 bind to the wild-type but not to two mutant p53 proteins identified in human tumors, suggesting that binding is dependent on p53 conformation. The characteristics of these interactions argue that 53BP1 and 53BP2 are involved in some aspect of p53-mediated tumor suppression.
Assuntos
Proteínas de Transporte/metabolismo , Mutagênese Insercional , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Anquirinas/química , Sequência de Bases , Sítios de Ligação , Northern Blotting , Proteínas de Transporte/química , DNA/metabolismo , Glutationa Transferase/biossíntese , Glutationa Transferase/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismoRESUMO
We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins. Plasmids are constructed to encode two hybrid proteins. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. We used this method with the yeast SIR4 protein, which is involved in the transcriptional repression of yeast mating type information. (i) We used the two-hybrid system to demonstrate that SIR4 can form homodimers. (ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction. (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library. This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.
Assuntos
Clonagem Molecular/métodos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Fatores de Transcrição , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Vetores Genéticos , Biblioteca Genômica , Substâncias Macromoleculares , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning the actI, actIII, actIV, and actVII genes (pANT12) of actinorhodin biosynthesis pathway from Streptomyces coelicolor in anthracycline producing streptomycetes. Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kb XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus 31 133. When S. galilaeus 31 671 was transformed with the actI, actVII, and actIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-O-methyldesoxyerythrolaccin. When S. galilaeus 31 671 was transformed with only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci, dauG, dnrR1, and asaA, on the production of natural and hybrid polyketides were also shown.
Assuntos
Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Streptomycetaceae/genética , Clonagem Molecular , Genes Bacterianos , Mapeamento por Restrição , Streptomycetaceae/metabolismoRESUMO
Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and 2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIII, actIV, and actVII, encoding early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobase-pair XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus ATCC 31133. Aloesaponarin II was synthesized via the condensation of 8 acetyl coenzyme A equivalents, followed by a decarboxylation reaction as demonstrated by [1,2-13C2]acetate feeding experiments. S. coelicolor B22 and B159, actVI blocked mutants, also formed aloesaponarin II as an apparent shunt product. Mutants of S. coelicolor blocked in several other steps in actinorhodin biosynthesis did not synthesize aloesaponarin II or other detectable anthraquinones. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actIII, and actVII loci, the recombinant strain produced both aloesaponarin II and aklavinone, suggesting that the actinorhodin biosynthesis DNA encoded a function able to deoxygenate 2-hydroxyaklavinone to aklavinone. When S. galilaeus ATCC 31671 was transformed with a plasmid carrying only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled polyketide to the corresponding secondary alcohol. In the presence of the actIII gene, anthraquinones or anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on which the keto reductase operated. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actVII, and actIV loci, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin, the 3-hydroxy analog of aloesaponarin II, and 1-O-methyldesoxyerythrolaccin. The results obtained in these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related compounds by S. coelicolor.