993 resultados para Motifs
Resumo:
The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.
Resumo:
Host Cell Factor-1 (HCF-1, C1) was first identified as a cellular target for the herpes simplex virus transcriptional activator VP16. Association between HCF and VP16 leads to the assembly of a multiprotein enhancer complex that stimulates viral immediate-early gene transcription. HCF-1 is expressed in all cells and is required for progression through G1 phase of the cell cycle. In addition to VP16, HCF-1 associates with a cellular bZIP protein known as LZIP (or Luman). Both LZIP and VP16 contain a four-amino acid HCF-binding motif, recognized by the N-terminal β-propeller domain of HCF-1. Herein, we show that the N-terminal 92 amino acids of LZIP contain a potent transcriptional activation domain composed of three elements: the HCF-binding motif and two LxxLL motifs. LxxLL motifs are found in a number of transcriptional coactivators and mediate protein–protein interactions, notably recognition of the nuclear hormone receptors. LZIP is an example of a sequence-specific DNA-binding protein that uses LxxLL motifs within its activation domain to stimulate transcription. The LxxLL motifs are not required for association with the HCF-1 β-propeller and instead interact with other regions in HCF-1 or recruit additional cofactors.
Resumo:
In the last decade, two tools, one drawn from information theory and the other from artificial neural networks, have proven particularly useful in many different areas of sequence analysis. The work presented herein indicates that these two approaches can be joined in a general fashion to produce a very powerful search engine that is capable of locating members of a given nucleic acid sequence family in either local or global sequence searches. This program can, in turn, be queried for its definition of the motif under investigation, ranking each base in context for its contribution to membership in the motif family. In principle, the method used can be applied to any binding motif, including both DNA and RNA sequence families, given sufficient family size.
Resumo:
We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson–Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert (∼20 bp) containing the modification or non-Watson–Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields (∼60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid–protein interactions.
Resumo:
The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ∼7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.
Resumo:
We present a method for discovering conserved sequence motifs from families of aligned protein sequences. The method has been implemented as a computer program called emotif (http://motif.stanford.edu/emotif). Given an aligned set of protein sequences, emotif generates a set of motifs with a wide range of specificities and sensitivities. emotif also can generate motifs that describe possible subfamilies of a protein superfamily. A disjunction of such motifs often can represent the entire superfamily with high specificity and sensitivity. We have used emotif to generate sets of motifs from all 7,000 protein alignments in the blocks and prints databases. The resulting database, called identify (http://motif.stanford.edu/identify), contains more than 50,000 motifs. For each alignment, the database contains several motifs having a probability of matching a false positive that range from 10−10 to 10−5. Highly specific motifs are well suited for searching entire proteomes, while generating very few false predictions. identify assigns biological functions to 25–30% of all proteins encoded by the Saccharomyces cerevisiae genome and by several bacterial genomes. In particular, identify assigned functions to 172 of proteins of unknown function in the yeast genome.
Resumo:
Mouse mast cells express gp49B1, a cell-surface member of the Ig superfamily encoded by the gp49B gene. We now report that by ALIGN comparison of the amino acid sequence of gp49B1 with numerous receptors of the Ig superfamily, a newly recognized family has been established that includes gp49B1, the human myeloid cell Fc receptor for IgA, the bovine myeloid cell Fc receptor for IgG2, and the human killer cell inhibitory receptors expressed on natural killer cells and T lymphocyte subsets. Furthermore, the cytoplasmic domain of gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs that are also present in killer cell inhibitory receptors; these motifs downregulate natural killer cell and T-cell activation signals that lead to cytotoxic activity. As assessed by flow cytometry with transfectants that express either gp49B1 or gp49A, which are 89% identical in the amino acid sequences of their extracellular domains, mAb B23.1 was shown to recognize only gp49B1. Coligation of mAb B23.1 bound to gp49B1 and IgE fixed to the high-affinity Fc receptor for IgE on the surface of mouse bone marrow-derived mast cells inhibited exocytosis in a dose-related manner, as defined by the release of the secretory granule constituent beta-hexosaminidase, as well as the generation of the membrane-derived lipid mediator, leukotriene C4. Thus, gp49B1 is an immunoreceptor tyrosine-based inhibition motif-containing integral cell-surface protein that downregulates the high-affinity Fc receptor for IgE-mediated release of proinflammatory mediators from mast cells. Our findings establish a novel counterregulatory transmembrane pathway by which mast cell activation can be inhibited.
Resumo:
A unique gene, RBP-MS, spanning over 230 kb in the human chromosome 8p11-12 near the Werner syndrome gene locus is described. The single-copy RBP-MS gene is alternatively spliced, resulting in a family of at least 12 transcripts (average length of 1.5 kb). Nine different types of cDNAs that encode an RNa-binding motif at the N terminus and helix-rich sequences at the C terminus have been identified thus far. Among the 16 exons identified, four 5'-proximal exons contained sequences homologous to the RNA-binding domain of Drosophila couch potato gene. Northern blot analysis showed that the RBP-MS gene was expressed strongly in the heart, prostate, intestine, and ovary, and poorly in the skeletal muscle, spleen, thymus, brain, and peripheral leukocytes. The possible role of this gene in RNA metabolism is discussed.
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Translation termination requires two codon-specific polypeptide release factors in prokaryotes and one omnipotent factor in eukaryotes. Sequences of 17 different polypeptide release factors from prokaryotes and eukaryotes were compared. The prokaryotic release factors share residues split into seven motifs. Conservation of many discrete, perhaps critical, amino acids is observed in eukaryotic release factors, as well as in the C-terminal portion of elongation factor (EF) G. Given that the C-terminal domains of EF-G interacts with ribosomes by mimicry of a tRNA structure, the pattern of conservation of residues in release factors may reflect requirements for a tRNA-mimicry for binding to the A site of the ribosome. This mimicry would explain why release factors recognize stop codons and suggests that all prokaryotic and eukaryotic release factors evolved from the progenitor of EF-G.
Resumo:
Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polygonal B-cell activation. This stimulatory motif is 20 times more common in the DNA of bacteria than higher vertebrates. The current work shows that the same motif induces the rapid and coordinated secretion of interleukin (IL) 6, IL-12, and interferon gamma (but not IL-2, IL-3, IL-4, IL-5, or IL-10) in vivo and in vitro. Stimulatory CpG DNA motifs induced B, T, and natural killer cells to secrete cytokine more effectively than did lipopolysaccharide. Thus, immune recognition of bacterial DNA may contribute to the cytokine, as well as the antibody production characteristic of an innate inflammatory response.
Resumo:
The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element.
Resumo:
HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.
Resumo:
Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-beta-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.
Resumo:
Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family.