Genetic and molecular biological analysis of protein-protein interactions in coronavirus assembly.
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Amino Acid Sequence↗
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A second protein kinase CK1-mediated step negatively regulates Wnt signalling by disrupting the lymphocyte enhancer factor-1/beta-catenin complex.
Deregulated activation of the canonical Wnt signalling pathway leads to stabilization of beta-catenin and is critically involved in carcinogenesis by an inappropriate induction of lymphocyte enhancer factor (LEF-1)/beta-catenin-dependent transcription of Wnt target genes. Phosphorylation of the pathway components beta-catenin, Dishevelled, Axin and APC (adenomatous polyposis coli) by glycogen synthase kinase-3beta, CK1 and CK2 is of central importance in the regulation of the beta-catenin destruction complex. Here, we identify CK1 and CK2 as major kinases that directly bind to and phosphorylate LEF-1 inducing distinct, kinase-specific changes in the LEF-1/DNA complex. Moreover, CK1-dependent phosphorylation in contrast to CK2 disrupts the association of beta-catenin and LEF-1 but does not impair DNA binding of LEF-1. Sequential phosphorylation assays revealed that for efficient disruption of the LEF-1/beta-catenin complex, beta-catenin also has to be phosphorylated. Consistent with these observations, CK1-dependent phosphorylation inhibits, whereas CK2 activates LEF-1/beta-catenin transcriptional activity in reporter gene assays. These data are in line with a negative regulatory function of CK1 in the Wnt signalling pathway, where CK1 in addition to the beta-catenin destruction complex at a second level acts as a negative regulator of the LEF-1/beta-catenin transcription complex, thereby protecting cells from development of cancer.
Binding Sites↗
nDsbD: a redox interaction hub in the Escherichia coli periplasm.
DsbD is a redox-active protein of the inner Escherichia coli membrane possessing an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain. nDsbD interacts with four different redox proteins involved in the periplasmic disulfide isomerization and in the cytochrome c maturation systems. We review here the studies that led to the structural characterization of all soluble DsbD domains involved and, most importantly, of trapped disulfide intermediate complexes of nDsbD with three of its four redox partners. These results revealed the structural features enabling nDsbD, a 'redox hub' with an immunoglobulin-like fold, to interact efficiently with its different thioredoxin-like partners.
Amino Acid Motifs↗
Expansion of amino acid homo-sequences in proteins: insights into the role of amino acid homo-polymers and of the protein context in aggregation.
Expansion of amino acid homo-sequences, such as polyglutamines or polyalanines, in proteins has been directly implicated in various degenerative diseases through a mechanism of protein misfolding and aggregation. However, it is still unclear how the nature of the expansion and the protein context influence the tendency of a protein to aggregate. Here, we have addressed these questions using spinocerebellar ataxia type-3 (ATX3) protein, the best characterised of the polyglutamine proteins, chosen as a model system. Using a transfected mammalian cell line, we demonstrate that ATX3 aggregation is noticeably reduced by deletion or replacement of regions other than the polyglutamine tract. The nature of the amino acid homo-sequences also has a strong influence on aggregation. From our studies, we draw general conclusions on the effect of the protein architecture and of the amino acid homo-sequence on pathology.
Amino Acids↗
Stimulation of c-Rel transcriptional activity by PKA catalytic subunit beta.
Nuclear factor kappaB (NF-kappaB) is a eukaryotic transcription factor which responds to different extracellular signals. It is involved in immune response, inflammation, and cell proliferation. Increased expression of c-Rel (or its viral homolog v-Rel), one component of the NF-kappaB factors, induces tumorigenesis in different systems. The activity of NF-kappaB can be regulated by protein kinase A (PKA) in a cAMP-independent manner. Our previous results showed that c-MYC induces the activity of PKA by inducing the transcription of the gene encoding the PKA catalytic subunit beta (PKA-Cbeta). Constitutive expression of PKA-Cbeta in Rat1a cells induces their transformation. Here we show that CREB is unlikely to be a phosphorylation target of PKA-Cbeta as characterized by different cell lines. Electrophoretic mobility shift assays showed that c-Rel is present as a significant component of the NF-kappaB factors in c-MYC overexpressing status. The transcriptional activity of c-Rel was significantly stimulated by PKA-Cbeta. Coactivators p300/CBP are at least partially responsible for the enhanced activation mediated by c-Rel and PKA-Cbeta. Interaction between c-Rel and PKA-Cbeta was demonstrated using coimmunoprecipitation assays. Immunoprecipitation-in vitro phosphorylation assays showed the direct phosphorylation of c-Rel by PKA-Cbeta. These results indicate that c-Rel is a reasonable phosphorylation target of PKA-Cbeta, and that the transcriptional activity of c-Rel is stimulated by PKA-Cbeta possibly through the interaction with p300/CBP.
Animals↗
The IA-2 interactome.
AIMS/HYPOTHESIS: Islet antigen-2 (IA-2), a major autoantigen in type 1 diabetes, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase (PTP) family. IA-2 is located in dense-core secretory vesicles and is involved in the regulation of insulin secretion. The present experiments were initiated to identify those proteins that interact with IA-2 (i.e. the IA-2 interactome) as a first step towards elucidating the mechanism(s) by which IA-2 influences insulin secretion and serves as an autoantigen. MATERIALS AND METHODS: To determine the proteins with which IA-2 interacts, a yeast two-hybrid system was used to screen a human foetal library, and deletion mutants were used to determine the binding sites. Positive interactions were confirmed by immunoprecipitation pull-down experiments using cell lysate from transfected mammalian cell lines. RESULTS: Six new interacting proteins were identified by this approach: mitogen-activated protein kinase-activating death domain (MADD), the MADD isoform IG20, PTPrho, PTPsigma, sorting nexin 19 (SNX19) and cyclophilin A. Using a series of IA-2 deletion mutants, we identified the regions on the IA-2 molecule to which five of the interacting proteins bound. Amino acids 744-979 of IA-2 were required for the maximum binding of MADD, IG20 and SNX19, whereas amino acids 602-907 of IA-2 were required for the maximum binding of PTPrho and PTPsigma. Pull-down experiments with cell lysate from transfected mammalian cells confirmed the binding of the interacting proteins to IA-2. CONCLUSIONS/INTERPRETATION: The IA-2 interactome based on, pull-down experiments, currently consists of 12 proteins. The identification of these interacting proteins provides clues as to how IA-2 exerts its biological functions.
Animals↗
The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16.
The H(2)-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H(2)-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H(2) sensing and H(2) oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.
Amino Acid Substitution↗
Demonstration of interactions among Myxococcus xanthus Dif chemotaxis-like proteins by the yeast two-hybrid system.
The Myxococcus xanthus dif locus encodes several bacterial chemotaxis homologues that are crucial for fibril exopolysaccharide (EPS) production, social gliding motility, and fruiting body development. In primary sequence, DifA is homologous to methyl-accepting chemotaxis protein, DifC to CheW, DifD to CheY, DifE to CheA, and DifG to CheC. In this study, the interactions among the Dif chemotaxis-like proteins were investigated using the yeast two-hybrid (Y2H) system. DifC was found to interact with both DifA and DifE. Using a modified Y2H or a "three-hybrid" system, it was demonstrated that DifC is capable of mediating the formation of DifA, DifC, and DifE ternary protein complexes. The conserved domains of DifE, based on sequence analysis, likely reflect functional conservations of CheA-type kinases, because its P2 domain interacts with DifD, P5 with DifC, and the P3 domain appears to dimerize. Similarly, C-terminal regions of DifA appear to dimerize as well. In addition, DifG was found to interact with DifD, which is consistent with the hypothesis that DifG is a phosphatase of DifD-phosphate. These findings support the models in which Dif proteins constitute a unique chemotaxis-like signal transduction pathway with central functions in regulating EPS production in M. xanthus.
Bacterial Proteins↗
Mutations in the heterochromatin protein 1 (HP1) hinge domain affect HP1 protein interactions and chromosomal distribution.
Heterochromatin Protein 1 (HP1) is a conserved component of the highly compact chromatin found at centromeres and telomeres. A conserved feature of the protein is multiple phosphorylation. Hyper-phosphorylation of HP1 accompanies the assembly of cytologically distinct heterochromatin during early embryogenesis. Hypo-phosphorylated HP1 is associated with the DNA-binding activities of the origin recognition complex (ORC) and an HMG-like HP1/ORC-Associated Protein (HOAP). Perturbations in HP1 localization in pericentric and telomeric heterochromatin in mutants for Drosophila ORC2 and HOAP, respectively, indicate roles for these HP1 phosphoisoforms in heterochromatin assembly also. To elucidate the roles of hypo- and hyper-phosphophorylated HP1 in heterochromatin assembly, we have mutated consensus Protein Kinase-A phosphorylation sites in the HP1 hinge domain and examined the mutant proteins for distinct in vitro and in vivo activities. Mutations designed to mimic hyper-phosphorylation render the protein incapable of binding HOAP and the DmORC1 subunit but confer enhanced homo-dimerization and lysine 9-methylated histone H3-binding to the protein. Mutations rendering the protein unphosphorylatable, by contrast, do not affect homo-dimerization or binding to lysine 9-di-methylated histone H3, HOAP, or DmORC1 but do confer novel DmORC2-binding activity to the protein. This mutant protein is ectopically localized throughout the chromosomes when overexpressed in vivo in the presence of a full dose of DmORC2. This ectopic targeting is accompanied by ectopic targeting of lysine 9 tri-methylated histone H3. The distinct activities of these mutant proteins could reflect distinct roles for HP1 phosphoisoforms in heterochromatin structure and function.
Amino Acid Sequence↗
Protein-protein interaction and functionTRPC channels.
Since their identification in the concluding years of the last century, the mammalian transient receptor potential (canonical) (TRPC) channels have remained in the limelight as the primary candidates for the Ca(2+) entry pathway activated by the hormones, growth factors, and neurotransmitters that exert their effect through activation of PLC. Although TRPC channels have been shown clearly to mediate, at least in part, receptor-activated Ca(2+) entry in literally all cell types, several of their central characteristics, as recorded in expression systems using recombinant channels, differ from those of the native receptor-dependent Ca(2+) influx channels. The present review attempts to highlight the interaction of TRPC channels with other proteins, which may explain the variability of TRPC channel activation and regulatory mechanisms observed with the native and recombinant channels. These include the homologous and heterotopous interactions of TRPC channel isoforms, the interaction of TRPC channels with calmodulin, PLCgamma, IP(3) receptors, and with scaffolding proteins like InaD, EBP50/NEHRF, caveolin, Janctate and Homers.
Animals↗
Functional role of TRPC channels in the regulation of endothelial permeability.
The endothelial cells (ECs) form a semipermeable barrier between the blood and the tissue. An important function of the endothelium is to maintain the integrity of the barrier function of the vessel wall. Ca(2+) signaling in ECs plays a key role in maintaining the barrier integrity. Transient receptor potential canonical (TRPC) channels are mammalian homologs of Drosophila TRP Ca(2+)-permeable channels expressed in EC. TRPC channels are thought to function as a Ca(2+) entry channel operated by store-depletion as well as receptor-activated channels in a variety of cell types, including ECs. Inflammatory mediators such as thrombin, histamine, bradykinin, and others increase endothelial permeability by actin polymerization-dependent EC rounding and formation of inter-endothelial gaps, a process critically dependent on the increase in EC cytosolic [Ca(2+)] ([Ca(2+)](i)). Increase in endothelial permeability depends on both intracellular Ca(2+) release and extracellular Ca(2+) entry through TRPC channels. This review summarizes recent findings on the role of TRPC channels in the mechanism of Ca(2+) entry in ECs, and, in particular, the role of TRPC channels in regulating endothelial barrier function.
Animals↗
The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein.
Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2.
Amino Acid Sequence↗
Phosphorylation and subsequent interaction with 14-3-3 proteins regulate plastid glutamine synthetase in Medicago truncatula.
In this report we demonstrate that plastid glutamine synthetase of Medicago truncatula (MtGS2) is regulated by phosphorylation and 14-3-3 interaction. To investigate regulatory aspects of GS2 phosphorylation, we have produced non-phosphorylated GS2 proteins by expressing the plant cDNA in E. coli and performed in vitro phosphorylation assays. The recombinant isoenzyme was phosphorylated by calcium dependent kinase(s) present in leaves, roots and nodules. Using an (His)6-tagged 14-3-3 protein column affinity purification method, we demonstrate that phosphorylated GS2 interacts with 14-3-3 proteins and that this interaction leads to selective proteolysis of the plastid located isoform, resulting in inactivation of the isoenzyme. By site directed mutagenesis we were able to identify a GS2 phosphorylation site (Ser97) crucial for the interaction with 14-3-3s. Phosphorylation of this target residue can be functionally mimicked by replacing Ser97 by Asp, indicating that the introduction of a negative charge contributes to the interaction with 14-3-3 proteins and subsequent specific proteolysis. Furthermore, we document that plant extracts contain protease activity that cleaves the GS2 protein only when it is bound to 14-3-3 proteins following either phosphorylation or mimicking of phosphorylation by Ser97Asp.
14-3-3 Proteins↗
Structure-based method for analyzing protein-protein interfaces.
Hydrogen bond, hydrophobic and vdW interactions are the three major non-covalent interactions at protein-protein interfaces. We have developed a method that uses only these properties to describe interactions between proteins, which can qualitatively estimate the individual contribution of each interfacial residue to the binding and gives the results in a graphic display way. This method has been applied to analyze alanine mutation data at protein-protein interfaces. A dataset containing 13 protein-protein complexes with 250 alanine mutations of interfacial residues has been tested. For the 75 hot-spot residues (deltadelta G > or = 1.5 kcal mol(-1)), 66 can be predicted correctly with a success rate of 88%. In order to test the tolerance of this method to conformational changes upon binding, we utilize a set of 26 complexes with one or both of their components available in the unbound form. The difference of key residues exported by the program is 11% between the results using complexed proteins and those from unbound ones. As this method gives the characteristics of the binding partner for a particular protein, in-depth studies on protein-protein recognition can be carried out. Furthermore, this method can be used to compare the difference between protein-protein interactions and look for correlated mutation.
Alanine↗
Evolutionary trace analysis of ionotropic glutamate receptor sequences and modeling the interactions of agonists with different NMDA receptor subunits.
The ionotropic N-methyl- d-aspartate (NMDA) receptor is of importance in neuronal development, functioning, and degeneration in the mammalian central nervous system. The functional NMDA receptor is a heterotetramer comprising two NR1 and two NR2 or NR3 subunits. We have carried out evolutionary trace (ET) analysis of forty ionotropic glutamate receptor (IGRs) sequences to identify and characterize the residues forming the binding socket. We have also modeled the ligand binding core (S1S2) of NMDA receptor subunits using the recently available crystal structure of NR1 subunit ligand binding core which shares approximately 40% homology with other NMDA receptor subunits. A short molecular dynamics simulation of the glycine-bound form of wild-type and double-mutated (D481N; K483Q) NR1 subunit structure shows considerable RMSD at the hinge region of S1S2 segment, where pore forming transmembrane helices are located in the native receptor. It is suggested that the disruption of domain closure could affect ion-channel activation and thereby lead to perturbations in normal animal behavior. In conclusion, we identified the amino acids that form the ligand-binding pocket in many ionotropic glutamate receptors and studied their hydrogen bonded and nonbonded interaction patterns. Finally, the disruption in the S1S2 domain conformation (of NR1 subunit- crystal structure) has been studied with a short molecular dynamics simulation and correlated with some experimental observations. [figure: see text]. The figure shows the binding mechanism of glutamate with NR2B subunit of the NMDA receptor. Glutamate is shown in cpk, hydrogen bonds in dotted lines and amino acids in blue. The amino acids shown here are within a 4-A radius of the ligand (glutamate).
Amino Acid Sequence↗
Analysis of the interactions of ribonuclease inhibitor with kanamycin.
The interaction of ribonuclease inhibitor (RI) with kanamycin was studied by molecular modeling. The preliminary binding model was constructed using the Affinity module of the Insight II molecular modeling program and the key residues involved in the combination of RI binding to kanamycin were determined. Meanwhile, we determined relevant surface characteristics determining the interaction behavior. The modeling results indicated that electrostatic interactions and H-bond forces may work as major factors for the molecular interaction between kanamycin and RI. The above results are useful for elucidating the molecular principles upon which the selectivity of a kanamycin is based. The quartz-crystal microbalance (QCM) is a new method usually used to monitor the binding function of macromolecules with samples online in a flow-injection analysis (FIA) system. The experimental results demonstrate that kanamycin has an extraordinary affinity to the basic protein RI, and our result is consistent with the molecular modeling results. These principles can in turn be used to study the molecular recognition mechanism and design a mimic of kanamycin for the development of new RI binders.
Binding Sites↗
Data mining for protein-protein interactions in invertebrate model organisms.
Well-annotated genome databases are available for many invertebrate species, notably the fruitfly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. An adequate interpretation of this information at the biological level requires the exploration of the interactions between the gene products. Knowledge of protein interactions and the components of cell signalling pathways in the fly and worm are particularly valuable as hypotheses can be rapidly tested using the powerful genetic toolkits available. Invertebrates offer additional experimental advantages when attempting to characterise protein-protein interactions (PPIs). Their relatively small genome size compared to mammals helps to reduce missed interactions due to redundancy, and their function can be addressed using forward (mutants) and reverse (RNA interference) genetics. However, the researcher looking for evidence of PPIs for a protein of interest is faced with the challenge of extracting interaction data from sources that are highly varied, such as the results of microarray experiments in the unstructured text of research papers. This challenge is greatly reduced by a range of public databases of curated information, as well as publicly available, enhanced search engines, which can provide either direct experimental evidence for a PPI, or valuable clues for generating new hypotheses.
Animals↗
Role of N-terminal helix in interaction of ribosomal protein S15 with 16S rRNA.
The position and conformation of the N-terminal helix of free ribosomal protein S15 was earlier found to be modified under various conditions. This variability was supposed to provide the recognition by the protein of its specific site on 16S rRNA. To test this hypothesis, we substituted some amino acid residues in this helix and assessed effects of these substitutions on the affinity of the protein for 16S rRNA. The crystal structure of the complex of one of these mutants (Thr3Cys S15) with the 16S rRNA fragment was determined, and a computer model of the complex containing another mutant (Gln8Met S15) was designed. The available and new information was analyzed in detail, and the N-terminal helix was concluded to play no significant role in the specific binding of the S15 protein to its target on 16S rRNA.
Bacillus subtilis↗