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At least 1,621 records · Page 90Linked to original sources
PubMed2005

Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box.

The anaphase-promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators. Cdh1p is an APC coactivator that directly binds APC substrates. A genetic screen in budding yeast identified residues within Cdh1p critical for its function. Cdh1p proteins containing mutations within the "C box" or the "IR" motif could bind substrate, but not the APC, whereas mutants that only bound the APC were not identified, suggesting an ordered assembly of the ternary APC-Cdh1p-substrate complex. Supporting this hypothesis, we found that substrate binding to wild-type Cdh1p enhanced its association with the APC in yeast cells. We used peptide competition assays to demonstrate that Cdh1p interacts directly with the D box and the KEN box, two motifs within APC substrates known to be required for APC-mediated degradation. Moreover, an intact D box domain within a substrate was required to stimulate the association between the Cdh1p-substrate complex and the APC.

Amino Acid Motifs↗
PubMed2006

Life is degrading--thanks to some zomes.

Three structurally related protein complexes, the COP9 signalosome, the proteasome lid, and the eukaryotic translation initiation factor 3, are revealing new insights into developmental processes and into cell cycle control in healthy cells and cells exposed to genotoxic stress. Newly discovered cullin-RING E3 ubiquitin ligases assembled on the CUL4 platform may provide links between DNA replication, chromatin, and proteolysis.

Animals↗
PubMed2005

Purification method of the COP9 signalosome from human erythrocytes.

The COP9 signalosome (CSN) is a multimeric protein complex that occurs in all eukaryotic cells. Originally described in plants as a regulator of photomorphogenesis, its purification and characterization from mammalian cells revealed significant sequence homologies to subunits of the 26S proteasome lid complex, as well as of the eukaryotic translation initiation factor 3. Recent studies disclosed its participation in processes such as DNA repair, cell cycle regulation, development, and angiogenesis. At the moment, the pleiotropic effects of the CSN point to a regulatory role in the ubiquitin/26S proteasome system, but its exact function still remains to be clarified. This chapter describes the method to purify human CSN from red blood cells. Two outdated erythrocyte concentrates are sufficient to prepare approximately 0.5 mg of CSN. Washed cells are first lysed and then proteins are separated by a DEAE anion-exchange column. The CSN-containing fractions are pooled and subjected to an ammonium sulfate precipitation followed by dialysis. The concentrated proteins are then loaded onto a glycerol density gradient and ultracentrifugation is performed. The purification procedure is continued using two succeeding anion-exchange columns, resulting in a sufficiently pure CSN complex. Optionally, an additional density gradient centrifugation can be attached. The purified CSN complex possesses kinase, deneddylase, and deubiquitinase activities and can be stored for at least 2 months on ice at 4 degrees .

Blotting, Western↗
PubMed1999

The COP9 signalosome is essential for development of Drosophila melanogaster.

The COP9 signalosome (originally described as the COP9 complex) is an essential multi-subunit repressor of light-regulated development in plants [1] [2]. It has also been identified in mammals, though its role remains obscure [3] [4] [5]. This complex is similar to the regulatory lid of the proteasome and eIF3 [5] [9] [10] [11] [12] and several of its subunits are known to be involved in kinase signaling pathways [4] [6] [7] [8]. No proteins homologous to COP9 signalosome components were identified in the Saccharomyces cerevisiae genome, suggesting that the COP9 signalosome is specific for multi-cellular differentiation [13]. In order to reveal the developmental function of the COP9 signalosome in animals, we have isolated Drosophila melanogaster genes encoding eight subunits of the COP9 signalosome, and have shown by co-immunoprecipitation and gel-filtration analysis that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of one of the subunits by either a P-element insertion or deletion of the gene caused lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. Our results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development.

Animals↗
PubMed2003

COP9 signalosome: a provider of DNA building blocks.

In fission yeast, the COP9 signalosome is required to activate ribonucleotide reductase for DNA synthesis. This is mediated via the ubiquitin ligase Pcu4, activation of which leads to degradation of the scaffold protein Spd1, which anchors the small ribonucleotide reductase subunit in the nucleus away from the large subunit in the cytoplasm.

COP9 Signalosome Complex↗
PubMed2005

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Amino Acid Sequence↗
PubMed2002

Deletion mutants in COP9/signalosome subunits in fission yeast Schizosaccharomyces pombe display distinct phenotypes.

The COP9/signalosome complex is highly conserved in evolution and possesses significant structural similarity to the 19S regulatory lid complex of the proteasome. It also shares limited similarity to the translation initiation factor eIF3. The signalosome interacts with multiple cullins in mammalian cells. In the fission yeast Schizosaccharomyces pombe, the Csn1 subunit is required for the removal of covalently attached Nedd8 from Pcu1, one of three S. pombe cullins. It remains unclear whether this activity is required for all the functions ascribed to the signalosome. We previously identified Csn1 and Csn2 as signalosome subunits in S. pombe. csn1 and csn2 null mutants are DNA damage sensitive and exhibit slow DNA replication. Two further putative subunits, Csn4 and Csn5, were identified from the S. pombe genome database. Herein, we characterize null mutations of csn4 and csn5 and demonstrate that both genes are required for removal of Nedd8 from the S. pombe cullin Pcu1 and that their protein products associate with Csn1 and Csn2. However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes. Our data suggest that the subunits of the signalosome cannot be considered as a distinct functional unit and imply that different subunits of the signalosome mediate distinct functions.

COP9 Signalosome Complex↗
PubMed2005

Numerical study of a binary Yukawa model in regimes characteristic of globular proteins in solutions.

The main goal of this paper is to assess the limits of validity, in the regime of low concentration and strong Coulomb coupling (high molecular charges), of a simple perturbative approximation to the radial distribution functions (RDF's), based upon a low-density expansion of the potential of mean force and proposed to describe protein-protein interactions in a recent small-angle-scattering (SAS) experimental study. A highly simplified Yukawa (screened Coulomb) model of monomers and dimers of a charged globular protein (beta-lactoglobulin) in solution is considered. We test the accuracy of the RDF approximation, as a necessary complementary part of the previous experimental investigation, by comparison with the fluid structure predicted by approximate integral equations and exact Monte Carlo (MC) simulations. In the MC calculations, an Ewald construction for Yukawa potentials has been used to take into account the long-range part of the interactions in the weakly screened cases. Our results confirm that the perturbative first-order approximation is valid for this system even at strong Coulomb coupling, provided that the screening is not too weak (i.e., for Debye length smaller than monomer radius). A comparison of the MC results with integral equation calculations shows that both the hypernetted-chain (HNC) and Percus-Yevick closures have a satisfactory behavior under these regimes, with the HNC being superior throughout. The relevance of our findings for interpreting SAS results is also discussed.

Binding Sites↗
PubMed2005

Consequences of COP9 signalosome and 26S proteasome interaction.

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr-Pad1-N-terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the formation of an intact Flag-CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull-downs also precipitated cullins indicating the existence of super-complexes consisting of the CSN, the 26S proteasome and cullin-based Ub ligases. Permanent expression of a chimerical subunit (Flag-CSN2-Rpn6) consisting of the N-terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag-CSN2 overexpression was de-novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c-Jun in B8 cells. The possible role of super-complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.

Animals↗
PubMed2002

The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis.

Recently the COP9 signalosome (CSN) has become a focus of interest for many researchers, because of its function at the interface between signal transduction and ubiquitin-dependent proteolysis. It is required for the proper progression of the cell cycle in Schizosaccharomyces pombe and is essential for development in plants and Drosophila. However, its function in mammalian cells remains obscure. Although the CSN shares structural similarities with the 26S proteasome lid complex (LID), its functions seem to be different from that of the LID. A variety of CSN-specific protein-protein interactions have been described in mammalian cells. However, it is currently unclear how many reflect true functions of the complex. Two activities associated with the CSN have been identified so far: a protein kinase and a deneddylase. The CSN-associated kinase phosphorylates transcription factors, which determines their stability towards the ubiquitin system. The associated deneddylase regulates the activity of specific SCF E3 ubiquitin ligases. The CSN thus appears to be a platform connecting signalling with proteolysis.

Animals↗
PubMed2005

The human U5 snRNP 52K protein (CD2BP2) interacts with U5-102K (hPrp6), a U4/U6.U5 tri-snRNP bridging protein, but dissociates upon tri-snRNP formation.

The U5 snRNP plays an essential role in both U2- and U12-dependent splicing. Here, we have characterized a 52-kDa protein associated with the human U5 snRNP, designated U5-52K. Protein sequencing revealed that U5-52K is identical to the CD2BP2, which interacts with the cytoplasmic portion of the human T-cell surface protein CD2. Consistent with it associating with an snRNP, immunofluorescence studies demonstrated that the 52K protein is predominantly located in the nucleoplasm of HeLa cells, where it overlaps, at least in part, with splicing-factor compartments (or "speckles"). We further demonstrate that the 52K protein is a constituent of the 20S U5 snRNP, but is not found in U4/U6.U5 tri-snRNPs. Thus, it is the only 20S U5-specific protein that is not integrated into the tri-snRNP and resembles, in this respect, the U4/U6 di-snRNP assembly factor Prp24p/p110. Yeast two-hybrid screening and pulldown assays revealed that the 52K protein interacts with the U5-specific 102K and 15K proteins, suggesting that these interactions are responsible for its integration into the U5 particle. The N-terminal two-thirds of 52K interact with the 102K protein, whereas its C-terminal GYF-domain binds the 15K protein. As the latter lacks a proline-rich tract, our data indicate that a GYF-domain can also engage in specific protein-protein interactions in a polyproline-independent manner. Interestingly, the U5-102K protein has been shown previously to play an essential role in tri-snRNP formation, binding the U4/U6-61K protein. The interaction of 52K with a tri-snRNP bridging protein, coupled with its absence from the tri-snRNP, suggests it might function in tri-snRNP assembly.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Characterization and purification of the mammalian COP9 complex, a conserved nuclear regulator initially identified as a repressor of photomorphogenesis in higher plants.

The COP9 complex has been identified as a repressor of photomorphogenesis in Arabidopsis. Here we demonstrate that the COP9 complex is also present in mammals. Specific antibodies were generated against human counterparts of the Arabidopsis COP9 and COP11, the two known subunits of plant COP9 complex. Using these antibodies, we showed that indeed mammalian COP9 and COP11, also known as GPS1, could be coimmuno-precipitated using either of the two specific antibodies, definitively confirming that they are physically part of the same complex. Further, the mammalian COP9 and COP11/GPS1 were cofractionated in the same large molecular weight fractions of about 500 kDa and were absent from the monomeric fractions. The mammalian COP9 complex was present in all organs examined but abundances vary. Indirect immunofluorescence studies suggested that the mammalian COP9 complex is largely nuclear localized. Both conventional biochemical and affinity purifications of the COP9 complex from pig spleen indicated that the mammalian COP9 complex consists of eight distinct subunits. These findings indicate that mammals also have a COP9 complex with conserved molecular composition and biochemical and cellular properties similar to the higher plant counterpart.

Animals↗
PubMed2005

T cell receptor/peptide/MHC molecular characterization and standardized pMHC contact sites in IMGT/3Dstructure-DB.

One of the key elements in the adaptive immune response is the presentation of peptides by the major histocompatibility complex (MHC) to the T cell receptors (TR) at the surface of T cells. The characterization of the TR/peptide/MHC trimolecular complexes (TR/pMHC) is crucial to the fields of immunology, vaccination and immunotherapy. In order to facilitate data comparison and cross-referencing between experiments from different laboratories whatever the receptor, the chain type, the domain, or the species, IMGT, the international ImMunoGeneTics information system (http://imgt.cines.fr), has developed IMGT-ONTOLOGY, the first ontology in immunogenetics and immunoinformatics. In IMGT/3Dstructure-DB, the IMGT three-dimensional structure database, TR/pMHC molecular characterization and pMHC contact analysis are made according to the IMGT Scientific chart rules, based on the IMGT-ONTOLOGY concepts. IMGT/3Dstructure-DB provides the standardized IMGT gene and allele names (CLASSIFICATION), the standardized IMGT labels (DESCRIPTION) and the IMGT unique numbering (NUMEROTATION). As the IMGT structural unit is the domain, amino acids at conserved positions always have the same number in the IMGT databases, tools and Web resources. For the TR alpha and beta chains, the amino acids in contact with the peptide/MHC (pMHC) are defined according to the IMGT unique numbering for V-DOMAIN. The MHC cleft that binds the peptide is formed by two groove domains (G-DOMAIN), each one comprising four antiparallel beta strands and one alpha helix. The IMGT unique numbering for G-DOMAIN applies both to the first two domains (G-ALPHA1 and G-ALPHA2) of the MHC class I alpha chain, and to the first domain (G-ALPHA and G-BETA) of the two MHC class II chains, alpha and beta. Based on the IMGT unique numbering, we defined eleven contact sites for the analysis of the pMHC contacts. The TR/pMHC contact description, based on the IMGT numbering, can be queried in the IMGT/StucturalQuery tool, at http://imgt.cines.fr.

Amino Acid Sequence↗
PubMed2005

SUMOylation and PPARgamma: wrestling with inflammatory signaling.

The molecular mechanisms whereby PPARgamma inhibits inflammatory gene expression in macrophages are poorly understood. In a recent Nature paper, provide a new model for trans-repression in which ligand-dependent SUMOylation of PPARgamma results in its recruitment to the promoters of inflammatory genes where it inhibits transcription by preventing clearance of corepressor complexes.

Animals↗
PubMed1999

EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains.

Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with "reverse" signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, which we have termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. Our findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The cellular C1 factor of the herpes simplex virus enhancer complex is a family of polypeptides.

The alpha/immediate early genes of herpes simplex virus are regulated by the specific assembly of a multiprotein enhancer complex containing the Oct-1 POU domain protein, the viral alpha-transinduction factor alpha TIF, (VP16, ICP25), and the C1 cellular factor. The C1 factor from mammalian cells is a heterogeneous but related set of polypeptides that interact directly with the alpha-transinduction factor to form a heteromeric protein complex. The isolation of cDNAs encoding the polypeptides of the C1 factor suggests that these proteins are proteolytic products of a novel precursor. The sequence of the amino termini of these polypeptide products indicate that the proteins are generated by site-specific cleavages within a reiterated 20-amino acid sequence. Although the C1 factor appears to be ubiquitously expressed, it is localized to subnuclear structures in specific cell types.

Amino Acid Sequence↗
PubMed2002

Regulation of histone acetylation and transcription by nuclear protein pp32, a subunit of the INHAT complex.

Histone acetylation by p300/CBP and PCAF coactivators is considered to be a key mechanism of chromatin modification and transcriptional regulation. A multiprotein cellular complex, INHAT (inhibitor of acetyltransferases), containing the Set/TAF-Ibeta oncoprotein and pp32 strongly inhibits the HAT activity of p300/CBP and PCAF by histone masking. Here we report that the INHAT complex and its subunits have overlapping but distinct HAT inhibitory and histone binding characteristics. We provide evidence suggesting that the histone binding and INHAT activity of pp32 can be regulated by its physical association with other INHAT subunits. In vivo colocalization and transfection studies show that pp32 INHAT domains are responsible for histone binding, HAT inhibitory activity, and repression of transcription. We propose that INHAT and its subunits may function by modulating histone acetyltransferases through a histone-masking mechanism and may play important regulatory roles in the establishment and maintenance of the newly proposed "histone code" of chromatin.

3T3 Cells↗