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PubMed2006

Head module control of mediator interactions.

Yeast Mediator proteins interacting with Med17(Srb4) have been expressed at a high level with the use of recombinant baculoviruses and recovered in homogeneous form as a seven subunit, 223 kDa complex. Electron microscopy and single-particle analysis identify this complex as the Mediator head module. The recombinant head module complements "headless" Mediator for the initiation of transcription in vitro. The module interacts with an RNA polymerase II-TFIIF complex, but not with the polymerase or TFIIF alone. This interaction is lost in the presence of a DNA template and associated RNA transcript, recapitulating the release of Mediator that occurs upon the initiation of transcription. Disruption of the head module in a temperature-sensitive mutant in vivo leads to the release of middle and tail modules from a transcriptionally active promoter. The head module evidently controls Mediator-RNA polymerase II and Mediator-promoter interactions.

Baculoviridae↗
PubMed2005

The Arabidopsis inositol 1,3,4-trisphosphate 5/6 kinase, AtItpk-1, is involved in plant photomorphogenesis under red light conditions, possibly via interaction with COP9 signalosome.

The COP9 signalosome (CSN) plays important roles in multifaceted cellular processes. Study has shown that inositol 1,3,4-trisphosphate 5/6 kinase (5/6 kinase) interacts with CSN in mammalian cells. However, the biological function of the interaction still remains unknown. Here, we report that the Arabidopsis inositol 1,3,4-trisphosphate 5/6 kinase (AtItpk-1) is also associated with CSN and involved in photomorphogenesis under red light (RL) conditions, as demonstrated by co-immunoprecipitation of AtItpk-1 with CSN and characterization of the atitpk-1 mutants. Expression analysis showed that AtItpk-1 had the same sub-cellular localization and organ expression pattern as CSN. Furthermore, autophosphorylation analysis showed that AtItpk-1 has protein kinase activity. Under RL, the atitpk-1 mutants exhibited phenotype slightly similar with that of the csn mutants, indicating that 5/6 kinase might be involved in the same developmental pathway as CSN. This study suggests that AtItpk-1 may function as a protein kinase that is involved in photomorphogenesis possibly via interaction with COP9 signalosome under red light.

Arabidopsis↗
PubMedDate not supplied

COPing with hypoxia.

To understand how cells respond to altered oxygenation, a frequent experimental paradigm is to isolate known components of bona fide oxygen responsive proteins. Recent studies have shown that a protein known as CSN5 or JAB1 interacts with both the HIF-1alpha oxygen-responsive transcription factor and its oxygen-dependent regulator, the Von Hippel-Lindau (pVHL) tumor suppressor. CSN5 is a component of the COP9 Signalosome (CSN) which is a multi-subunit protein that has high homology to the lid of the 19S lid of 26S proteasome. The exact function of the CSN5 interaction with pVHL and HIF-1alpha remains to be fully elucidated, but it is clear that the interaction is both oxygen dependent and that CSN5 may play different roles under oxic and hypoxic responses. Further, evidence has also been published indicating that pVHL can be potentially post-translationally modified by CSN5 (de-neddylation) and that CSN5 transcription is regulated by hypoxia as are many of the key pVHL/HIF-1alpha regulatory genes such as the PHDs and OS-9. This review will give a broad overview of known CSN5 and COP9 Signalosome functions and how these functions impact the pVHL/HIF-1alpha signaling complex and potentially other oxygen-sensitive response networks.

Amino Acid Sequence↗
PubMed2003

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.

Animals↗
PubMed2002

Inositol 1,3,4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1.

The COP9 signalosome (CSN) is a complex of eight proteins first identified as a repressor of plant photomorphogenesis. A protein kinase activity associated with the COP9 signalosome has been reported but not identified; we present evidence for inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) as a protein kinase associated with the COP9 signalosome. We have shown that 5/6-kinase exists in a complex with the eight-component COP9 signalosome both when purified from bovine brain and when transfected into HEK 293 cells. 5/6-kinase phosphorylates the same substrates as those of the COP9 signalosome, including IkappaBalpha, p53, and c-Jun but fails to phosphorylate several other substrates, including c-Jun 1-79, which are not substrates for the COP9-associated kinase. Both the COP9 signalosome- associated kinase and 5/6-kinase are inhibited by curcumin. The association of 5/6-kinase with the COP9 signalosome is through an interaction with CSN1, which immunoprecipitates with 5/6-kinase. In addition, the inositol kinase activity of 5/6-kinase is inhibited when in a complex with CSN1. We propose that 5/6-kinase is the previously described COP9 signalosome-associated kinase.

Amino Acid Sequence↗
PubMed2004

MBD3L1 is a transcriptional repressor that interacts with methyl-CpG-binding protein 2 (MBD2) and components of the NuRD complex.

Methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) are transcriptional repressors that contain methyl-CpG binding domains and are components of a CpG-methylated DNA binding complex named MeCP1. Methyl-CpG-binding protein 3-like 1 (MBD3L1) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG binding domain. MBD3L1 interacts with MBD2 and MBD3 in vitro and in yeast two-hybrid assays. Gel shift experiments with a CpG-methylated DNA probe indicate that recombinant MBD3L1 can supershift an MBD2-methylated DNA complex. In vivo, MBD3L1 associates with and colocalizes with MBD2 but not with MBD3 and is recruited to 5-methylcytosine-rich pericentromeric heterochromatin in mouse cells. In glutathione S-transferase pull-down assays MBD3L1 is found associated with several known components of the MeCP1.NuRD complex, including HDAC1, HDAC2, MTA2, MBD2, RbAp46, and RbAp48, but MBD3 is not found in the MBD3L1-bound fraction. MBD3L1 enhances transcriptional repression of methylated DNA by MBD2. The data are consistent with a role of MBD3L1 as a methylation-dependent transcriptional repressor that may interchange with MBD3 as an MBD2-interacting component of the NuRD complex. MBD3L1 knockout mice were created and were found to be viable and fertile, indicating that MBD3L1 may not be essential or there is functional redundancy (through MBD3) in this pathway. Overall, this study reveals additional complexities in the mechanisms of transcriptional repression by the MBD family proteins.

Animals↗
PubMed2006

Phosphorylation of B14.5a subunit from bovine heart complex I identified by titanium dioxide selective enrichment and shotgun proteomics.

Shotgun proteomics was used to study the steady phosphorylation state of NADH:ubiquinone oxidoreductase (complex I) subunits from bovine heart mitochondria. A total tryptic digestion of enzymatically active complex I was performed, and the resulting peptide mixture was subjected to phosphopeptide enrichment by the use of titanium dioxide (TiO2). The phosphopeptide-enriched fraction was separated and analyzed with nanoscale reverse-phase HPLC-ESI-MS/MS in single information-dependent acquisition. Hence two phosphorylated complex I subunits were detected: 42 kDa and B14.5a. Phosphorylation of 42-kDa subunit at Ser-59 has already been determined with fluorescent phosphoprotein-specific gel staining and mass spectrometry (Schilling, B., Aggeler, R., Schulenberg, B., Murray, J., Row, R. H., Capaldi, R. A., and Gibson, B. W. (2005) Mass spectrometric identification of novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I. FEBS Lett. 579, 2485-2490). In our work, this finding was confirmed using a non-gel-based approach. In addition, we report novel phosphorylation on B14.5a nuclear encoded subunit. We demonstrated evidence of the phosphorylation site at Ser-95 residue by collision-induced dissociation experiments on three different molecular ions of two tryptic phosphopeptides of B14.5a.

Amino Acid Sequence↗
PubMed2002

COP9 signalosome components play a role in the mating pheromone response of S. cerevisiae.

A family of genetically and structurally homologous complexes, the proteasome lid, Cop9 signalosome (CSN) and eukaryotic translation initiation factor 3, mediate different regulatory pathways. The CSN functions in numerous eukaryotes as a regulator of development and signaling, yet until now no evidence for a complex has been found in Saccharomyces cerevisiae. We identified a group of proteins, including a homolog of Csn5/Jab1 and four uncharacterized PCI components, that interact in a manner suggesting they form a complex analogous to the CSN in S. cerevisiae. These newly identified subunits play a role in adaptation to pheromone signaling. Deletants for individual subunits enhance pheromone response and increase mating efficiency. Overexpression of individual subunits or a human homolog mitigates sst2-induced pheromone sensitivity. Csi1, a novel CSN interactor, exhibits opposite phenotypes. Deletants also accumulate Cdc53/cullin in a Rub1-modified form; however, this role of the CSN appears to be distinct from that in the mating pathway.

COP9 Signalosome Complex↗
PubMed2004

The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B.

Pure hereditary spastic paraplegia is characterized by length-dependent degeneration of the distal ends of long axons. Mutations in spastin are the most common cause of the condition. We set out to investigate the function of spastin using a yeast two-hybrid approach to identify interacting proteins. Using full-length spastin as bait, we identified CHMP1B, a protein associated with the ESCRT (endosomal sorting complex required for transport)-III complex, as a binding partner. Several different approaches confirmed the physiological relevance of the interaction in mammalian cells. Epitope-tagged CHMP1B and spastin showed clear cytoplasmic co-localization in Cos-7 and PC12 cells. CHMP1B and spastin interacted specifically in vitro and in vivo in beta-lactamase protein fragment complementation assays, and spastin co-immunoprecipitated with CHMP1B. The interaction was mediated by a region of spastin lying between residues 80 and 196 and containing a microtubule interacting and trafficking domain. Expression of epitope-tagged CHMP1B in mammalian cells prevented the development of the abnormal microtubule phenotype associated with expression of ATPase-defective spastin. These data point to a role for spastin in intracellular membrane traffic events and provide further evidence to support the emerging recognition that defects in intracellular membrane traffic are a significant cause of motor neuron pathology.

Adenosine Triphosphatases↗
PubMed2005

The Hrs/STAM complex in the downregulation of receptor tyrosine kinases.

Cell surface receptor proteins that have undergone endocytosis are transported to the endosome. From the endosome, ligand-activated receptor tyrosine kinases are further transported to the lysosome for degradation, a process called "receptor downregulation." By contrast, nutrient receptors, such as those for low-density lipoprotein and transferrin, are recycled back to the plasma membrane. Sorting of these two types of receptors occurs at the endosome, where ubiquitination of receptor proteins serves as the sorting signal. Namely, ubiquitinated receptors are incorporated into the lysosomal degradation pathway, whereas those that are not ubiquitinated are returned to the cell surface. Hrs and STAM are proteins that form a complex on the endosomal membrane. Recent studies have shown that the Hrs/STAM complex binds ubiquitin moieties and acts as sorting machinery that recognizes ubiquitinated receptors and transfers them to further sequential lysosomal sorting/trafficking processes.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Arabidopsis FUSCA5 encodes a novel phosphoprotein that is a component of the COP9 complex.

The COP9 complex is a regulator essential for repression of light-mediated development in Arabidopsis. Using partial amino acid sequence data generated from purified COP9 complexes, we cloned the Arabidopsis cDNA encoding the 27-kD subunit of the COP9 complex and showed that it is encoded by the previously identified FUSCA5 (FUS5) locus. fus5 mutants exhibit constitutive photomorphogenic phenotypes similar to those of cop9 and fus6. Point mutations in FUS5 that led to a loss of FUS5 protein were detected in four fus5 allelic strains. FUS5 contains the PCI/PINT and mitogen-activated protein kinase kinase activation loop motifs and is highly conserved with the mammalian COP9 complex subunit 7 and the Aspergillus nidulans AcoB proteins. FUS5 is present in both complex and monomeric forms. In the COP9 complex, FUS5 may interact directly with FUS6 and COP9. Mutations in FUS6 and COP9 result in a shift in the electrophoretic mobility of FUS5. This shift can be mimicked by in vitro phosphorylation of FUS5 by plant extracts. These findings further support the hypothesis that the COP9 complex is a central and common regulator that may interact with multiple signaling pathways.

Amino Acid Sequence↗
PubMed2003

Signal-induced transcriptional activation by Dif requires the dTRAP80 mediator module.

The Mediator complex is the major multiprotein transcriptional coactivator complex in Drosophila melanogaster. Mediator components interact with diverse sets of transcriptional activator proteins to elicit the sophisticated regulation of gene expression. The distinct phenotypes associated with certain mutations in some of the Mediator genes and the specific in vitro interactions of Mediator gene products with transcriptional activator proteins suggest the presence of activator-specific binding subunits within the Mediator complex. However, the physiological relevance of these selective in vitro interactions has not been addressed. Therefore, we analyzed dTRAP80, one of the putative activator-binding subunits of the Mediator, for specificity of binding to a number of natural transcriptional activators from Drosophila. Among the group of activator proteins that requires the Mediator complex for transcriptional activation, only a subset of these proteins interacted with dTRAP80 in vitro and only these dTRAP80-interacting activators were defective for activation under dTRAP80-deficient in vivo conditions. In particular, activation of Drosophila antimicrobial peptide drosomycin gene expression by the NF-kappa B-like transcription factor Dif during induction of the Toll signaling pathway was dependent on the dTRAP80 module. These results, and the indirect support from the dTRAP80 artificial recruitment assay, indicate that dTRAP80 serves as a genuine activator-binding target responsible for a distinct group of activators.

Animals↗
PubMed2002

Orc6 involved in DNA replication, chromosome segregation, and cytokinesis.

Origin recognition complex (ORC) proteins serve as a landing pad for the assembly of a multiprotein prereplicative complex, which is required to initiate DNA replication. During mitosis, the smallest subunit of human ORC, Orc6, localizes to kinetochores and to a reticular-like structure around the cell periphery. As chromosomes segregate during anaphase, the reticular structures align along the plane of cell division and some Orc6 localizes to the midbody before cells separate. Silencing of Orc6 expression by small interfering RNA (siRNA) resulted in cells with multipolar spindles, aberrant mitosis, formation of multinucleated cells, and decreased DNA replication. Prolonged periods of Orc6 depletion caused a decrease in cell proliferation and increased cell death. These results implicate Orc6 as an essential gene that coordinates chromosome replication and segregation with cytokinesis.

Animals↗
PubMed2006

A close-up of the ESCRTs.

The ESCRT protein complexes assemble on the endosome into a protein network that sorts ubiquitinated transmembrane proteins into lumenal vesicles of the compartment. New structural information highlights the complexity of the ESCRT network.

Endosomes↗
PubMed2005

The structural interactions between T cell receptors and MHC-peptide complexes place physical limits on self-nonself discrimination.

The activation and expansion of T cells in an antimicrobial immune response is based on the ability of T cell receptors (TCR) to discriminate between MHC-bound peptides derived from different microbial agents as well as self-proteins. However, the specificity of T cells is constrained by the limited number of peptide side chains that are available for TCR binding. By considering the structural requirements for peptide binding to MHC molecules and TCR recognition of MHC-peptide complexes, we demonstrated that human T cell clones could recognize a number of peptides from different organisms that were remarkably distinct in their primary sequence. These peptides were particularly diverse at those sequence positions buried in pockets of the MHC binding site, whereas a higher degree of similarity was present at a limited number of peptide residues that created the interface with the TCR. These T cell clones had been isolated from multiple sclerosis patients with human myelin basic protein, demonstrating that activation of such autoreactive T cells by microbial peptides with sufficient structural similarity may contribute to the disease process. Similar findings have now been made for a variety of human and murine T cell clones, indicating that specificity and cross-reactivity are inherent properties of TCR recognition. The observations that particular TCR are highly sensitive to changes at particular peptide positions but insensitive to many other changes in peptide sequence are not contradictory, but rather the result of structural interactions in which a relatively flat TCR surface contacts a limited number of side chains from a peptide that is deeply embedded in the MHC binding site.

Amino Acid Sequence↗
PubMed2006

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples.

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.

Cytochrome c Group↗
PubMed2004

An improved method for the solution cyclization of peptides under pseudo-high dilution conditions.

Depending on the ring size, the cyclization of peptides often is accompanied by dimerization or cyclodimerization. Hence, these macrocyclizations have to be performed under high dilution conditions. Efficient cyclization of peptides in solution with a minimum amount of solvent succeeds, when a dual syringe pump is used to simultaneously add the linear peptide precursor and a coupling reagent from two separate syringes.

Complex Mixtures↗