PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Multiprotein Complexes”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,531 records · Page 85Linked to original sources
PubMed2000

Structure of the TPR domain of p67phox in complex with Rac.GTP.

p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac.GTP bound to the N-terminal TPR (tetratricopeptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.

Amino Acid Sequence↗
PubMed2003

A nuclear protein complex containing high mobility group proteins B1 and B2, heat shock cognate protein 70, ERp60, and glyceraldehyde-3-phosphate dehydrogenase is involved in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues.

Thiopurine treatment of human leukemia cells deficient in components of the mismatch repair system (Nalm6) initiated apoptosis after incorporation into DNA, as revealed by caspase activation and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. To elucidate the cellular sensor(s) responsible for recognition of DNA damage in cells with an inactive mismatch repair system, we isolated a multiprotein nuclear complex that preferentially binds DNA with thioguanine incorporated. The components of this nuclear multiprotein complex, as identified by protein mass spectroscopy, included high mobility group proteins 1 and 2 (HMGB1, HMGB2), heat shock protein HSC70, protein disulfide isomerase ERp60, and glyceraldehyde 3-phosphate dehydrogenase. The same complex was also shown to bind synthetic oligodeoxyribonucleotide duplexes containing the nonnatural nucleosides 1-beta-D-arabinofuranosylcytosine or 5-fluoro-2'-deoxyuridine. Fibroblast cell line derived from Hmgb1(-/-) murine embryos had decreased sensitivity to thiopurines, with an IC(50) 10-fold greater than Hmgb1-proficient cells (P < 0.0001) and exhibited comparable sensitivity to vincristine, a cytotoxic drug that is not incorporated into DNA. These findings indicate that the HMGB1-HMGB2-HSC70-ERp60-glyceraldehyde 3-phosphate dehydrogenase complex detects changes in DNA structure caused by incorporation of nonnatural nucleosides and is a determinant of cell sensitivity to such DNA modifying chemotherapy.

Antimetabolites, Antineoplastic↗
PubMed1996

Estrogen induces the assembly of a multiprotein messenger ribonucleoprotein complex on the 3'-untranslated region of chicken apolipoprotein II mRNA.

UV cross-linking was used to identify estrogen-induced hepatocyte proteins that bind to apoII mRNA. Probes spanning the entire message revealed the presence of eight estrogen-induced proteins cross-linked to the 3'-untranslated region (UTR), but not to the coding region or the 5'-UTR. Two estrogen-induced proteins of 132 and 50 kDa were either absent or barely detectable in control animals, whereas six additional proteins of 93, 83, 74, 65, 58, and 45 kDa were clearly present in control animals and increased 2-5-fold by estrogen. A similar profile of estrogen-induced proteins was seen with the 3'-UTRs of the estrogen-regulated mRNAs for apoB and vitellogenin II, but not with the 3'-UTRs of the non-estrogen-regulated mRNAs for apoA-I and glyceraldehyde-phosphate dehydrogenase. These findings indicate that the estrogen-induced proteins discriminate among mRNAs and suggest that they interact selectively with the family of estrogen-regulated mRNAs. The estrogen-induced proteins are found in the cytoplasmic fraction of liver extracts, and a subset of them are also found in adrenal glands, testes, heart, brain, and kidneys, but they are estrogen-induced only in the liver. Deletion analysis defined a 150-nucleotide region of the apoII 3'-UTR that is necessary for maximal binding of the estrogen-induced proteins. An internal deletion of endonucleolytic cleavage sites previously identified within the apoII 3'-UTR selectively reduced the binding of the 58-kDa protein. These findings reveal remarkable complexity in estrogen-stimulated protein-RNA interactions within the 3'-UTRs of estrogen-regulated mRNAs. These proteins may participate in the mRNA degradation process or in other aspects of cytoplasmic mRNA metabolism that accompany estrogen-stimulated vitellogenesis.

Animals↗
PubMed2003

Association of the Mediator complex with enhancers of active genes.

The multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro. Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo. We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells.

Enhancer Elements, Genetic↗
PubMed2002

c-Cbl is a negative regulator of GH-stimulated STAT5-mediated transcription.

We have previously demonstrated that cellular stimulation with GH results in the formation of a multiprotein signaling complex. One component of this multiprotein signaling complex is the adapter molecule c-Cbl. Here we have examined the role of c-Cbl in the mechanism of GH signal transduction. Forced expression of c-Cbl in NIH3T3 cells did not alter GH-stimulated Janus kinase 2 tyrosine phosphorylation nor GH-stimulated p44/42 MAPK activation and consequent Elk-1- mediated transcription. c-Cbl overexpression did, however, result in enhanced and prolonged GH-stimulated activation of phosphatidylinositol 3-kinase. Forced expression of c-Cbl did not affect GH-stimulated STAT5 tyrosine phosphorylation, nuclear translocation, nor binding to DNA but markedly abrogated GH-stimulated STAT5-mediated transactivation. c-Cbl overexpression resulted in increased ubiquitination and proteosomal degradation of STAT5 and increased degradation of GH-stimulated tyrosine phosphorylated STAT5. Cellular pretreatment with the proteosomal inhibitor MG132 reversed the effect of c-Cbl overexpression with prolonged duration of GH-stimulated STAT5 tyrosine phosphorylation and restoration of STAT5-mediated transcription. Thus, c-Cbl is a negative regulator of GH-stimulated STAT5-mediated transcription by direction of STAT5 for proteosomal degradation.

3T3 Cells↗
PubMed2002

A candidate X-linked mental retardation gene is a component of a new family of histone deacetylase-containing complexes.

Eukaryotic genes are under the control of regulatory complexes acting through chromatin structure to control gene expression. Here we report the identification of a family of multiprotein corepressor complexes that function through modifying chromatin structure to keep genes silent. The polypeptide composition of these complexes has in common a core of two subunits, HDAC1,2 and BHC110, an FAD-binding protein. A candidate X-linked mental retardation gene and the transcription initiation factor II-I (TFII-I) are components of a novel member of this family of complexes. Other subunits of these complexes include polypeptides associated with cancer causing chromosomal translocations. These findings not only delineate a novel class of multiprotein complexes involved in transcriptional repression but also reveal an unanticipated role for TFII-I in transcriptional repression.

Blotting, Western↗
PubMed1997

Cancer cells release a covalent complex containing disulfide-linked domains from urinary plasminogen activator, neural cell adhesion molecule, and haptoglobin alpha and beta chains.

We have previously reported on the secretion of a family of high Mr plasminogen activators (PAs) by a human lung cancer cell line [Harvey et al. (1991) Biochim. Biophys. Acta 1078, 360-368]. We have now extended these studies to several human cancer cell lines and a human embryonic lung cell line. In the present study with HPL-SK-1 lung cancer, A431 epidermoid cancer, ovarian carcinoma, and embryonic lung cell lines, we show that the 900- and the 660-kDa PAs are disulfide-bonded multiprotein oligomeric complexes. They are functionally and immunologically related to human urinary PA (uPA). Their size and PA activity are not destroyed by strong denaturants such as 8 M urea or 2% sodium dodecyl sulfate (SDS), suggesting that the uPA moiety is covalently associated with the rest of the molecule. It is only under strong denaturing conditions with 1.4 M beta-mercaptoethanol and 2% SDS that the uPA moiety could be released as a 21- to 23-kDa fragment along with two major polypeptide chains of 70 and 40 kDa, respectively. The presence of the uPA active center in the reduced PA660 was demonstrated by [3H]diisopropylphosphorofluoridate labeling and by Western blot using a monoclonal antibody to uPA B chain. N-terminal amino acid sequencing of the 70- and 40-kDa polypeptides, respectively, showed homology to the neural cell adhesion molecule and the beta chain of haptoglobin. A minor fragment of 18 kDa obtained under strong reduction conditions was also sequenced and shown to share homology with the alpha chain of haptoglobin. Western blot analysis of the reduced PAs with monoclonal antibody to the neural cell adhesion molecule and rabbit anti-haptoglobin confirmed the homologies obtained by the sequence data. Further, immobilized monoclonal antibodies to the neural cell adhesion molecule, uPA B chain, and rabbit anti-haptoglobin bound the multiprotein complexes with uPA activity, from A431, ovarian cancer, and embryonic lung cell lines. The bound material, after dissociation, exhibited PA activity that was inhibited by monoclonal antibody to the uPA B chain. These data suggest that in tumor and embryonal cell lines, in addition to proper folding and assembly of proteins by intramolecular disulfide bond formation in the endomembrane compartment, intermolecular disulfide bonds could also occur, producing multiprotein oligomers as in the present case. Formation of such oligomers may have a selective advantage for such cells in the focalization of proteolytic activity through the interaction of the neural cell adhesion molecule domain with the extracellular matrix and in immunosuppression of lymphocytes by the haptoglobin portion of the complex.

Binding Sites↗
PubMed2005

Poxvirus multiprotein entry-fusion complex.

Poxviruses have evolved elaborate mechanisms for cell entry, assembly, and exocytosis. Recently, four vaccinia virus membrane proteins, namely A21, A28, H2 and L5, were reported to be necessary for cell entry and virus-induced cell-cell fusion but not for virion morphogenesis or attachment of virus particles to cells. Using immunoaffinity purification followed by mass spectrometry, we now show that these four proteins as well as four additional previously uncharacterized putative membrane proteins (A16, G3, G9, and J5) form a stable complex. These proteins fall into two groups: A21, A28, G3, H2, and L5 have an N-terminal transmembrane domain, 0-2 intramolecular disulfide bonds, and no sequence similarity, whereas A16, G9, and J5 have a C-terminal transmembrane domain and 4-10 predicted disulfide bonds and are homologous. Studies with conditional-lethal null mutants indicated that the viral membrane was crucial for assembly of the complex and that the absence of individual polypeptide components profoundly decreased complex formation or stability, suggesting a complicated interaction network. Analysis of purified virions, however, demonstrated that the polypeptides of the complex trafficked independently to the viral membrane even under conditions in which the complex itself could not be isolated. All eight proteins comprising the entry-fusion complex are conserved in all poxviruses, suggesting that they have nonredundant functions and that the basic entry mechanism evolved before the division between vertebrate and invertebrate poxvirus species.

Cell Line↗
PubMed2005

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells.

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.

3T3-L1 Cells↗
PubMed1989

Phosphorylation of HeLa cell multiprotein DNA polymerase alpha complex: impact on activity and partial purification of the associated kinase.

Phosphorylation is a major post-translational regulatory mechanism and plays a key role in transduction of mitogenic signals in cell proliferation. The role of phosphorylation and dephosphorylation in regulating the activities of a multiprotein DNA polymerase alpha complex was examined. Treatment of the HeLa cell multiprotein DNA polymerase alpha with calf intestinal alkaline phosphatase resulted in the inactivation of DNA polymerase alpha and DNA primase but had no effect on deoxyribonuclease- and primer-recognition proteins. A protein kinase co-purified with the multiprotein DNA polymerase alpha and was partially purified from HeLa cells. The partially purified kinase was active in phosphorylating dephosphorylated polymerase alpha and used casein and histones as exogenous substrates. This study demonstrates that phosphorylation-dephosphorylation may have modulated the activities of DNA replicative enzymes and suggests a role for specific phosphatases and kinases in this process.

DNA↗
PubMed2003

A mammalian mediator subunit that shares properties with Saccharomyces cerevisiae mediator subunit Cse2.

The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193 open reading frame. In this study, we present direct biochemical evidence that the FLJ10193 protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex.

Amino Acid Sequence↗
PubMed2006

SUMO modification enhances p66-mediated transcriptional repression of the Mi-2/NuRD complex.

Human p66alpha and p66beta are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66alpha and one in p66beta. Both p66alpha and p66beta are SUMO-modified in vivo: p66alpha at two sites (Lys-30 and Lys-487) and p66beta at one site (Lys-33). Expression of SUMO1 enhanced the transcriptional repression activity of Gal-p66alpha and Gal-p66beta. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase resulted in a significant decrease of the transcriptional repression of p66alpha and p66beta. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 were found to bind to both p66alpha and p66beta in vivo. Most of the interactions were not affected by the SUMO site mutations in p66alpha or p66beta, with two exceptions. HDAC1 binding to p66alpha was lost in the case of a p66alphaK30R mutant, and RbAp46 binding was reduced in the case of a p66betaK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation.

Animals↗
PubMed2006

Phosphoinositide 3-Kinase C2beta regulates cytoskeletal organization and cell migration via Rac-dependent mechanisms.

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein-protein and protein-lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2beta (PI3KC2beta) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2beta stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2beta reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2beta-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2beta regulates the migration and survival of human tumor cells by distinct molecular mechanisms.

Adaptor Proteins, Signal Transducing↗
PubMed1996

A histone octamer-like structure within TFIID.

The general transcription factor TFIID nucleates initiation complex formation through direct core promoter binding, commits promoters within chromatin to transcription, and mediates the action of transcriptional activators, a phenomenon that may correlate with enhanced TFIID recruitment or conformational changes in TFIID-promoter complexes. Molecular studies of the multiprotein TFIID complex have identified a primary TATA binding subunit (TBP), TBP-associated factors (TAFs) that interact with and mediate the function of activators and intersubunit interactions but have yielded relatively little insight into the structural organization of the complex or the actual mechanism of transcriptional activation. Here we present biochemical evidence for the structural relevance of histone homologies in the human TFIID subunits hTAF80, hTAF31 and hTAF20/15. Together with analyses of native TFIID complexes and accompanying crystallographic studies, the results suggest that there is a histone octamer-like TAF complex within TFIID.

Amino Acid Sequence↗
PubMed1998

The first nuclear-encoded complex I mutation in a patient with Leigh syndrome.

Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.

Amino Acid Sequence↗
PubMed2005

The LIM protein Ajuba influences interleukin-1-induced NF-kappaB activation by affecting the assembly and activity of the protein kinase Czeta/p62/TRAF6 signaling complex.

The Zyxin/Ajuba family of cytosolic LIM domain-containing proteins has the potential to shuttle from sites of cell adhesion into the nucleus and thus can be candidate transducers of environmental signals. To understand Ajuba's role in signal transduction pathways, we performed a yeast two-hybrid screen with the LIM domain region of Ajuba. We identified the atypical protein kinase C (aPKC) scaffold protein p62 as an Ajuba binding partner. A prominent function of p62 is the regulation of NF-kappaB activation in response to interleukin-1 (IL-1) and tumor necrosis factor signaling through the formation of an aPKC/p62/TRAF6 multiprotein signaling complex. In addition to p62, we found that Ajuba also interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and PKCzeta. Ajuba recruits TRAF6 to p62 and in vitro activates PKCzeta activity and is a substrate of PKCzeta. Ajuba null mouse embryonic fibroblasts (MEFs) and lungs were defective in NF-kappaB activation following IL-1 stimulation, and in lung IKK activity was inhibited. Overexpression of Ajuba in primary MEFs enhances NF-kappaB activity following IL-1 stimulation. We propose that Ajuba is a new cytosolic component of the IL-1 signaling pathway modulating IL-1-induced NF-kappaB activation by influencing the assembly and activity of the aPKC/p62/TRAF6 multiprotein signaling complex.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Inflammation modulates the interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) I/polypyrimidine tract binding protein and hnRNP L with the 3'untranslated region of the murine inducible nitric-oxide synthase mRNA.

Interaction of two members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with the 3'untranslated region (UTR) of the murine inducible nitric-oxide synthase (iNOS) mRNA is demonstrated in this study. An iNOS RNA-protein complex is formed using protein extracts from untreated and septic shock treated mouse liver. UV cross-linking reveals that the complex consists of at least two proteins, with apparent molecular masses of 60 and 70 kDa, respectively. The 60-kDa protein binding site lies within a 112-nt pyrimidine-rich sequence, approximately 160 nt from the coding sequence, and the RNA-protein complex can be precipitated by a monoclonal antibody directed against hnRNP I [also named polypyrimidine tract binding protein (PTB)]. The 70-kDa protein binds a 43-nt sequence near the 3'end of the 3'UTR and is immunoprecipitated by a monoclonal antibody against hnRNP L. A computer-simulated conformation of the 3'UTR suggests that both binding sites reside in regions easily accessible for a protein. Supershifts of the native RNA-protein complex could only be achieved with anti-hnRNP L, suggesting that within this multiprotein RNA complex, only hnRNP L is exposed to the antibodies, whereas the hnRNP I/PTB is mainly responsible for its interaction with the mRNA. Up-regulation of iNOS by septic shock reduces the RNA-protein complex formation, thus showing that hnRNP I/PTB and hnRNP L binding to the iNOS mRNA is modulated by inflammation. This suggests a novel function for the two previously described proteins as regulators of the iNOS gene.

3' Untranslated Regions↗