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Results for “Multiprotein Complexes”

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PubMed2000

JAB1 interacts with both the progesterone receptor and SRC-1.

JAB1 (Jun activation domain-binding protein-1) has previously been described as a coactivator of AP1 transcription factor. We show here, by yeast and mammalian two-hybrid analyses and by pull-down experiments, that JAB1 also interacts with both the progesterone receptor (PR) and the steroid receptor coactivator 1 (SRC-1) and that it stabilizes PR-SRC-1 complexes. We also show that JAB1 potentiates the activity of a variety of transcription factors known to associate with SRC-1 (nuclear receptors, activator protein-1, and nuclear factor kappaB). This occurs without any modification of PR or SRC-1 concentration. JAB1 is a subunit of a large multiprotein complex that has been called the COP9 signalosome. The latter is present in plant and animal cells and has been shown to be involved in a variety of cellular mechanisms including transcription regulation, cell cycle control, and phosphorylation cascades. We now show that it is also involved in the mechanisms of action of nuclear receptors and of their coactivators.

Binding Sites↗

PubMed2000

Control of beta-catenin signaling in tumor development.

The wnt signal transduction pathway is involved in various differentiation events during embryonic development and leads to tumor formation when aberrantly activated. The wnt signal is transmitted to the nucleus by the cytoplasmic component beta-catenin: in the absence of wnts, beta-catenin is constitutively degraded in proteasomes, whereas in the presence of wnts beta-catenin is stabilized and can associate with HMG box transcription factors of the LEF/TCF family. The LEF/TCF/beta-catenin complexes activate specific wnt target genes. In tumors, beta-catenin degradation is blocked by mutations of beta-catenin or of the tumor suppressor gene product APC. As a consequence, beta-catenin is stabilized, constitutive complexes with LEF/TCF factors are formed, and oncogenic target genes, such as c-myc, cyclin D1, and c-jun, are activated. Thus, control of beta-catenin is a major regulatory event in normal wnt signaling and during tumor formation. It has been found that a multiprotein complex assembled by the cytoplasmic component conductin induces degradation of cytoplasmic beta-catenin. The complex includes APC, the serine/threonine kinase GSK3 beta, and beta-catenin, which bind to conductin at distinct domains. In colon carcinoma cells, forced expression of conductin downregulates beta-catenin, whereas in normal cells mutants of conductin that are deficient in complex formation stabilize beta-catenin. Fragments of APC that contain a conductin-binding domain also block beta-catenin degradation. In Xenopus embryos, conductin inhibits the wnt pathway. In situ hybridization analysis shows that conductin is expressed in various embryonal tissues known to be regulated by wnts, such as the developing brain, mesenchyme below the epidermis, lung mesenchyme, and kidney. It is suggested that conductin controls wnt signaling by assembling the essential components of the beta-catenin degradation pathway. Alterations of conductin function may lead to tumor formation.

Cell Transformation, Neoplastic↗

PubMed1990

The transcription complex of the Xenopus somatic 5 S RNA gene. A functional analysis of protein-DNA interactions outside of the internal control region.

We have defined protein-DNA interactions associated with the transcription complex of a Xenopus somatic 5 S RNA gene under efficient in vitro transcription conditions. Transcription factor IIIA, the internal control region of the 5 S RNA gene and specific DNA sequences 5' and 3' of the internal control region are all involved in the formation of a multiprotein complex. Specific protein-DNA interactions outside of the 5 S RNA gene itself, revealed by DNase I footprinting, have no apparent role in the transcription process. Sequences within the 5 S RNA gene, 5' of the internal control region, are not essential for transcription by RNA polymerase III in vitro, but do contribute to its efficiency.

Animals↗

PubMed2000

Characterization of the multimeric Eps complex required for cholera toxin secretion.

Vibrio cholerae causes diarrheal disease through colonization of the small intestine. A critical aspect of V. cholerae pathogenesis is its ability to actively secrete cholera toxin to the extracellular environment. This occurs via the type II secretion pathway, where the toxin subunits are first transported to the periplasm through the Sec pathway. Following folding and assembly the toxin is then translocated across the outer membrane by a specialized Extracellular Protein Secretion (Eps) machinery encoded by at least 13 genes. Although the Eps proteins are believed to form a secretion apparatus that spans both membranes, cholera toxin is thought to engage this complex first in the periplasm. In order to determine the organization of the Eps apparatus and to understand the mechanism of secretion, the Eps apparatus has been dissected and three of the components, EpsE, EpsL and EpsM, have been purified and characterized. They were shown to form a stable, multiprotein complex spanning the cytoplasmic membrane.

Bacterial Proteins↗

PubMed2001

A Drosophila Polycomb group complex includes Zeste and dTAFII proteins.

A goal of modern biology is to identify the physical interactions that define 'functional modules' of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate 'on' or 'off' state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes, but these complexes have not been described owing to inherent difficulties in purification. We previously fractionated a major PcG complex, PRC1, to 20-50% homogeneity from Drosophila embryos. Here, we identify 30 proteins in these preparations, then further fractionate the preparation and use western analyses to validate unanticipated connections. We show that the known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery.

Animals↗

PubMed2001

Fibrillary co-deposition of laminin-5 and large unspliced tenascin-C in the invasive front of oral squamous cell carcinoma in vivo and in vitro.

PURPOSE: Invasion of oral squamous cell carcinoma (OSCC) is associated with laminin-5 (Ln-5) synthesis, focal Ln-5 loss from the basement membrane (BM), and Ln-5 depositions in the stroma beneath invading carcinoma cell complexes. METHODS: The study is focused on the laminin-5 matrix reorganisation within the stroma of the OSCC invasive front outside the basement membrane region as well as in OSCC-fibroblast co-culture in relation to unspliced tenascin-C (Tn-CL) and ED-B+ fibronectin (ED-B+ fn) using confocal laser scanning microscopy. RESULTS: In vivo, Ln-5 was demonstrated as fibrillary deposition in the invasive front. It was co-localised to Tn-CL. In pure OSCC cultures, Ln-5 was synthesised and deposited as a spot-like matrix. Fibrillary structures were not found. In contrast, in the OSCC-fibroblast co-culture, a fibrillary Ln-5 matrix organisation was revealed within the interface of OSCC cell-fibroblast complexes exclusively in co-distribution with Tn-CL and ED-B+ fn. CONCLUSION: At least in vitro, a carcinoma cell-stroma fibroblast interaction is indispensable for fibrillary Ln-5/Tn-CL matrix organisation. Behind the parallels to the initial basement membrane formation in organotypic cultures, the fibrillary multiprotein complexes at the OSCC cell-fibroblast interface are suggested as provisional basement membrane fragments with a possible supportive role for invasive tumour behaviour.

Carcinoma, Squamous Cell↗

PubMed2003

Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes.

Multi-protein complexes are emerging as important entities of biological activity inside cells that serve to create functional diversity by contextual combination of gene products and, at the same time, organize the large number of different proteins into functional units. Many a time, when studying protein complexes rather than individual proteins, the biological insight gained has been fundamental, particularly in cases in which proteins with no previous functional annotation could be placed into a functional context derived from their 'molecular environment'. In this minireview, we summarize the current state of the art for the retrieval of multiprotein complexes by affinity purification and their analysis by mass spectrometry. The advances in technology made over the past few years now enable the study of protein complexes on a proteomic scale and it can be anticipated that the knowledge gathered from such projects will fuel drug target discovery and validation pipelines and that the technology is also going to prove valuable in the emerging field of systems biology.

Animals↗

PubMed1995

Transcription regulation by inflexibility of promoter DNA in a looped complex.

The gal operon of Escherichia coli is negatively regulated by repressor binding to bipartite operators separated by 11 helical turns of DNA. Synergistic binding of repressor to separate sites on DNA results in looping, with the intervening DNA as a topologically closed domain containing the two promoters. A closed DNA loop of 11 helical turns, which is in-flexible to torsional changes, disables the promoters either by resisting DNA unwinding needed for open complex formation or by impeding the processive DNA contacts by an RNA polymerase in flux during transcription initiation. Interaction between two proteins bound to different sites on DNA modulating the activity of the intervening segment toward other proteins by allostery may be a common mechanism of regulation in DNA-multiprotein complexes.

Allosteric Regulation↗

PubMed1999

DP1 phosphorylation in multimeric complexes: weaker interaction with cyclin A through the E2F1 cyclin A binding domain leads to more efficient phosphorylation than stronger interaction through the p107 cyclin A binding domain.

Stable enzyme-substrate interaction has been recognized as a major mechanism underlying the substrate preferences of cyclin-dependent kinases (Cdks). To learn the relationship between stability of physical association and efficiency of phosphorylation, we studied DP1 phosphorylation by cyclin A-Cdk2 in multiprotein complexes. When DP1 was connected to cyclin A-Cdk2 through E2F4 and p107, its phosphorylation was very inefficient, although its association with cyclin A-Cdk2 was stable. In contrast, DP1 was efficiently phosphorylated when weakly connected to cyclin A-Cdk2 via E2F1 or E2F4 with a fused cyclin A binding domain of E2F1. The transactivation activity of E2F4-DP1 heterodimers was reduced when DP1 was phosphorylated, while a phosphorylation deficient mutant of DP1 resisted this down-regulation. Phosphorylation and functional regulation of DP1 were not due to nuclear localization. Thus, stronger physical association between the kinase and the substrate does not necessarily lead to more efficient phosphorylation than weaker interaction does.

Amino Acid Sequence↗

PubMed1997

A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-erbB signaling to actin stress fiber disassembly.

The avian erythroblastosis viral oncogene (v-erbB) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v-erbB in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v-erbB-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc-Grb2-Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.

Actins↗

PubMed2001

SH3 domains: complexity in moderation.

The SH3 domain is perhaps the best-characterized member of the growing family of protein-interaction modules. By binding with moderate affinity and selectivity to proline-rich ligands, these domains play critical roles in a wide variety of biological processes ranging from regulation of enzymes by intramolecular interactions, increasing the local concentration or altering the subcellular localization of components of signaling pathways, and mediating the assembly of large multiprotein complexes. SH3 domains and their binding sites have cropped up in many hundreds of proteins in species from yeast to man, which suggests that they provide the cell with an especially handy and adaptable means of bringing proteins together. The wealth of genetic, biochemical and structural information available provides an intimate and detailed portrait of the domain, serving as a framework for understanding other modular protein-interaction domains. Processes regulated by SH3 domains also raise important questions about the nature of specificity and the overall logic governing networks of protein interactions.

Actins↗

PubMed2026

Targeting the Disease Response With NlpD and LytM for Effective Nonantibiotic Treatment of Urinary Tract Infections.

BACKGROUND: Finding new ways of treating bacterial infections is essential. The NlpD protein, which inhibits RNA polymerase II (Pol II), has shown therapeutic efficacy against urinary tract infection. This study investigated the mechanism of Pol II inhibition and protection by NlpD and its LytM peptide. METHODS: Recombinant NlpD and LytM were screened for interactions with constituents of the Pol II complex, using AlphaFold predictions and protein interaction technology. Treatment effects were quantified in infected tissues and regulated host response pathways identified by genome-wide transcriptomics analysis in models of acute pyelonephritis and acute cystitis in Irf3-/- and Asc-/- mice, respectively. RESULTS: LytM was shown to interact with constituents of the Pol II multiprotein complex, inhibiting the CDK12 kinase from phosphorylating the Pol II subunit RPB1 and disrupting Pol II complex formation by interfering with the interaction between PAF1C and RPB1. The protection by LytM against acute pyelonephritis was accompanied by a reduction in gene expression in infected kidneys from >1900 significantly regulated genes (fold change >6) in the placebo group to about 150 in LytM-treated mice. The inhibition of gene expression in infected kidneys particularly targeted the excessive innate immune response. A similar effect was observed in acute cystitis. Bacterial clearance was accelerated in both model by LytM treatment, with effects against antibiotic-sensitive and resistant Escherichia coli strains. CONCLUSIONS: The results suggest that inhibiting the disease response of the host, using NlpD or LytM, may offer an efficient alternative to antibiotics in these models.

Animals↗

PubMed2004

Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts.

The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade.

Cell Line↗

PubMedDate not supplied

Structure and control of a cell-cell adhesion complex associated with spermiation in rat seminiferous epithelium.

Spermiation, the release of late spermatids from the Sertoli cell, is disrupted by a number of toxicants. Control of the spermiation process, and the proteins that interact to adhere mature spermatids to Sertoli cells, is poorly understood. In these studies we used immunohistochemistry, coimmunoprecipitation/Western blotting, and mass spectrometry to refine an earlier model of sperm adhesion proposed by our laboratory. We have identified specific proteins linked together as part of a multiprotein complex, as well as several additional proteins (cortactin, ERK1/2, and 14-3-3 zeta) that may be functioning in both structural and signal transduction roles. The current and prior data suggest that protein phosphorylation is central to the control of spermiation. We also present and characterize an in vitro tubule culture system that allowed functional testing of the spermiation model by pharmacologic manipulation, and yielded data consistent with the importance of protein phosphorylation in spermiation.

Animals↗

PubMed1997

MPc2, a new murine homolog of the Drosophila polycomb protein is a member of the mouse polycomb transcriptional repressor complex.

The evolutionarily conserved polycomb and trithorax-group genes are required to maintain stable expression patterns of homeotic genes and other target genes throughout development. Here, we report the cloning and characterization of a novel mouse polycomb homolog, MPc2, in addition to the previously described M33 polycomb gene. Co-immunoprecipitations and subnuclear co-localization studies show that MPc2 interacts with the mouse polycomb-group oncoprotein Bmi1 and is a new member of the mouse polycomb multiprotein complex. Gal4DB-MPc2 or -M33 fusion proteins mediate a five- to tenfold repression of stably integrated reporter constructs carrying GAL4 binding sites, demonstrating that these proteins are transcriptional repressors. The MPc2 gene is localized on chromosome 11, in close proximity to the classical mouse mutations tail short (Ts) and rabo torcido (Rbt). Ts and Rbt hemizygous mice display anemia and transformations of the axial skeleton reminiscent of phenotypes observed in mice with mutated polycomb or trithorax-group genes, suggesting that MPc2 is a candidate gene for Ts and Rbt.

Amino Acid Sequence↗

PubMed1991

Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.

A discontinuous electrophoretic system for the isolation of membrane proteins from acrylamide gels has been developed using equipment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie dyes were introduced to induce a charge shift on the proteins and aminocaproic acid served to improve solubilization of membrane proteins. Solubilized mitochondria or extracts of heart muscle tissue, lymphoblasts, yeast, and bacteria were applied to the gels. From cells containing mitochondria, all the multiprotein complexes of the oxidative phosphorylation system were separated within one gel. The complexes were resolved into the individual polypeptides by second-dimension Tricine-SDS-PAGE or extracted without SDS for functional studies. The recovery of all respiratory chain complexes was almost quantitative. The percentage recovery of functional activity depended on the respective protein complex studied and was zero for some complexes, but almost quantitative for others. The system is especially useful for small scale purposes, e.g., separation of radioactively labeled membrane proteins, N-terminal protein sequencing, preparation of proteins for immunization, and diagnostic studies of inborn neuromuscular diseases.

Aminocaproates↗

PubMed2006

Probing the in vivo dynamics of type I protein secretion complex association through sensitivity to detergents.

The Serratia marcescens hemophore is secreted by a type I secretion system consisting of three proteins: a membrane ABC protein, an adaptor protein, and the TolC-like outer membrane protein. Assembly of these proteins is induced by substrate binding to the ABC protein. Here we show that a hemophore mutant lacking the last 14 C-terminal amino acids is not secreted but rather interacts with the ABC protein and promotes a stable multiprotein complex. Strains expressing the transporter and the mutant protein are sensitive to detergents (sodium dodecyl sulfate [SDS]). TolC is trapped in the transporter jammed by the truncated substrate and therefore is not present at sufficient concentrations to allow the efflux pumps to expel detergents. Using an SDS sensitivity assay, we showed that the hemophore interacts with the ABC protein via two nonoverlapping sites. We also demonstrated that the C-terminal peptide, which functions as an intramolecular signal sequence in the complete substrate, may also have intermolecular activity and triggers complex dissociation in vivo when it is provided as a distinct peptide. The SDS sensitivity test on plates enables workers to study type I secretion protein association and dissociation independent of the secretion process itself.

ATP-Binding Cassette Transporters↗

PubMed2006

VirB8: a conserved type IV secretion system assembly factor and drug target.

Type IV secretion systems are used by many gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA-protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8-12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure-function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein-protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.

Amino Acid Sequence↗