Cognate Binding Proteins Research Articles

GERp95 belongs to a family of signal-transducing proteins and requires Hsp90 activity for stability and Golgi localization.

GERp95 (Golgi-endoplasmic reticulum protein 95 kDa) is part of a large family of highly conserved proteins found in all metazoans and the fission yeast Schizosaccharomyces pombe. Genetic studies suggest that homologs of GERp95 are components of signaling pathways that regulate cellular differentiation, development, and RNA interference. However, the precise molecular functions of these proteins remain unknown. Genetic analysis of GERp95 homologs has been complicated by the presence of multiple genes with overlapping functions in most organisms. Binding partners for members of this protein family have not been identified. The purpose of this study was to identify proteins that associate with GERp95. Glutathione S-transferase-GERp95 fusions were expressed in transfected cells, and proteins that bound to GERp95 were co-purified using glutathione-agarose beads. The amino-terminal region of GERp95 was found to interact with the specialized chaperone Hsp90 and a number of its cognate binding proteins. Inhibition of Hsp90 activity with geldanamycin or radicicol resulted in rapid degradation of newly synthesized GERp95. The membrane-associated pool of GERp95 was not bound to Hsp90, although activity of this chaperone was required for stable association of GERp95 with the Golgi in normal rat kidney cells. These results indicate that GERp95 engages an Hsp90 chaperone complex prior to association with intracellular membranes.

Unzipping ion channels.

The functions of ion channels can be regulated by their phosphorylation state. Protein kinases and protein phosphatases tightly control the activity of channels, thereby regulating the flow of ions across cell membranes. Channel proteins and kinases or phosphatases can associate directly or through intermediate adaptor proteins. An interaction domain termed the leucine zipper (LZ), once thought to be unique to some families of transcription factors, has been identified in channel proteins and their cognate binding proteins. MacFarlane and Levitan discuss what roles LZ-containing proteins might have in controlling channel function.

Accessibility and Activity of the Promoter for a Dioxin-Inducible Ecto-ATPase Gene

We have analyzed the core promoter for a dioxin-inducible ecto-ATPase gene in mouse hepatoma cells. The transcriptional initiation site maps to a region that contains neither a TATA sequence nor a consensus initiator sequence nor a downstream promoter element. The core promoter has constitutive activity that does not require either the aromatic hydrocarbon receptor or its heterodimerization partner Arnt. Two GC-rich regions contribute approximately equally to the constitutive activity. Proteins constitutively occupy the GC-rich regions in chromatin. The promoter assumes a non-nucleosomal configuration in its native chromosomal setting in both uninduced and dioxin-induced cells. Our findings imply that the GC-rich regions together with their cognate binding proteins carry out core promoter functions for the ecto-ATPase gene. The promoter is constitutively accessible in situ, and chromatin structure is not a limiting factor for dioxin-inducible ecto-ATPase transcription in intact cells.

Regulation of human coagulation factor X gene expression by GATA-4 and the Sp family of transcription factors

AbstractSerine protease factor Xa plays a critical role in the coagulation cascade. Zymogen factor X is synthesized and modified in the liver. To understand the mechanisms governing the liver-specific expression of factor X, the proximal promoter of human factor X was previously characterized. Two crucial cis elements at −73 and −128 and their cognate binding proteins, HNF-4 and NF-Y, respectively, were identified. In this report, studies are extended to 3 additionalcis elements within the factor X promoter. Using gel mobility shift assays, the liver-enriched protein GATA-4 was identified as the protein binding to the GATA element at −96. GATA-4 transactivates the factor X promoter 28-fold in transient transfection experiments. It was also determined that the Sp family of transcription factors binds 2 DNase I–footprinted sites at −165 and −195. Disruption of Sp protein binding at either site reduces the promoter activity by half. Simultaneous disruption of both sites reduces the promoter activity 8-fold. This is the first report indicating the involvement of GATA-4 in the regulation of clotting factor expression. These observations provide novel insight into mechanisms by which the vitamin K–dependent coagulation factors are regulated.

34] Chloroplast p54 Endoribonuclease

Much of the regulation of chloroplast gene expression has been assigned to stability determinants at the level of RNA precursors, including interactions of sequence and/or structural elements with their cognate binding proteins. A number of these proteins are known to assemble at the 5′-untranslated region (UTR) of chloroplast mRNAs, protecting them from exonucleolytic degradation and forming ribonucleoprotein (RNP) complexes involved in translational control. One of the proteins known to be controlled by phosphorylation is a 3′-endoribonuclease, which was first found to specifically bind to a conserved U-rich sequence element (UUUAUCU) of chloroplast precursor transcripts. This protein, which purifies as a monomeric polypeptide of an apparent molecular size of 54 kDa, was subsequently termed p54. This chapter presents the purification and properties of the p54 endoribonuclease from mustard (Sinapis alba), which might turn out to be a key player in posttranscriptional regulation of chloroplast gene expression. As a first step toward the purification of the p54 endonuclease, chloroplast protein extracts from mustard (S. alba) are prepared. Although the procedure has been developed for mustard, it has been found suitable for other higher plant species as well without significant modifications.

Regulation of gene expression by GC-rich DNA cis-elements.

GC-rich DNA cis elements are important transcriptional regulatory elements present in the promoter, enhancer and locus control regions of many eukaryotic genes from several species. This review attempts to examine the structure, function and biological significance of GC-rich cis -regulatory elements and their cognate binding proteins, with a view to understanding their role in regulation of gene expression.

The period E-box is sufficient to drive circadian oscillation of transcription in vivo.

The minimum element from the Drosophila period promoter capable of driving in vivo cycling mRNA is the 69 bp circadian regulatory sequence (CRS). In cell culture, an 18 bp E-box element from the period promoter is regulated by five genes that are involved in the regulation of circadian expression in flies. This E-box is a target for transcriptional activation by bHLH-PAS proteins dCLOCK (dCLK) and CYCLE (CYC), this activation is inhibited by PERIOD (PER) and TIMELESS (TIM) together, and inhibition of dCLK/CYC by PER and TIM is blocked by CRYPTOCHROME (CRY) in the presence of light. Here, the same 18 bp E-box region generated rhythmic expression of luciferase in flies under both light-dark cycling and constant conditions. Flies heterozygous for the Clke(jrk) mutation maintained rhythmic expression from the E-box although at a lower level than wild type. Homozygous mutant Clk(jrk) animals had drastically lowered and arrhythmic expression. In a per01 background, expression from the E-box was high and not rhythmic. Transcription mediated by the per E-box was restricted to the same spatial pattern as the CRS. The per E-box DNA element and cognate binding proteins can confer per-like temporal and spatial expression. This demonstrates in vivo that the known circadian genes that form the core of the circadian oscillator in Drosophila integrate their activities at a single DNA element.

Regulation of Competence Development in Haemophilus influenzae: Proposed Competence Regulatory Elements are CRP-Binding Sites

Development of competence for DNA uptake by the bacterium Haemophilus influenzae is tightly regulated, and expression of the cell's complement of competence genes is absolutely dependent on the cAMP–CRP complex. A second regulator of competence may maximize competence under starvation conditions. Several investigators have recently identified a consensus sequence (competence regulatory element, CRE) in the promoter regions of some competence genes and have proposed that this may be a binding site for Sxy (TfoX), a putative positive regulator of competence. However, a scoring method that reliably ranks candidate binding sites according to affinity for the cognate binding protein predicts that the cAMP–CRP complex will bind CRE sequences with high affinity. Moreover, the predicted Sxy protein lacks recognizable DNA-binding motifs and has not been shown to bind DNA. No other consensus sequences (putative binding sites) were identified in the promoter regions of competence genes. These observations suggest that the proposed competence-specific regulatory elements are in fact CRP-binding sites, and highlight the central role of cAMP—an established bacterial mediator of the response to nutritional stress—in competence regulation. Minor sequence elements uniquely conserved in the set of CRE sequences are predicted to reduce CRP affinity, and a model is suggested in which a secondary regulator of competence genes may interact with CRP under certain conditions to stabilize the initiation complex.

Interplay between bone morphogenetic proteins and cognate binding proteins in bone and cartilage development: noggin, chordin and DAN

This commentary is a concise discussion of the interactions between bone morphogenetic proteins (BMPs) and their binding proteins in bone and cartilage morphogenesis. BMPs are a family of growth and differentiation factors, and they act on mesenchymal cells to induce cartilage and bone differentiation in concentration-dependent thresholds. The BMP–BMP receptor binding leads to a cascade of signaling and transcription of BMP response genes. BMP binding proteins, noggin, chordin and DAN, act as antagonists and determine the bioavailability of BMPs for binding to cognate receptors to elicit the biological response. Noggin null mice with unrestricted action of BMPs exhibit defects in joint morphogenesis. BMPs and their binding proteins may reciprocally regulate the dynamic topography of joints, muscle, tendons and ligaments during morphogenesis of the skeleton. In addition, BMP actions may be potentiated by twisted gastrulation. BMPs and their binding proteins may play a critical role in regeneration of cartilage in osteoarthritis.

A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins

Background: Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. Results: The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. Conclusions: These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

Regulation of c-myc Transcription by Interleukin-2 (IL-2): IDENTIFICATION OF A NOVEL IL-2 RESPONSE ELEMENT INTERACTING WITH STAT-4

Regulation of c-myc expression is known to occur at the level of transcription initiation. However, the participating promoter elements and their cognate binding proteins have not been fully characterized. c-myc transcription can be stimulated by a number of cytokines including interleukin-2 (IL-2). We have identified a novel IL-2-responsive element, located in the 5'-flanking region of the c-myc gene, between nucleotides -1406 and -1387 (relative to the P2 promoter). This element belongs to the family of interferon-gamma activation site-like responsive elements and has the core sequence TTCCAATAA. We confirmed that IL-2-mediated signaling involves activation by phosphorylation of Jak2 tyrosine kinase and subsequently STAT4. The transcription factor STAT4 binds the TTCCAATAA motif within this responsive element and, therefore, is probably involved in enhancing c-myc transcription upon IL-2 stimulation. Our results propose participation of Jak2 and STAT4 in IL-2-induced up-regulation of c-myc.

Transcriptional repression by neuron-restrictive silencer factor is mediated via the Sin3-histone deacetylase complex.

A large number of neuron-specific genes characterized to date are under the control of negative transcriptional regulation. Many promoter regions of neuron-specific genes possess the repressor element repressor element 1/neuron-restrictive silencing element (RE1/NRSE). Its cognate binding protein, REST/NRSF, is an essential transcription factor; its null mutations result in embryonic lethality, and its dominant negative mutants produce aberrant expression of neuron-specific genes. REST/NRSF acts as a regulator of neuron-specific gene expression in both nonneuronal tissue and developing neurons. Here, we shown that heterologous expression of REST/NRSF in Saccharomyces cerevisiae is able to repress transcription from yeast promoters engineered to contain RE1/NRSEs. Moreover, we have taken advantage of this observation to show that this repression requires both yeast Sin3p and Rpd3p and that REST/NRSF physically interacts with the product of the yeast SIN3 gene in vivo. Furthermore, we show that REST/NRSF binds mammalian SIN3A and HDAC-2 and requires histone deacetylase activity to repress neuronal gene transcription in both nonneuronal and neuronal cell lines. We show that REST/NRSF binding to RE1/NRSE is accompanied by a decrease in the acetylation of histones around RE1/NRSE and that this decrease requires the N-terminal Sin3p binding domain of REST/NRSF. Taken together, these data suggest that REST/NRSF represses neuronal gene transcription by recruiting the SIN3/HDAC complex.

Collaborative interactions between MEF‐2 and Sp1 in muscle‐specific gene regulation

Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase–Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation. J. Cell. Biochem. 70:366–375, 1998. © 1998 Wiley-Liss, Inc.

Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation

Previous investigations have demonstrated synergistic interactions in vivo between CCAC and A/T-rich nucleotide sequence motifs as functional components of muscle-specific transcriptional enhancers. Using CCAC and A/T-rich elements from the myoglobin and muscle creatine kinase (MCK) gene enhancers, Sp1 and myocyte-specific enhancer factor-2 (MEF-2) were identified as cognate binding proteins that recognize these sites. Physical interactions between Sp1 and MEF-2 were demonstrated by immunological detection of both proteins in DNA binding complexes formed in vitro by nuclear extracts in the presence of only the A/T sequence motif, by coprecipitation of recombinant MEF-2 in the presence of a glutathione-S-transferase-Sp1 fusion protein bound to glutathione beads, and by a two-hybrid assay in Saccharomyces cerevisiae. The interaction with Sp1 in vitro and in vivo is specific for MEF-2 and was not observed with serum response factor, a related MADS domain protein. Forced expression of Sp1 and MEF-2 in insect cells otherwise lacking these factors promotes synergistic transcriptional activation of a promoter containing binding sites for both proteins. These data expand the repertoire of functional and physical interactions between lineage-restricted (MEF-2) and ubiquitous (Sp1) transcription factors that may be important for myogenic differentiation.

A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo.

RNA-protein interactions are central to many aspects of cellular metabolism, cell differentiation, and development as well as the replication of infectious pathogens. We have devised a versatile, broadly applicable in vivo system for the analysis of RNA-protein interactions in yeast. TRAP (translational repression assay procedure) is based on the translational repression of a reporter mRNA encoding green fluorescent protein by an RNA-binding protein for which a cognate binding site has been introduced into the 5' untranslated region. Because protein binding to the 5' untranslated region can sterically inhibit ribosome association, expression of the cognate binding protein causes significant reduction in the levels of green fluorescent protein fluorescence. By using RNA-protein interactions with affinities in the micromolar to nanomolar range, we demonstrate the specificity of TRAP as well as its ability to recover the cDNA encoding a specific RNA-binding protein, which has been diluted 500,000-fold with unrelated cDNAs, by using fluorescence-activated cell sorting. We suggest that TRAP offers a strategy to clone RNA-binding proteins for which little else than the binding site is known, to delineate RNA sequence requirements for protein binding as well as the protein domains required for RNA binding, and to study effectors of RNA-protein interactions in vivo.

Induction of Amelogenin and Ameloblastin by Insulin and Insulin-like Growth Factors (IGF-I and IGF-II) during Embryonic Mouse Tooth Development In Vitro

Insulin and insulin-like growth factors (IGF-I and IGF-II) are considered pleiotropic, acting as both mitogen and differentiation factors. Several investigators have demonstrated the expression of insulin, IGFs, their cognate receptors and IGF binding proteins during tooth morphogenesis. Previous work done in our laboratory indicated that exogenous insulin and IGFs induce the accumulation of enamel extracellular matrix on mouse mandibular molars cultured in a serumless, chemically defined medium. In order to determine the level of control of these factors in the induction of enamel biomineraliza-tion, we designed experiments to quantitate mRNAs for enamel specific-gene products. Mandibular first molars (M1) obtained from E15 Swiss Webster mice were placed in organ culture in the presence of insulin (1000ng/ml), IGF-I (100ng/ml) or IGF-II (100ng/ml) for 6,12 and 18-days. At termination date, the RNA was extracted and the concentration of mRNAs for amelogenin, tuftelin and ameloblastin were determined using a quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) technique (PCR mimic). Our results showed that after 6-days in culture; treatment with insulin, IGF-I and IGF-II increased the synthesis of amelogenin and ameloblastin. In contrast, the expression of tuftelin mRNA was not affected by either factor. In conclusion, our studies showed that the increase in enamel matrix formation by overexpression of IGFs is the result of transcriptional regulation of enamel specific proteins like amelogenin and ameloblastin but not tuftelin. These studies also suggest that the regulatory mechanisms controlling tuftelin gene expression are different than the mechanisms regulating ameloblastin and amelogenin transcription.

The role of cell adhesion molecules in placental development and barrier function: A workshop report

There is a striking evolutionary, structural and functional homology between epithelial and endothelial adhesion molecules. Different cell populations express overlapping repertoires of molecules, with the major classes of CAMs (cell adhesion molecules), immunoglobulin superfamily, integrins, cadherins, occludin and selectins, represented. John Aplin (Manchester) pointed out that the regulatory biology of the cell surface is unique: in addition to changing receptor numbers by altered transcription and translation, regulation can be achieved by altering density at the surface (insertion, internalization, shedding), redistribution into specialized domains such as junctional complexes, association with components of the submembranous cytoskeleton and activation mechanisms such as phosphorylation. Signaling events affecting receptor function are directional, either inside-out or outside-in. Therefore, immunolocalization in tissue is no more than a first step towards understanding function. Aplin took as an opening paradigm the apical plasma membrane of endometrial epithelium which, despite not being normally in contact with another plasma membrane (except with trophoectoderm at implantation), contains several adhesion molecules: integrins ~v[33 and cw~5, cad-11, trophinin, heparan sulphate and its cognate cell surface binding proteins, and the glycans H type 1 and Lewis y (Aplin, 1997). It also contains the cell surface-intercalated mucin MUC1, which has been shown to have non-specific antiadhesive activities as well as to mediate adhesion by interaction with ICAM-1. Hans-Werner Denker (Essen) showed morphological evidence of the close apposition between plasma membranes of the trophectoderm and uterine epithelium at attachment. For studies in the human, model systems are necessary; Denker's group is using the interaction between choriocarcinoma and endometrial carcinoma cells. JAr cells are cultured on Sephadex beads to model the trophectodermal surface of the blastocyst. The beads are allowed to settle onto monolayer cultures of carcinoma cells as a model of the uterine luminal epithelium. He described the use of the atomic force microscope to measure the energy of interaction. The data suggest significantly different interactions in the two endometrial cell lines HEC and RL95, perhaps reflecting their marked differences in polarization (Thie et aL, 1995). Denker also showed data

Identification of an Endothelial Cell-specific Regulatory Region in the Murine Endothelin-1 Gene

Endothelin-1 is a 21-amino acid peptide first characterized as a potent vasoactive compound synthesized by endothelial cells. Because of its high level cell-restricted pattern of expression, we have employed this gene as a model for investigating the DNA and protein elements that mediate endothelial cell-specific gene expression. In this study we have identified a complex positive regulatory region located at base pairs -364 to -320 in the murine endothelin-1 gene. This region consists of three functionally dependent elements, ETE-C, ETE-D, and ETE-E, which are all required for full activity. When a 43-base pair fragment containing these three elements was employed in heterologous promoter experiments, this sequence was capable of increasing transcriptional activity in an endothelial cell-specific fashion. None of the elements contains a recognized consensus sequence known to bind transcriptional regulatory proteins in higher eukaryotes; however, each element does appear to mediate protein binding. The combination of all three elements promotes binding of a protein complex that is endothelial cell-specific. This is the first evidence for an endothelial cell-specific DNA regulatory element and cognate binding proteins.

Regulation of Eukaryotic Messenger RNA Turnover

We have demonstrated the existence of multiple mRNA binding proteins that interact specifically with defined regions in posttranscriptionally regulated mRNAs. These domains appear to be destabilizers whose function can be attenuated by the interaction with the specific binding proteins. Thus, the ability to alter mRNA decay rates on demand, given different environmental or intracellular conditions, appears to be mediated by controlling the localization, activity, and overall function of the cognate binding protein. Based on our limited experience, we predict that most, if not all, of similarly regulated mRNAs will ultimately be found to interact with regulatory mRNA binding proteins. Under conditions whereby the mRNA binding proteins are constitutively active (e.g., tumor cell lines), abnormal mRNA decay will result, with accumulation and overtranslation. Such appears to be the case for cytokines and possibly amyloid protein precursor mRNAs in cancer and Alzheimer's disease, respectively. Conversely, mutagenesis of these critical 3' untranslated region elements will likely have comparable deleterious effects on the regulation of gene expression. To the extent that such derangements exist in human disease, attention to understanding the mechanistic detail at this level may provide insights into the development of appropriate therapeutics or treatment strategies.

Characterization of three promoter elements and cognate DNA binding protein(s) necessary for IFN-gamma induction of gp91-phox transcription.

The cytochrome b558 heavy chain (gp9l-phox) is expressed in terminally differentiated myelomonocytic cells. Three cis-elements located between -450 and -100 bp of the gp91-phox promoter are required for IFN-gamma induced transcription. Mutations that disrupt individual cis-elements incrementally decrease gp9l-phox promoter activity, and one of the two proximal elements must be present for an IFN-gamma response. The DNA-binding activities that interact with each of the cis-elements exhibit similar gel mobility and binding site specificity, although a consensus binding site common to the three elements is not apparent. An increased level of each DNA/protein complex is observed in myeloid cells following treatment with PMA, retinoic acid/dimethylformamide, or IFN-gamma, but not in similarly treated HeLa cells. The myeloid-specific increase in the intensity of each complex is delayed 12 to 24 h following IFN-gamma treatment, and the complexes are not immunoreactive with antisera directed against IFN-responsive factors such as IRF-1, IRF-2, IFN consensus sequence binding protein, Stat1, and IFN-stimulated gene factor-3 gamma, although IRF-2 is additionally detected as binding to the middle cis-element. These results reveal cis-elements and a DNA-binding factor(s) that participate in a common pathway in response to various stimuli that induce gp9l-phox transcription.