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C A Panagiotidis

Publications and source records attributed to C A Panagiotidis.

15 recordsLinked to original sources
PubMed1997

Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27.

The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4.

Binding Sites↗
PubMed1996

Repression of the alpha0 gene by ICP4 during a productive herpes simplex virus infection.

During a productive infection by herpes simplex virus type 1 (HSV-1), ICP4, the major regulatory protein encoded by the alpha4 gene, binds to its transcription initiation site and represses the accumulation of alpha4 RNA. Evidence suggests that the degree of repression by ICP4 is a function of the absolute distance of an ICP4 binding site 3' from a TATA box. However, repression of HSV-1 gene expression by ICP4 through binding sites located 5' of TATA boxes, as in the case of the alpha0 gene, has not been adequately addressed. To this end, recombinant alpha0 promoters with various arrays of ICP4 binding sites flanking the alpha0 TATA box were constructed and recombined into the HSV-1 genome. Our results demonstrate the following. (i) Destruction of the endogenous alphaO ICP4 binding site, located 5' of the TATA box, results in derepression of alpha0 protein and RNA accumulation in infected Vero cells. (ii) The degree of alpha0 derepression is equivalent to that reported for the alpha4 gene following destruction of the ICP4 binding site at the alpha4 mRNA cap site in HSV-1. (iii) Introduction of an ICP4 binding site at the alpha0 mRNA cap site represses the accumulation of alpha0 RNA greater than threefold relative to the wild type. (iv) Changes in the abundance of alpha0 protein and RNA in infected cells do not affect replication or growth of HSV-1 in tissue culture. Our findings are consistent with the conclusion that alpha0 transcription is repressed by ICP4. These results demonstrate that repression by ICP4 can occur through binding sites located 5' of virus gene TATA boxes in HSV-1. Thus, models addressing repression of HSV-1 gene expression by ICP4 should incorporate the role of binding sites located 5', as well as 3', of virus gene TATA boxes.

Animals↗
PubMed1995

pALEX, a dual-tag prokaryotic expression vector for the purification of full-length proteins.

pALEX, a prokaryotic expression vector, was constructed in which the multiple cloning site (MCS, polylinker) is flanked by sequences encoding glutathione S-transferase (GST) at the 5' end and a His6 residue tag at the 3' end. Open reading frames cloned into this vector can direct production of fusion proteins with GST at their N terminus and a His6 tag at their C terminus. This allows for the purification of full-size fusion proteins by a sequential two-step procedure on glutathione-agarose and Ni(2+)-agarose columns.

Amino Acid Sequence↗
PubMed1995

Polyamines alter sequence-specific DNA-protein interactions.

The polyamines are abundant biogenic cations implicated in many biological processes. Despite a plethora of evidence on polyamine-induced DNA conformational changes, no thorough study of their effects on the activities of sequence-specific DNA binding proteins has been performed. We describe the in vitro effects of polyamines on the activities of purified, representative DNA-binding proteins, and on complex protein mixtures. Polyamines at physiological concentrations enhance the binding of several proteins to DNA (e.g. USF, TFE3, Ig/EBP, NF-IL6, YY1 and ICP-4, a herpes simplex virus gene regulator), but inhibit others (e.g. Oct-1). The degree of enhancement correlates with cationic charge; divalent putrescine is ineffective whereas tetravalent spermine is more potent than trivalent spermidine. Polyamine effects on USF and ICP-4 result from increased rate of complex formation rather than a decreased rate of dissociation. DNAse I footprint analysis indicated that polyamines do not alter DNA-protein contacts. Polyamines also facilitate formation of complexes involving binding of more than one protein on a DNA fragment.

Base Sequence↗
PubMed1995

Relationship of the expression of the S20 and L34 ribosomal proteins to polyamine biosynthesis in Escherichia coli.

Polyamine biosynthesis in Escherichia coli is regulated transcriptionally and post-translationally. Antizyme and ribosomal proteins S20 and L34 participate in post-translational inhibition of the polyamine biosynthetic enzymes ornithine and arginine decarboxylase. The aim of the present study was to investigate the significance of S20 and L34 in polyamine regulation in vivo. In vivo overexpression of S20 and L34 lowered the activities of ornithine and arginine decarboxylases and decreased total polyamine production. The levels of cadaverine, a related diamine whose synthesis is not regulated by S20 and L34, did not decrease but increased. The diminished ornithine and arginine decarboxylase activities are shown to result from reversible post-translational inhibition since the enzymes could be reactivated to normal levels upon titration of the inhibitors. The effects were specific as overexpression of eight other ribosomal proteins had no influence. Overexpression of ornithine decarboxylase results in elevated polyamine production and it increases S20 and L34 levels but not those of other ribosomal proteins. Ornithine depletion decreases S20 and L34 to normal levels in the ornithine decarboxylase overproducing cells. Immunoprecipitation experiments coupled with immunoblots indicated that ornithine and arginine decarboxylases physically interact with S20 and L34. This study shows that ribosomal proteins S20 and L34 can inhibit ornithine and arginine decarboxylases and polyamine biosynthesis in vivo. It is concluded that, unlike other basic ribosomal proteins and polycationic compounds which inhibit the activities of these enzymes only in vitro, S20 and L34 are biologically relevant in the regulation of the polyamine biosynthetic pathway.

Carboxy-Lyases↗
PubMed1994

Isolation and characterization of an Escherichia coli DnaK mutant with impaired ATPase activity.

A temperature-sensitive mutant of DnaK, the principal Escherichia coli member of the 70 kDa heat shock protein family, has been isolated. The mutation, dnaK25, lies in the putative ATP binding pocket of DnaK. It consists of a C to T transition that changes the highly conserved proline 143 to serine. Mutant strains do not support the propagation of bacteriophage lambda or of plasmids that require DnaA for replication. They are also defective in the utilization of mannose and sorbitol. ATPase activity of the mutant protein is reduced 20-fold relative to wild-type, while autophosphorylation is unaffected. DnaK25 has a fourfold faster rate of nucleotide exchange than wild-type DnaK; nucleotide exchange by both proteins is markedly increased by GrpE. The DnaK25 ATPase is still stimulated by DnaJ and GrpE and by peptide substrates. However, the affinity of most peptides tested for stimulating the DnaK25 ATPase is reduced significantly. These results indicate that a mutation in the N-terminal nucleotide binding domain can alter substrate interactions with the C-terminal substrate binding site. Nucleotide exchange by both wild-type DnaK and DnaK25 proceeds at a much faster rate than ATP hydrolysis, and therefore cannot be the rate limiting step of ATP hydrolysis under the conditions used in these experiments. Consistent with this, peptides, which stimulate ATP hydrolysis, have no effect on nucleotide exchange. Peptides thus appear to stimulate the ATPase by acting at another step, such as increasing the rate of phosphate bond cleavage.

Adenosine Triphosphatases↗
PubMed1994

Inhibition of DnaK autophosphorylation by heat shock proteins and polypeptide substrates.

DnaK, the Hsp70 of Escherichia coli, autophosphorylates in vitro. Of the two heat shock proteins that interact with DnaK, GrpE inhibits DnaK phosphorylation, whereas DnaJ has no effect on the reaction. Three synthetic peptides are shown to inhibit DnaK phosphorylation. The potency of a given peptide correlates with its affinity for the DnaK protein. A truncated DnaK that lacks the carboxyl-terminal peptide-binding domain autophosphorylates; this reaction is resistant to the inhibitory peptides. Phosphorylation of the truncated DnaK is still inhibited by GrpE, indicating that the GrpE-binding site resides in the DnaK amino-terminal domain. Thus, DnaK phosphorylation is regulated in vitro, and possibly in vivo, by physiologically relevant substrates and cofactors.

Adenosine Triphosphatases↗
PubMed1994

Barley beta-glucosidase: expression during seed germination and maturation and partial amino acid sequences.

Unlike most of the hydrolytic enzymes that participate in endosperm mobilization, beta-glucosidase of barley (Hordeum vulgare) seeds does not increase during germination, even in the presence of exogenously added gibberellic acid. However, the germination process affects the physical properties of beta-glucosidase in terms of charge and apparent molecular weight. Analysis of developing barley grains shows that the enzyme is synthesized two weeks before maturation and is stored in the endosperm of the dry dormant seed. Partial amino acid sequencing of the purified beta-glucosidase demonstrates significant similarity between the barley enzyme and beta-glycosidases that belong to family 1 of glycosyl hydrolases.

Amino Acid Sequence↗
PubMed1994

Post-translational and transcriptional regulation of polyamine biosynthesis in Escherichia coli.

Ornithine and arginine decarboxylases (ODC and ADC) of Escherichia coli are inhibited post-translationally by antizyme and ribosomal proteins S20 and L34. The inhibition of either enzyme is relieved when excess of the other decarboxylase is added. Using this approach, in vitro as well as in vivo, we demonstrate that the extent of the post-translational inhibition of ODC and ADC in E. coli is at least 65 and 50%, respectively. The inhibited enzyme levels increase even further upon exposure of cells to polyamines. The post-translational mode of regulation can counteract a 4-fold increase of ODC protein in the cell. The negative transcriptional regulation of ODC and ADC expression by polyamines is mediated by transcription factors and not by direct polyamine effects on the promoters of their genes. Three proteins interacting with the ODC promoter region were found by southwestern blot analysis.

Carboxy-Lyases↗
PubMed1993

Identification, cloning, and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli.

The ornithine decarboxylase antizyme gene of Escherichia coli was identified by immunological screening of an E. coli genomic library. A 6.4-kilobase fragment containing the antizyme gene was subcloned and sequenced. The open reading frame encoding the antizyme was identified on the basis of its ability to direct the synthesis of immunoreactive antizyme. Antizyme shares significant homology with bacterial transcriptional activators of the two-component regulatory system family; these systems consist of a "sensor" kinase and a transcriptional regulator. The open reading frame next to antizyme is homologous to sensor kinases. Antizyme overproduction inhibits the activities of both ornithine and arginine decarboxylases without affecting their protein levels. Extracts from E. coli bearing an antizyme gene-containing plasmid exhibit increased antizyme activity. These data strongly suggest that (i) the cloned gene encodes the ornithine decarboxylase antizyme and (ii) antizyme is a bifunctional protein serving as both an inhibitor of polyamine biosynthesis as well as a transcriptional regulator of an as yet unknown set of genes.

Amino Acid Sequence↗
PubMed1992

Modulation of ribonuclease P expression in Escherichia coli by polyamines.

1. The presence of polyamines in the growth medium of Escherichia coli can modulate the activity of the RNA-processing enzyme, ribonucleoprotein ribonuclease P (RNase P), by altering the expression of the rnpA and rnpB genes, which encode its C5 protein and M1 RNA subunits, respectively. 2. Following growth in the presence of 1 mM spermidine the levels of C5 protein mRNA and catalytic M1 RNA were significantly elevated in the wild type E. coli K-12 strain MG1655. 3. The rnpA mRNA, together with the ribosomal protein L34 (rpmH) mRNA, was found to constitute a dicistronic rpmH-rnpA message whose half-life did not change upon Escherichia coli growth in the presence of spermidine. 4. This suggests that the spermidine effect is on the transcriptional level. 5. Increased expression of the rnpA and rnpB genes was reflected in the activity of RNase P, which almost doubled. 6. These results identify yet another component of the protein synthetic machinery which is specifically affected by polyamines.

Blotting, Northern↗
PubMed1990

Transcriptional effects of polyamines on ribosomal proteins and on polyamine-synthesizing enzymes in Escherichia coli.

We find that the transcription of various ribosomal proteins can be differentially affected by polyamines and by changes in growth rates. Using strain MG1655 of Escherichia coli K-12 (F-, lambda-), we have determined the effects of polyamines and changes in growth rate on the transcription of several ribosomal genes and the polyamine-synthesizing enzymes ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17) and arginine decarboxylase (L-arginine carboxylyase; EC 4.1.1.19). Ribosomal proteins S20 and L34 can be differentiated from the other ribosomal proteins studied; the transcription of S20 and L34 is especially sensitive to polyamines and less sensitive to changes in growth rates. In contrast, the transcription of S10, S15, S19, L2, L4, L20, L22, and L23 is insensitive to polyamines although it is particularly sensitive to changes in growth rates. Like S20 and L34, the transcription of ornithine decarboxylase and arginine decarboxylase is especially sensitive to polyamines. Polyamines specifically enhance the transcription of ribosomal proteins S20 and L34, and decrease that of ornithine decarboxylase and arginine decarboxylase. It is evident that polyamines can exert both positive and negative regulation of gene expression in E. coli that can be differentiated from the effects caused by changes in growth rates.

Biogenic Polyamines↗
PubMed1987

Biosynthesis of polyamines in ornithine decarboxylase, arginine decarboxylase, and agmatine ureohydrolase deletion mutants of Escherichia coli strain K-12.

Escherichia coli K-12 mutants that carry deletions in their genes for ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (speC), arginine decarboxylase (L-arginine carboxy-lyase, EC 4.1.1.19) (speA), and agmatine ureohydrolase (agmatinase or agmatine amidinohydrolase, EC 3.5.3.11) (speB) can still synthesize very small amounts of putrescine and spermidine. The putrescine concentration in these mutants was found to be 1/2500th that in spe+ cells. The pathway of putrescine synthesis appears to be through the biodegradative arginine decarboxylase, which converts arginine to agmatine, in combination with a low agmatine ureohydrolase activity--1/2000th that in spe+ strains. These results suggest that even such low levels of polyamines permit a low level of protein synthesis. Evidence is presented that the polyamine requirement for the growth of the polyamine-dependent speAB, speC deletion mutants, which are also streptomycin resistant, is not due to a decreased ability to synthesize polyamines.

Bacterial Proteins↗
PubMed1984

Comparison of the basic Escherichia coli antizyme 1 and antizyme 2 with the ribosomal proteins S20/L26 and L34.

The two basic Escherichia coli proteins that inhibit ornithine and arginine decarboxylase and were named provisionally antizyme 1 and antizyme 2 (Heller, J.S., Rostomily, R., Kyriakidis, D.A., and Canellakis, E.S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5181-5184) are shown to have long identical sequences with the ribosomal proteins S20/L26 and L34, respectively. We have also isolated ribosomal proteins from purified E. coli ribosomes by established methodology and further purified them by our purification procedure for antizymes 1 and 2. Of the various basic ribosomal proteins, two were found to have the same properties as antizyme 1 and 2. These results indicate that these two basic E. coli antizymes are ribosomal proteins. The nature of the acidic antizyme remains to be elucidated.

Amino Acid Sequence↗