Cytoplasmic DNA-binding Protein Research Articles

Inhibition of the RNA polymerase III-mediated dsDNA-sensing pathway of innate immunity by vaccinia virus protein E3

The vaccinia virus E3 protein is an important intracellular modulator of innate immunity that can be split into distinct halves. The C terminus contains a well defined dsRNA-binding domain, whereas the N terminus contains a Z-DNA-binding domain, and both domains are required for virulence. In this study, we investigated whether the E3 Z-DNA-binding domain functions by sequestering cytoplasmic dsDNA thereby preventing the induction of type I interferon (IFN). In line with this hypothesis, expression of E3 ablated both IFN-β expression and NF-κB activity in response to the dsDNA, poly(dA–dT). However, surprisingly, the ability of E3 to block poly(dA–dT) signalling was independent of the N terminus, whereas the dsRNA-binding domain was essential, suggesting that the Z-DNA-binding domain does not bind immunostimulatory dsDNA. This was confirmed by the failure of E3 to co-precipitate with biotinylated dsDNA, whereas the recruitment of several cytoplasmic DNA-binding proteins could be detected. Recently, AT-rich dsDNA was reported to be transcribed into 5′-triphosphate poly(A-U) RNA by RNA polymerase III, which then activates retinoic acid-inducible gene I (RIG-I). Consistent with this, RNA from poly(dA–dT) transfected cells induced IFN-β and expression of the E3 dsRNA-binding domain was sufficient to ablate this response. Given the well documented function of the E3 dsRNA-binding domain we propose that E3 blocks signalling in response to poly(dA–dT) by binding to transcribed poly(A-U) RNA preventing RIG-I activation. This report describes a DNA virus-encoded inhibitor of the RNA polymerase III-dsDNA-sensing pathway and extends our knowledge of E3 as a modulator of innate immunity.

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To understand the determinants of membrane protein interactions, we have developed an in vivo genetic assay system for detecting homodimerization of transmembrane (TM) segments from integral membrane proteins. Our approach is to generate gene fusions between potentially dimerizing TM segments and a cytoplasmic DNA-binding protein that lacks its intrinsic dimerization domain. This genetic approach allows us to screen and distinguish among known dimerizing domains and weakly dimerizing mutants, as well as non-dimerizing TM segments. We replaced the bacteriophage λ cI repressor C-terminal dimerization domain with the human erythrocyte glycophorin A transmembrane segment (GpA TM). GpA TM forms SDS-resistant homodimers in vitro. Expression of this membrane-associated fusion in Escherichia coli conferred the same degree of immunity to λcI phages as the wild-type, intact λ repressor. Single amino acid substitutions that disrupt the GpA TM dimer interface were introduced into the λ-GpA TM fusion proteins. These mutations dramatically reduced immunity of E. coli to λcI, such that the efficiency of plating these phages increased by greater than 10,000-fold over that conferred by the wild-type λ-GpA TM fusion. Introduction of the putatively non-dimerizing first TM from E. coli MalF into the λ-TM fusion vector resulted in no immunity to λcI phages. Fusion of the homodimeric, periplasmically localized, mature alkaline phosphatase domain to the C terminus of the λ-TM fusion proteins containing weakly to non-dimerizing TM segments restored immunity to λcI phages. Results from this in vivo genetic assay system demonstrate that (1) dimerization of the λ cI DNA-binding domain can be promoted by dimerizing TM segments, (2) strongly, weakly, and non-dimerizing TM segments can be distinguished on the basis of their ability to confer immunity to λcI phages, and (3) introduction of a dimerizing periplasmic domain can provide functionality to λ-TM fusions containing weakly to non-dimerizing TM segments.

Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interactions in Vivo

To understand the determinants of membrane protein interactions, we have developed an in vivo genetic assay system for detecting homodimerization of transmembrane (TM) segments from integral membrane proteins. Our approach is to generate gene fusions between potentially dimerizing TM segments and a cytoplasmic DNA-binding protein that lacks its intrinsic dimerization domain. This genetic approach allows us to screen and distinguish among known dimerizing domains and weakly dimerizing mutants, as well as non-dimerizing TM segments. We replaced the bacteriophage λ cI repressor C-terminal dimerization domain with the human erythrocyte glycophorin A transmembrane segment (GpA TM). GpA TM forms SDS-resistant homodimers in vitro. Expression of this membrane-associated fusion in Escherichia coli conferred the same degree of immunity to λcI phages as the wild-type, intact λ repressor. Single amino acid substitutions that disrupt the GpA TM dimer interface were introduced into the λ-GpA TM fusion proteins. These mutations dramatically reduced immunity of E. coli to λcI, such that the efficiency of plating these phages increased by greater than 10,000-fold over that conferred by the wild-type λ-GpA TM fusion. Introduction of the putatively non-dimerizing first TM from E. coli MalF into the λ-TM fusion vector resulted in no immunity to λcI phages. Fusion of the homodimeric, periplasmically localized, mature alkaline phosphatase domain to the C terminus of the λ-TM fusion proteins containing weakly to non-dimerizing TM segments restored immunity to λcI phages. Results from this in vivo genetic assay system demonstrate that (1) dimerization of the λ cI DNA-binding domain can be promoted by dimerizing TM segments, (2) strongly, weakly, and non-dimerizing TM segments can be distinguished on the basis of their ability to confer immunity to λcI phages, and (3) introduction of a dimerizing periplasmic domain can provide functionality to λ-TM fusions containing weakly to non-dimerizing TM segments.

The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro.

The cytoplasmic protein TraM is one of four essential gene products of the F factor which are involved in DNA transfer after mating pair formation. TraM binds to three specific sites within the oriT region. Besides regulation of its own synthesis, the precise function of TraM during conjugation is not yet known. In the present work, the affinity of TraM to TraD was studied in vitro by an overlay assay and by affinity chromatography. Whether the interaction between TraM and TraD causes a transient or permanent anchoring of the F factor to the site of transfer is discussed. A 35-kDa host membrane protein of yet unknown function also shows affinity to TraM and may be involved in this anchoring process as well.

Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli.

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.

Changes in cytoplasmic DNA-binding phosphoproteins during the cell cycle of Physarum polycephalum

The cytoplasmic DNA-binding proteins of Physarum polycephalum were examined during two successive cell cycles for alterations in the phosphorylated species. Plasmodia were grown continuously in [ 3H]leucine and [ 32P]orthophosphate in order to label proteins with 3H and phosphoproteins additionally with 32P. Cytosol proteins were passed through columns of double-and single-stranded DNA cellulose. Proteins with affinity for DNA-cellulose were eluted with increasing NaCl concentrations and were examined by polyacrylamide gel electrophoresis and gel autoradiography. A general decrease in phosphorus content of approx. 30% was found in both DNA-binding and non-binding proteins during the G2 phase of the cell cycle. When compared among the cytoplasmic DNA-binding proteins this decreased phosphorus content was attributable to the attenuation or disappearance of several phosphorylated DNA-binding proteins. In addition, several differences in the polypeptide profiles of phosphorylated and non-phosphorylated DNA-binding proteins were found at the same point in two successive cell cycles. In general, there was a decreased phosphate content among the DNA-binding proteins during the second successive cell cycle. It is suggested that this decrease is related to a depletion of nutrients, and that the coordinated alterations in the phosphate content of most cytoplasmic DNA-binding proteins is indicative of their functional relatedness.

Changes in synthesis of DNA-binding proteins during the onset of transformation in NRK cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Synthesis of cytoplasmic DNA-binding proteins was investigated after a shift from the nonpermissive to the permissive temperature in NRK cells transformed by a temperature-sensitive mutant of Rous sarcoma virus [ts339(RSV)]. Cells were labeled for several generations in [3H]leucine and were pulse-labeled with [35S]methionine for 1 h at the nonpermissive temperature (39 degrees C) and at the permissive temperature (33 degrees C, 5 h after shift from 39 degrees C). Proteins binding to sequential columns of double-stranded and single-stranded DNA-cellulose were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the 35S/3H ratios were obtained for each column fraction and for individual polypeptides. The protein fractions binding to single-stranded, but not double-stranded, DNA and eluting at high salt concentrations (greater than 0.60 M NaCl) showed elevated 35S/3H ratios. This indicated increased synthesis of these proteins within 5 h after the onset of transformation. The majority of the polypeptides in these fractions showed increased synthesis as a consequence of transformation. One prominent polypeptide among them constituted 0.1% of the cytosol protein and had a molecular weight of 93,000. We conclude that the synthesis of proteins binding tightly to single-stranded DNA is increased early after the onset of transformation.

Cytoplasmic DNA-binding phosphoproteins of Physarum polycephalum

The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH 2[ 32P]O 4 and [ 3H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins.

The effect of 5-bromodeoxyuridine on the synthesis of nuclear and cytoplasmic DNA-binding proteins isolated from sea urchin embryos

Newly synthesized DNA-binding proteins were isolated from the nuclei and, separately, from the cytoplasm of sea urchin morula stage embryos. The presence of 5-bromode-oxyuridine during embryogenesis did not appear to alter the synthesis of either class of DNA-binding proteins. This result tends to argue that cell differentiation in early embryos is not regulated by differential synthesis of DNA-binding proteins. Sea urchin morulae synthesize a broad range, by molecular weight, of cytoplasmic DNA-binding proteins which dissociate from sea urchin DNA-cellulose at relatively high salt concentrations (0.6–2.0 M NaCl). The most prominant of these apparently high-binding-affinity proteins has an approximate molecular weight of 33,000.

Synthesis of DNA-binding proteins during the cell cycle of WI-38 cells

The DNA-binding proteins synthesized by WI-38 cells at specific periods during the cell cycle were chromatographed on calf thymus DNA-cellulose and analyzed by SDS-polyacrylamide gel electrophoresis. The DNA-binding proteins which were predominantly labeled in G 1 had approximate mol wts of 20 000, 30 000 and 68 000. During the S phase, in addition to the G 1 proteins, some large proteins and histones were labeled. Proteins of approx. 45 000 D seemed to be unique to the S period. The profile of DNA-binding proteins synthesized in G 2 is very similar to that of G 1. The labeling of DNA-binding proteins in the early G1 was markedly reduced in the presence of 5 μg/ml of actinomycin D indicating that synthesis of these G 1 proteins is dependent on new transcription. In order to determine the relationship between the DNA-binding proteins prepared from the whole cell sonicates and nuclear proteins, DNA-binding proteins of cytoplasm and nuclear fractions were examined. DNA-binding proteins are found to exist both in nuclear and cytoplasmic fractions of WI-38 cells. Nucleocytoplasmic migration of DNA-binding proteins was also studied by pulse labeling G 1 cells with 3H-leucine and determining the amount of radioactivity incorporated into cytoplasmic and nuclear DNA-binding protein fractions after chasing for various periods of time. Furthermore, incubation of unlabeled isolated nuclei with the 3H-DNA-binding proteins in vitro revealed that fractions of these cytoplasmic DNA-binding proteins are transported in nuclei. Present experiments suggest that DNA-binding proteins synthesized in the late G 1 are correlated with the onset of S and that a fraction of DNA-binding proteins synthesized in the cytoplasm migrates into the nuclei.

Cytoplasmic DNA-binding proteins

Cytoplasmic DNA-binding proteins were isolated from Chinese hamster liver, kidney and tissue culture cells by DNA-polyacrylamide chromatography. With homologous Chinese hamster DNA, and with calf thymus DNA, 1.4% of the proteins were bound to the column. With single-stranded DNA and with heterologous Micrococcus lysodeikticus DNA there was only 0.3% binding, suggesting the proteins preferentially bind to double-stranded DNA and show some sequence specificity. By a nitrocellulose filter assay the bound proteins had at least a 4- to 7-fold greater affinity for DNA than bulk cytoplasmic protein. SDS gel electrophoresis showed that specific proteins were being markedly concentrated by the column and it was primarily the high molecular weight proteins of 65 000 D and over which showed sequence specificity. Some proteins appeared in common with different organs, others were unique. These studies thus define a group of high molecular weight, cytoplasmic proteins which bind to native DNA with a degree of sequence specificity. Their possible relationship to gene regulation is discussed.