R1 Domain Research Articles

A NEW MOLECULAR MODEL FOR REGULATING THE TGFβ RECEPTOR II PROMOTER IN PANCREATIC CELLS

KLF11, a TGFβ-inducible transcription factor that contains three distinct repressor domains, R1, R2, and R3, acts as a tumor suppressor for pancreatic cancer, at least in part, via the interaction of the R1 domain and the corepressor Sin3a-Histone deacetylase complex (HDAC). Although R1 has been credited with the gene repression activity of KLF11, previous results from our lab were seemingly paradoxical, such that KLF11 appears to silence certain promoters as a default long-term mechanism. Even oncogenic cascades, such as the K-ras-MEK-ERK pathway, inactivate the R1-Sin3 interaction, yet KLF11 is still active. Thus, other corepressors are clearly required to provide KLF11 with full gene silencing activity. In this study, we identified a consensus PxVxL HP1-interacting domain within the C-terminus of KLF11. HP1 proteins are "gatekeepers" of epigenetic gene silencing that is mediated by histone H3 lysine-9 methylation, and therefore a good candidate for involvement in KLF11-associated gene repression. Indeed, KLF11 interacts with HP1α in vitro, which is also dysregulated in pancreatic cancer. In addition, we observe an interaction between KLF11 and HP1 in vivo with several pancreatic cancer cells, using immunoprecipitation and biomolecular fluorescence complementation. Deletion of the PxVxL interacting domain eliminates HP1 binding to KLF11, without disrupting interaction with other co-repressor/co-activators. ChIP assays show that both KLF11 and HP1 bind the same promoter regions of important TGFβ pathway genes that KLF11 represses, such as SMAD3 and TGFβRII, suggesting that these proteins interact to regulate key promoters involved in pancreatic homeostasis and diseases, such as pancreatitis and pancreatic cancer. A KLF11 mutant in HP1 binding abolishes the repression activity of KLF11 on the TGFβRII promoter, facilitating activation of the promoter. Thus, these results offer a new model for the function of KLF11 which may not only work in transient repression via HDAC, but also in long term repression via histone methylation/HP1.

Wnt-mediated Down-regulation of Sp1 Target Genes by a Transcriptional Repressor Sp5

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.

ISOLATION OF WD40 PROTEINS AS POTENTIAL CO-REPRESSORS FOR THE TGF-??-INDUCIBLE TUMOR SUPPRESSOR GENE KLF11

KLF11/TIEG2 was isolated by our group in 1998 as a TGFβ-inducible transcription factor that contains three distinct repressor domains, RI, R2, and R3. KLF11 acts as a tumor suppressor for pancreatic cancer, at least in part, via the interaction of the R1 domain (also called SID) and the corepressor Sin3a-Histone deacetylase complex (HDAC). Recent reports indicate that this gene correlates with either the development or aggressiveness of additional malignancies including head and neck, leukemia, and esophageal cancer. Unfortunately, however, the corepressor molecules for either the R2 or the R3 domain have remained unknown. Identifying these proteins is important since the K-ras-MEK-ERK pathway inactivates the R1 domain but not the R2 and R3 leaving the tumor suppressor protein KLF11 impaired but still active. To search for these proteins, we have used the KLF11 fragment lacking the DNA binding domain but including R2 and R3 as a bait for yeast two hybrid experiments. From these studies, we find that KLF11 interacts with several proteins but in particular with three WD40 proteins. Clone WD40.a encodes a protein that transmits signal from the membrane to the nucleus and has recently been shown to also function as a corepressor. Clone WD40.b is member of a protein family, which other members have been shown to bind to methylated histones (a common regulator of repression). Clone WD40.c encodes a protein with similarities to a sea urchin protein of unknown function. Therefore, these proteins are optimal candidates to function as corepressor for the KLF11 R2 and R3. Understanding how these corepressors regulate the function of KLF11 may shed light into the mechanism used by this TGFβ-inducible transcription factor in pancreatic tumor suppression.

Differential Utilization of Transcription Activation Subdomains by Distinct Coactivators Regulates Pit-1 Basal and Ras Responsiveness

The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1beta, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1-45), and a basal and Ras-responsive region, R2 (AA46-80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1betaTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50-70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60-80. In contrast, GAL4-Pit-1betaTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the beta-domain, we inserted the beta-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1beta, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, beta-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61-71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1beta TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a beta-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.

Egr-1 Induces the Expression of Its Corepressor Nab2 by Activation of the Nab2 Promoter Thereby Establishing a Negative Feedback Loop

The transcription factor Egr-1 regulates the expression of numerous genes involved in differentiation, growth, and in response to environmental signals. Egr-1 activity is modulated in part through the binding of corepressors Nab1 and Nab2. Nab2 appears crucial for controlling Egr-1-mediated transactivation because it is a delayed early response gene, induced by the same stimuli that induce the immediate early gene Egr-1. To identify important elements regulating Nab2 expression, we cloned the human Nab2 gene and investigated the 5'-region. The TATA- and initiator-less Nab2 promoter, located from -679 to -74 bp, contains a total of 11 Egr binding sites, including a cluster of multiple overlapping Egr/Sp1 sites between -329 and -260 bp. This region is critical for basal promoter activity as well as for maximum induction by phorbol esters. Electromobility shifts show that Sp1 binds to this region in normal and stimulated cells, whereas stimulation induces binding of Egr-1. In addition Egr-1 activates the Nab2 promoter in a pattern similar to phorbol esters, suggesting that Egr-1 is a major inducer of protein kinase C-mediated Nab2 induction. Depletion of Egr-1 by each of two distinct Egr-1 short-interfering RNAs reduces Nab2 expression and inducibility, confirming that Egr-1 is an important regulator of Nab2 expression. Transfection experiments show that Egr-1-induced Nab2 promoter activity is itself repressed by Nab2. These results indicate that Egr-1 mediates the induction of its own repressor, thereby preventing a permanent transactivation of Egr-1 target genes and a damaging overreaction in response to environmental signals.

Studies of Partial Amino Acid Sequences of γ‐40k Secalins of Rye

ABSTRACTThough γ‐40k secalins are a major protein type within rye storage proteins, total amino acid sequences are not as well known as the gluten proteins of wheat. Well‐reputed structural features such as amino acid compositions and molecular masses indicated a close relationship between γ‐40k secalins and γ‐gliadins of wheat, but the degree of homology of amino acid sequences and the positions of intramolecular disulfide bonds are unknown. Therefore, two major components of γ‐40k secalins (R1, R2) were analyzed for partial amino acid sequences. The R1 and R2, derivatized with 4‐vinylpyridine, were isolated from the prolamin fraction of rye cultivar Danko by means of a two‐step RP‐HPLC on C18 silica gel. The proteins were digested in parallel with trypsin and thermolysin, and the partial hydrolyzates were separated by RP‐HPLC. Simultaneous measurement of UV absorbance at 210 and 254 nm allowed the detection of all peptides eluted as well as the specific detection of pyridylethylated cysteine peptides. Isolated peptides were characterized by sequence analysis, and in parts by mass spectrometry, and assigned to known sequences of γ‐gliadins. The results demonstrated that the N‐terminal domain of R1 and R2 remained undigested after tryptic hydrolysis; they were in agreement with the N‐terminal domain of γ‐gliadins in their molecular masses and in the absence of cysteine residues. Most of the isolated peptides originated from the C‐terminal domains, they covered 83% (R1) and 77% (R2), respectively, of the C‐terminal domain of a known γ‐gliadin (clone pW1020). Comparison of R1 and R2 revealed differences only in a few sequence positions. The degree of homology between the C‐terminal domains present in γ‐40k secalins and γ‐gliadins was ≈85%. All eight cysteine residues of γ‐gliadins were found in R1 and R2 sequences. Remarkably, sequences close to corresponding cysteine residues were identical for γ‐40k secalins and γ‐gliadins. Therefore, it can be assumed that the positions of intramolecular disulfide links are homologous.

Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.

Phosphorylation by Protein Kinase CK2 Modulates the Activity of the ATP Binding Cassette A1 Transporter

In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.

Ribosomal protein S1 binds mRNA and tmRNA similarly but plays distinct roles in translation of these molecules.

Ribosomes stalled during protein synthesis can be rescued by tmRNA, which acts first as a tRNA and then as an mRNA to direct addition of a C-terminal degradation tag to the nascent polypeptide. Ribosomal protein S1 binds tmRNA, but its functional role in tmRNA-mediated tagging is uncertain. To probe interactions between S1 and tmRNA, truncated variants missing one or more of the six contiguous S1 domains were studied. The third S1 domain (R1) plays a critical role in binding tmRNA and mRNA but requires additional N- or C-terminal S1 domains. The binding of S1 and its fragments to tmRNA and mRNA is positively cooperative, and the essential role of the R1 domain may be to mediate protein-protein interactions. Overproduction of N-terminal fragments of S1 in Escherichia coli displaces endogenous S1 from ribosomes, inhibits general protein synthesis, and slows growth but causes little if any disruption of tmRNA-mediated tagging. Moreover, tagging of proteins translated from model mRNAs with either no or an increased requirement for S1 is indistinguishable. These results raise the possibility that S1 plays little or no role in tmRNA-mediated tagging.

Modulation of the ABCA1 transporter activity by protein kinase CK2

We identified putative protein kinase CK2 phosphorylation sites in the cytoplasmic R1 and R2 domains, downstream of nucleotide binding domains (NBD) of the ABCA1 transporter, and tested their functional significance. In vitro, the recombinant ABCA1 NBD1+R1 domain, and not the NBD2+R2 domain, was phosphorylated by CK2. We identified T1242, T1243 and S1255 as the phosphorylated residues in the R1 domain. In HEK-293 cells, ABCA1 flippase activity, apolipoproteins AI and AII binding to the cell, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine residues. Mutants mimicking threonine phosphorylation had activities close to that of WT ABCA1. Our data therefore suggest that protein kinase CK2 might regulate the transport activity of ABCA1 in vivo.

Co-crystallization of the yeast phosphorelay protein YPD1 with the SLN1 response-regulator domain and preliminary X-ray diffraction analysis.

The SLN1, YPD1 and SSK1 proteins function in a multi-step phosphorelay signal transduction pathway in yeast. YPD1, a histidine-containing phosphotransfer (HPt) protein, mediates the transfer of a phosphoryl group between the two response-regulator domains associated with SLN1 and SSK1, the R1 and R2 domains, respectively. Co-crystallization of the SLN1-R1 domain with YPD1 is reported here. Two different crystal forms were obtained by the hanging-drop vapor-diffusion method using 2.6 M ammonium sulfate as a precipitant. X-ray diffraction analysis indicates that crystal form I belongs to a trigonal space group P3(2)/P3(1), with unit-cell parameters a = b = 91.4, c = 201.1 A, while crystal form II belongs to an orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.6, b = 74.2, c = 98.7 A. Complete data sets to 2.1 and 2.3 A resolution have been collected from trigonal and orthorhombic crystals, respectively. Protein gel analysis indicates that in both crystal forms YPD1 and the SLN1-R1 domain are present in a 1:1 stoichiometry suggestive of a complex.

The Human Papillomavirus 16 E6 Protein Binds to Tumor Necrosis Factor (TNF) R1 and Protects Cells from TNF-induced Apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that transfection of HPV 16 E6 into the tumor necrosis factor (TNF)-sensitive LM cell line protects expressing cells from TNF-induced apoptosis in a p53-independent manner, and the purpose of this study was to determine the molecular mechanism underlying this protection. Caspase 3 and caspase 8 activation were significantly reduced in E6-expressing cells, indicating that E6 acts early in the TNF apoptotic pathway. In fact, E6 binds directly to TNF R1, as shown both by co-immunoprecipitation and mammalian two-hybrid approaches. E6 requires the same C-terminal portion of TNF R1 for binding as does TNF R1-associated death domain, and TNF R1/TNF R1-associated death domain interactions are decreased in the presence of E6. HA-E6 also blocked cell death triggered by transfection of the death domain of TNF R1. Together, these results provide strong support for a model in which HPV E6 binding to TNF R1 interferes with formation of the death-inducing signaling complex and thus with transduction of proapoptotic signals. They also demonstrate that HPV, like several other viruses, has developed a method for evading the TNF-mediated host immune response.

Activation of lymphocyte signaling by the R1 protein of rhesus monkey rhadinovirus.

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that exhibits a considerable degree of similarity to the human Kaposi's sarcoma-associated herpesvirus (KSHV). The R1 protein of RRV is distantly related to the K1 protein of KSHV, and R1, like K1, can contribute to cell growth transformation. In this study we analyzed the ability of the cytoplasmic tail of R1 to function as a signal transducer. The cytoplasmic domain of the R1 protein contains several tyrosine residues whose phosphorylation is induced in cells expressing Syk kinase. Expression of a CD8 chimera protein containing the extracellular and transmembrane domains of CD8 fused to the cytoplasmic domain of R1 mobilized intracellular calcium and induced cellular tyrosine phosphorylation in B cells upon stimulation with anti-CD8 antibody. None of the CD8-R1 cytoplasmic deletion mutants tested were able to mobilize intracellular calcium or to induce tyrosine phosphorylation to a significant extent upon addition of anti-CD8 antibody. Expression of wild-type R1 protein activated nuclear factor of activated T lymphocytes (NFAT) eightfold in B cells in the absence of antibody stimulation; expression of the CD8-R1C chimera strongly induced NFAT activity (60-fold) but only upon the addition of anti-CD8 antibody. We conclude that the cytoplasmic domain of R1 is capable of transducing signals that elicit B-lymphocyte activation events. The signal-inducing properties of R1 appear to be similar to those of K1 but differ in that the required sequences are distributed over a much longer stretch of the cytoplasmic domain (>150 amino acids). In addition, the induction of calcium mobilization was considerably longer in duration and stronger with R1 than with K1.

A search within the IL-1 type I receptor reveals a peptide with hydropathic complementarity to the IL-1β trigger loop which binds to IL-1 and inhibits in vitro responses

In previous research, we were able to demonstrate that a seven amino acid residue peptide (VITFFSL), designed as an antisense peptide of the β-bulge trigger loop region of interleukin 1β (IL-1β) (QGEESND; residues 48–54 [mature protein sequence]), was able to interact with IL-1 specifically and inhibit the response to IL-1 in an in vitro bioassay. The evidence was consistent with a specific interaction ocurring between antisense peptide and the trigger loop region. On the basis that antisense peptides are able to interact with their corresponding sense peptide sequences as a result of their mutually complementary hydropathic profiles (Fassina G., Verdoliva, A., Cassani, G., Melli, M., 1994. Binding of type I IL-1 receptor fragment 151–162 to IL-1. Growth Factors 10, 99–106; Maier, C.C., Moseley, H.N.B., Zhou, S., Whitaker, J.N., Blalock, J.E., 1994. Indentification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles. Immunomethods 5, 107–113), we devised a computer program (FINDH) to search the amino acid residue sequence of interleukin-1 type 1 receptor (IL-1 R1) for peptide motifs possessing hydropathic complementarity to the trigger loop sequence. The most complementary “best-fit peptide” motif (LITVLNI) was located in the third extracellular domain of IL-1 R1. A best-fit peptide corresponding to this motif was synthesised and found to bind to IL-1β as well as inhibit the response to IL-1 in two independent in vitro bioassays (monitoring IL-1 dependent serum amyloid A synthesis and IL-1 dependent alkaline phosphatase activity, respectively). A second peptide motif (VIEFITL) was identified and the corresponding peptide synthesised along with a reordered version (LTILINV) of the best fit peptide. Both failed to bind measurably with IL-1β or inhibit the response to IL-1 in the two bioassays. This best fit peptide behaved very similarly, in terms of IL-1 binding and inhibition behaviour, to the original trigger loop antisense peptide. Reference to the recently released X-ray crystal structure of IL-1β and the IL1-R1 extracellular domain shows that the best fit peptide motif in IL-1 R1 is not apparantly interacting with the IL-1 trigger loop, although both are close in space. The intriguing possibility exists that the best fit peptide motif could represent an alternative site for IL-1β receptor interaction which has not thus far been identified.

Functional consequences of mutations in the early growth response 2 gene (EGR2) correlate with severity of human myelinopathies.

The early growth response 2 gene ( EGR2 ) is a Cys2His2zinc finger transcription factor which is thought to play a role in the regulation of peripheral nervous system myelination. This idea is based partly on the phenotype of homozygous Krox20 ( Egr2 ) knockout mice, which display hypomyelination of the PNS and a block of Schwann cells at an early stage of differentiation. Mutations in the human EGR2 gene have recently been associated with the inherited peripheral neuropathies Charcot-Marie-Tooth type 1, Dejerine-Sottas syndrome and congenital hypomyelinating neuropathy. Three of the four EGR2 mutations are dominant and occur within the zinc finger DNA-binding domain. The fourth mutation is recessive and affects the inhibitory domain (R1) that binds the NAB transcriptional co-repressors. A combination of DNA-binding assays and transcriptional analysis was used to determine the functional consequences of these mutations. The zinc finger mutations affect DNA binding and the amount of residual binding directly correlates with disease severity. The R1 domain mutation prevents interaction of EGR2 with the NAB co-repressors and thereby increases transcriptional activity. These data provide insight into the possible disease mechanisms underlying EGR2 mutations and the reason for varying severity and differences in inheritance patterns.

The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli.

Studies were performed to determine if the unique N-terminal domain of the R1 subunit from herpes simplex virus (HSV) type 1 ribonucleotide reductase is a substrate for casein kinase 2 (CK2). Transphosphorylation assays demonstrated that R1 was highly phosphorylated by this enzyme with multiple phosphorylation sites mapped to the N terminus between residues 1 and 245. Immunoprecipitation pull-down assays using R1-specific antisera failed to demonstrate a stable interaction between R1 and CK2 but residual amounts of CK2 present after immunoprecipitation efficiently transphosphorylated R1. Activity assays with a peptide substrate identified CK2 in R1 immunoprecipitated from infected-cell extracts but did not detect activity in R1 proteins immunoprecipitated from bacterial extracts. However, Western blotting identified potential E. coli homologues of the CK2 alpha and beta subunits. These results support conclusions that the N-terminal domain of HSV R1 is not a protein kinase and that all previous results can be explained by contaminating kinases, principally CK2.

Novel mutants of NAB corepressors enhance activation by Egr transactivators.

The NGFI-A binding corepressors NAB1 and NAB2 interact with a conserved domain (R1 domain) within the Egr1/NGFI-A and Egr2/Krox20 transactivators, and repress the transcription of Egr target promoters. Using a novel adaptation of the yeast two-hybrid screen, we have identified several point mutations in NAB corepressors that interfere with their ability to bind to the Egr1 R1 domain. Surprisingly, NAB proteins bearing some of these mutations increased Egr1 activity dramatically. The mechanism underlying the unexpected behavior of these mutants was elucidated by the discovery that NAB conserved domain 1 (NCD1) not only binds to Egr proteins but also mediates multimerization of NAB molecules. The activating mutants exert a dominant negative effect on NAB repression by multimerizing with native NAB proteins and preventing binding of endogenous NAB proteins with Egr transactivators. To examine NAB repression of a native Egr target gene, we show that NAB2 represses Egr2/Krox20-mediated activation of the bFGF/FGF-2 promoter, and that repression is reversed by coexpression of dominant negative NAB2. Because of their specific ability to alleviate NAB repression of Egr target genes, the dominant negative NAB mutants will be useful in elucidating the mechanism and function of NAB corepressors.

A peptide domain on gingipain R which confers immunity against Porphyromonas gingivalis infection in mice.

The cysteine proteinases referred to as gingipains R (gingipain R1 and gingipain R2) and gingipain K produced by Porphyromonas gingivalis are virulence factors of this periodontal pathogen which likely act by interrupting host defense mechanisms and by participating in the penetration and destruction of host connective tissue. To examine the effect of immunization with gingipains R on the ability of P. gingivalis to colonize and invade in the mouse chamber model, BALB/c mice were immunized intraperitoneally with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or multiple antigenic peptide (MAP)-conjugated gingipain R-derived peptides and then challenged with P. gingivalis. Immunization of mice with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or a peptide derived from the N-terminal sequence of the catalytic domain of gingipains R (peptide A) followed by challenge with P. gingivalis A7436 resulted in protection from P. gingivalis invasion. In contrast, immunization with peptides corresponding to either a sequence encompassing the catalytic cysteine residue of gingipains R (peptide B) or an identical sequence within the catalytic domains of gingipain R1 and gingipain K (peptide C), followed by challenge with P. gingivalis, did not protect animals, nor did immunization with a peptide corresponding to sequences within the adhesion/hemagglutinin domain of gingipain R1 (peptide D) which have been shown to be directly involved in the hemagglutinin activity of gingipain R1. However, the immunoglobulin G (IgG) titer obtained following immunization with peptide D was comparable to that obtained following immunization with the N-terminal peptide (peptide A). Competitive enzyme-linked immunosorbent assays, using either the 95-kDa gingipain R1 or gingipain K as the competing soluble antigen, indicated that 42 and 53% of the antibodies induced by immunization with heat-killed bacteria recognize gingipain R1 and gingipain K, respectively; however, even at very high concentrations, the 50-kDa gingipain R2 did not hinder IgG binding to P. gingivalis. These results indicate that antibodies directed to the amino-terminal region of the catalytic domain of gingipains R are capable of inducing a protective immune response against P. gingivalis infection in the mouse chamber model.

Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain.

Nab proteins constitute an evolutionarily conserved family of corepressors that specifically interact with and repress transcription mediated by three members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate-early gene transcription factors, which includes NGFI-C, Krox20, and Egr3. We explored the mechanism of Nab1 repression and identified structural domains required for Nab1 function. Nab1 does not act by blocking DNA binding or nuclear localization of NGFI-A. In fact, Nab1 repression is not unique to NGFI-A because multiple types of non-NGFI-A activation domains were repressed, as was a heterologous transcription factor carrying the NGFI-A R1 domain, which is required for Nab1 interaction. Additionally, Nab1 tethered directly to DNA repressed constitutively active promoters. Tethered repression was not dependent on the identity of the basal promoter elements, the presence of a distal enhancer, or the distance separating the binding sites from the promoter. These results suggest that Nab1 repression is not specific to particular activators and that Nab1 is an active repressor that works by a direct mechanism. We identified a bipartite-like nuclear localization sequence and localized the repression function to the Nab conserved domain 2 (NCD2), a region found in the carboxy-terminal half of all Nab proteins. Three small regions of homology between Nab1 and previously characterized corepressors, Dr1 and E1b 55-kDa protein, were identified within NCD2. Replacement mutagenesis of residues conserved between these proteins interfered with Nab1 repression, although Nab1 does not function by the same mechanism as Dr1. The human NAB1 genomic locus was mapped to chromosome 2q32.3-33.

NAB2, a Corepressor of NGFI-A (Egr-1) and Krox20, Is Induced by Proliferative and Differentiative Stimuli

Previous work had identified a corepressor, NAB1, which represses transcriptional activation mediated by NGFI-A (also known as Egr-1, zif268, and Krox24) and Krox20. These zinc finger transcription factors are encoded by immediate-early genes and have been implicated in a wide variety of proliferative and differentiative processes. We have isolated and characterized another corepressor, NAB2, which is highly related to NAB1 within two discrete domains. The first conserved domain of NAB2 mediates an interaction with the R1 domain of NGFI-A. NAB2 represses the activity of both NGFI-A and Krox20, and its expression is regulated by some of the same stimuli that induce NGFI-A expression, including serum stimulation of fibroblasts and nerve growth factor stimulation of PC12 cells. The human NAB2 gene has been localized to chromosome 12ql3.3-14.1, a region that is rearranged in several solid tumors, lipomas, uterine leiomyomata, and liposarcomas. Sequencing of the Caenorhabditis elegans genome has identified a gene that bears high homology to both NAB1 and NAB2, suggesting that NAB molecules fulfill an evolutionarily conserved role.