SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-I chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-gamma and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-gamma responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N76-114, each of which contained nonameric H2(d) binding domains with high binding scores for both class I and, except for N76-93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-gamma and IL-4 responses and strong memory CTL responses to the LAMP-N chimera. (C) 2005 Elsevier Inc. All rights reserved.

Identification and analysis of chromodomain-containing proteins encoded in the mouse transcriptome

The chromodomain is 40-50 amino acids in length and is conserved in a wide range of chromatic and regulatory proteins involved in chromatin remodeling. Chromodomain-containing proteins can be classified into families based on their broader characteristics, in particular the presence of other types of domains, and which correlate with different subclasses of the chromodomains themselves. Hidden Markov model (HMM)-generated profiles of different subclasses of chromodomains were used here to identify sequences encoding chromodomain-containing proteins in the mouse transcriptome and genome. A total of 36 different loci encoding proteins containing chromodomains, including 17 novel loci, were identified. Six of these loci (including three apparent pseudogenes, a novel HP1 ortholog, and two novel Msl-3 transcription factor-like proteins) are not present in the human genome, whereas the human genome contains four loci (two CDY orthologs and two apparent CDY pseuclogenes) that are not present in mouse. A number of these loci exhibit alternative splicing to produce different isoforms, including 43 novel variants, some of which lack the chromodomain. The likely functions of these proteins are discussed in relation to the known functions of other chromodomain-containing proteins within the same family.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha- D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (K-d = 0.15 muM) than mannose (K-d = 2.3 muM). Exploration of the binding affinities of alpha-D-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.

Human KLF17 is a new member of the Sp/KLF family of transcription factors

The Sp/KLF transcription factors perform a variety of biological functions, but are related in that they bind GC-box and CACCC-box sequences in DNA via a highly conserved DNA-binding domain. A database homology search, using the zinc finger DNA-binding domain characteristic of the family, has identified human KLF17 as a new family member that is most closely related to KLFs 1-8 and 12. KLF17 appears to be the human orthologue of the previously reported mouse gene, zinc finger protein 393 (Zfp393), although it has diverged significantly. The DNA-binding domain is the most conserved region, suggesting that both the murine and the human forms recognize the same binding sites in DNA and may retain similar functions. We show that human KLF17 can bind G/C-rich sites via its zinc fingers and is able to activate transcription from CACCC-box elements. This is the first report of the DNA-binding characteristics and transactivation activity of human KLF17, which, together with the homology it displays to other KLF proteins, put it in the Sp/KLF family. (c) 2006 Elsevier Inc. All rights reserved.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

Site-specific labelling of proteins with a rigid lanthanide-binding tag

This paper describes a generic method for the site-specific attachment of lathanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Echerichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisostropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

DNA Motifs Suppressing TLR9 Responses

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythernatosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleoti des (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.