Identification of a Strong Promoter of Bacteriophage MB78 that Lacks Consensus Sequence Around Minus 35 Region and Interacts with Phage Specific Factor

A strong promoter of bacteriophage MB78 does not have minus 35 consensus sequence although it has a TGn motif immediately upstream of minus 10 sequence as well as the AT rich UP element. It is efficiently recognised by the sigma 70 RNA polymerase, however, a phage-specific factor competes with sigma 70 RNA polymerase for binding to this region, the binding of the factor being stronger than that of the polymerase. Contrary to the reports in the literature the polymerase appears not to bind to the UP element whereas the phage-specific factor does. The latter seems to be involved in the regulation of the promoter activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA

Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 A resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3' overhangs and for juxtaposition of alternative 5' splice sites are proposed.     

Definition of the alpha 2 region of HLA-DR molecules involved in CD4 binding.

HLA class II molecules present antigenic peptides to the T cell receptor of CD4+ T lymphocytes and interact with CD4 during the antigen recognition process. A major CD4 binding site encompassing amino acids (aa) 134-148 in the beta 2 domain of HLA-DR has been previously identified and residues located within the alpha 2 subunit of murine MHC class II I-Ad molecules have been shown to contribute to CD4-class II interaction. To characterize the alpha 2 region of HLA-DR molecules involved in the binding of CD4, we have synthesized overlapping linear and cyclic peptides derived from a region encompassing aa 121-143. We demonstrate that two linear peptides (aa 124-138 and 130-143) and a cyclic one (aa 121-138) specifically bind to CD4-sepharose affinity columns. Although cyclic analogues exhibit more ordered populations as detected by circular dichroism measurements, cyclization did not improve the activity of some peptides. Peptide sequence positioning in HLA-DR1 dimer model indicates that alpha 2 residues 124 to 136 form a solvent-exposed loop which faces the beta 2 loop delimited by residues 134-148. These data suggest that one CD4 molecule contacts both alpha 2 and beta 2 loops of the HLA-DR homodimer.     

Identification of four new HLA-Cw alleles in the Sudanese population

The major histocompatibility complex (MHC) is the most polymorphic region of the human genome. Human leukocyte antigen-C (HLA-C) genes are located in the class I region of MHC. Most polymorphisms of HLA class I antigens are present in exons 2 and 3, which encode the alpha1 and alpha2 domains of the HLA-A heavy chain, involved in both peptide binding and HLA-restricted recognition by the T-cell receptor. Four new HLA-Cw alleles were identified in the Sudanese population during HLA class I and class II sequencing-based typing at the HLA-C locus of case-control study of Sudanese HIV patients, in individuals from different ethnic background. Based on the localization of the affected amino acid positions in an outer loop of the alpha-helix forming the side of the peptide-binding groove, we do not expect the replacement mutations to have an effect on peptide binding or T-cell receptor interaction.     

Role of aromatic amino acids in receptor binding activity and subunit assembly of the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans

The periodontal pathogen Aggregatibacter actinomycetemcomitans produces a cytolethal distending toxin (Cdt) that inhibits the proliferation of oral epithelial cells. Structural models suggest that the CdtA and CdtC subunits of the Cdt heterotrimer form two putative lectin domains with a central groove. A region of CdtA rich in heterocyclic amino acids (aromatic patch) appears to play an important role in receptor recognition. In this study site-specific mutagenesis was used to assess the contributions of aromatic amino acids (tyrosine and phenylalanine) to receptor binding and CdtA-CdtC assembly. Predominant surface-exposed aromatic residues that are adjacent to the aromatic patch region in CdtA or are near the groove located at the junction of CdtA and CdtC were studied. Separately replacing residues Y105, Y140, Y188, and Y189 with alanine in CdtA resulted in differential effects on binding related to residue position within the aromatic region. The data indicate that an extensive receptor binding domain extends from the groove across the entire face of CdtA that is oriented 180 degrees from the CdtB subunit. Replacement of residue Y105 in CdtA and residues Y61 and F141 in CdtC, which are located in or at the periphery of the groove, inhibited toxin assembly. Taken together, these results, along with the lack of an aromatic amino acid-rich region in CdtC similar to that in CdtA, suggest that binding of the heterotoxin to its cell surface receptor is mediated predominantly by the CdtA subunit. These findings are important for developing strategies designed to block the activity of this prominent virulence factor.     

人巨细胞病毒PPUL44蛋白核定位信号结合多肽氨基酸序列分析

目的 寻找与人巨细胞病毒（HCMV）PPUL44蛋白核定位信号（NLS）具有较强能力的多肽,研究这些多肽的氨基酸序列特点。方法 采用合成的HCMV PPUL44 NLSA和NLSB分别在一个随机多肽文库中筛选出与NLS具有结合能力的克隆,用四色荧光自动测序的方法测定上述克隆的DNA序列,对这些克隆的多肽氨基酸序列进行同源性比较,并与蛋白质文库中已知蛋白的氨基酸序列进行比较。结果 与HCMV PPUL44 NLSA具有结合能力的多肽（bNLSApep)的氨基酸序列与细胞输入蛋白Importin α亚单位的氨基酸具有较高的同源序列,其中bNLSApep48氨基酸序列AVVTPVLTEILK与Importin α亚单位的Arm7区的第18至29氨基酸序列ANIFPVLTEILQ具有极高的相似性;bNLSBpep39氨基酸序列则与Imporin α亚单位的全部8个Am区的第10至21氨基酸的同源序列相似。结论 HCMV PPUL44 NISA可能是Importin α亚单位针对HCMV PPUL44蛋白的特异性识别位点,与Importin α亚单位Arm7区结合;而HCMV PPUL44 NLSB则为Importin α亚单位的非特异性识别位点。本研究结果提供了Importin α亚单位的Arm重复区是NLS结构结合区的实验证据。     

The major fimbrial subunit of Bordetella pertussis binds to sulfated sugars.

Bordetella pertussis fimbriae are composed of major and minor subunits, and recently it was shown that the minor fimbrial subunit binds to Vla-5, a receptor located on monocytes (W. Hazenbos, C. Geuijen, B. van den Berg, F. Mooi, and R. van Furth, J. Infect. Dis. 171:924-929, 1995). Here we present evidence that the major subunits bind to sulfated sugars, which are ubiquitous in the respiratory tract. Binding was observed to chondroitin sulfate, heparan sulfate, and dextran sulfate but not to dextran. Removal of the minor subunit from fimbriae did not significantly affect binding to sulfated sugars, indicating that the major subunit alone is sufficient for this binding. Fimbriae were also able to bind HEp-2 cells, which are known to display glycoconjugates on their surface. This binding was not dependent on the presence of the minor subunit. However, binding was dependent on the sulfation state of the glycoconjugates, since inhibition of the sulfation resulted in a significant reduction of fimbria binding. The specificity of fimbria binding was further characterized by using heparan sulfate-derived disaccharides in inhibition assays. Two disaccharides were highly effective inhibitors, and it was observed that both the degree of sulfation and the arrangement of the sulfate groups on the disaccharides were important for binding to fimbriae. B. pertussis bacteria also bound to sulfated sugars and HEp-2 cells, and analysis of B. pertussis mutants indicated that both filamentous hemagglutinin and fimbriae were required for this binding. A host protein present in the extracellular matrix, fibronectin, has binding activities similar to those of B. pertussis fimbriae, binding to both Vla-5 and sulfated sugars. Two regions in the major fimbrial subunit were identified which showed similarity with fibronectin peptides which bind to sulfated sugars. Thus, B. pertussis fimbriae exemplify molecular mimicry and may co-opt host processes by mimicking natural ligand-receptor interactions.     

Contribution of tumour necrosis factor-alpha (TNF-α) in host defence mechanism against Cryptococcus neoformans

The prevalence of autoantibodies to the three RNA polymerase (RNAP) enzymes in the sera of 249 SSc patients was measured using the technique of immunoprecipitation of ³⁵S-methionine-labelled K562 cell extracts. Forty-six anti-RNAP sera were detected (18.5%) and three main groups were identified: anti-RNAP I/III sera (10; 4.0%), anti-RNAP I/II/III sera (15; 6.0%), and sera precipitating the phosphorylated (IIO) form of RNAP II (18; 7.2%). All sera in the third group also precipitated topoisomerase I (topo I), and six of them also precipitated the unphosphorylated (IIA) form of RNAP II. Although RNAP II/topo I multienzyme complexes may occur in cell extracts, autoreactive epitopes were shown to be located on both enzymes by a combination of antigen depletion studies, and in vitro assays which demonstrated functional inhibition of topo I activity. Furthermore, immunoblotting experiments using affinity-purified extracts demonstrated that all sera with anti-RNAP II antibodies recognized the largest RNAP II subunit in its phosphorylated form (IIo; 240 kD), whereas the unphosphorylated subunit (IIa; 220 kD) was only recognized by sera which also precipitated RNAP IIA. Therefore at least two different sites on the largest subunit of RNAP II are recognized by SSc sera, and one of these sites is unique to the phosphorylated (IIO) form.