Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosis

Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).     

Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP

The X-ray structure of the ionotropic GluR2 ligand-binding core (GluR2-S1S2J) in complex with the bicyclical AMPA analogue (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid [(S)-4-AHCP] has been determined, as well as the binding pharmacology of this construct and of the full-length GluR2 receptor. (S)-4-AHCP binds with a glutamate-like binding mode and the ligand adopts two different conformations. The K(i) of (S)-4-AHCP at GluR2-S1S2J was determined to be 185 +/- 29 nM and at full-length GluR2(R)o it was 175 +/- 8 nM. (S)-4-AHCP appears to elicit partial agonism at GluR2 by inducing an intermediate degree of domain closure (17 degrees). Also, functionally (S)-4-AHCP has an efficacy of 0.38 at GluR2(Q)i, relative to (S)-glutamate. The proximity of bound (S)-4-AHCP to domain D2 prevents full D1-D2 domain closure, which is limited by steric repulsion, especially between Leu704 and the ligand.     

Mechanisms of inactivation of HSV-2 during storage in frozen and lyophilized forms

The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.     

Analysis of the hypervariable region of the Salmonella enterica genome associated with tRNA(leuX)

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region.     

Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis

Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T(m)) of 37 degrees C, like mammalian collagen. However, rBclA trimers exhibited T(m) values of 84 degrees C and 95 degrees C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T(m) values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.     

Natural transposon mutagenesis of clinical isolates of Mycobacterium tuberculosis: how many genes does a pathogen need?

Transposable elements can affect an organism's fitness through the insertional inactivation of genes and can therefore be used to identify genes that are nonessential for growth in vitro or in animal models. However, these models may not adequately represent the genetic requirements during chains of human infection. We have therefore conducted a genome-wide survey of transposon mutations in Mycobacterium tuberculosis isolates from cases of human infection, identifying the precise, base-specific insertion sites of the naturally occurring transposable element IS6110. Of 294 distinct insertions mapped to the strain H37Rv genome, 180 were intragenic, affecting 100 open reading frames. The number of genes carrying IS6110 in clinical isolates, and hence apparently not essential for infection and transmission, is very much lower than the estimates of nonessential genes derived from in vitro studies. This suggests that most genes in M. tuberculosis play a significant role in human infection chains. IS6110 insertions were underrepresented in genes associated with virulence, information pathways, lipid metabolism, and membrane proteins but overrepresented in multicopy genes of the PPE family, genes of unknown function, and intergenic sequences. Population genomic analysis of isolates recovered from an organism's natural habitat is an important tool for determining the significance of genes or classes of genes in the natural biology of an organism.     

CD4 down-regulation by HIV-1 and simian immunodeficiency virus (SIV) Nef proteins involves both internalization and intracellular retention mechanisms

Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.     

Extended DNA binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres

The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions. The Pot1 amino-terminal DNA binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity. We have now examined DNA binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein. Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection. Specifically, full-length Pot1.DNA complexes are more stable, and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats. We provide evidence that Pot1 contains a second DNA binding motif that recognizes DNA with reduced sequence specificity compared with the domain present in Pot1N. The two DNA binding motifs cooperate, whereby the amino-terminal oligonucleotide/oligosaccharide binding fold determines the registry of binding, and the internal DNA binding motif stabilizes the complex and expands the protected region toward the 3' -end. Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3'-end and protects against exonucleolytic degradation.