Massive in Silico Study of Noble Gas Binding to the Structural Proteome.

Noble gases are chemically inert, and it was therefore thought they would have little effect on biology. Paradoxically, it was found that they do exhibit a wide range of biological effects, many of which are target-specific and potentially useful and some of which have been demonstrated in vivo. The underlying mechanisms by which useful pharmacology, such as tissue and neuroprotection, anti-addiction effects, and analgesia, is elicited are relatively unexplored. Experiments to probe the interactions of noble gases with specific proteins are more difficult with gases than those with other chemicals. It is clearly impractical to conduct the large number of gas-protein experiments required to gain a complete picture of noble gas biology. Given the simplicity of atoms as ligands, in silico methods provide an opportunity to gain insight into which noble gas-protein interactions are worthy of further experimental or advanced computational investigation. Our previous validation studies showed that in silico methods can accurately predict experimentally determined noble gas binding sites in X-ray structures of proteins. Here, we summarize the largest reported in silico reverse docking study involving 127 854 protein structures and the five nonradioactive noble gases. We describe how these computational screening methods are implemented, summarize the main types of interactions that occur between noble gases and target proteins, describe how the massive data set that this study generated can be analyzed (freely available at group18.csiro.au), and provide the NDMA receptor as an example of how these data can be used to understand the molecular pharmacology underlying the biology of the noble gases. We encourage chemical biologists to access the data and use them to expand the knowledge base of noble gas pharmacology, and to use this information, together with more efficient delivery systems, to develop "atomic drugs" that can fully exploit their considerable and relatively unexplored potential in medicine.

Characterization of the epstein-barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1.

The switch from latency to a productive cycle in Epstein-Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5' end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions -469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.

Epstein-Barr virus EB2 protein exports unspliced RNA via a Crm-1-independent pathway.

Human herpesviruses encode posttranscriptional activators that are believed to up-regulate viral replication by facilitating early and late gene expression. We have reported previously that the Epstein-Barr virus protein EB2 (also called M or SM) promotes nuclear export of RNAs that are poor substrates for spliceosome assembly, an effect that closely resembles the human immunodeficiency virus type 1 Rev-dependent nuclear export of unspliced viral RNA. Here we present experimental data showing that EB2 efficiently promotes the nuclear export of unspliced RNA expressed from a Rev reporter construct. Site-directed mutagenesis as well as domain swapping experiments indicate that a leucine-rich region found in the EB2 protein, which matches the consensus sequence for the leucine-rich nuclear export signal, is not a nuclear export signal per se. Accordingly, leptomycin B (LMB), a specific Crm-1 inhibitor, impairs Rev- but not EB2-dependent nuclear export of unspliced RNA. Moreover, EB2 nucleocytoplasmic shuttling visualized by a heterokaryon assay is, unlike Rev shuttling, not affected by LMB. We also show that overexpression of an N-terminal deletion mutant of Nup214/can, a major nucleoporin of the nuclear pore complex involved in several aspects of nuclear transport, blocks both Rev- and EB2-dependent nuclear export of RNA. These results strongly suggest that EB2 nuclear export of unspliced RNA is mediated by a Crm-1-independent pathway.

A new nucleoporin-like protein interacts with both HIV-1 Rev nuclear export signal and CRM-1.

The HIV-1 Rev is a shuttling protein required for the nuclear export of unspliced and partially spliced viral mRNA. In this study, we have identified a new Rev-interacting protein, that specifically interacts with the Rev nuclear export signal both in yeast and mammalian cells. This protein has features found in nucleoporins including many phenylalanine-glycine repeats, a very high serine content, a putative zinc finger, and a coiled-coil domain; we thus called it NLP-1 (nucleoporin-like protein 1). In addition, gene expression analysis and wheat germ agglutinin chromatography experiments suggested that NLP-1 is an ubiquitous O-glycosylated nuclear protein. Recently, a cellular factor called CRM-1 has been shown to be an essential nuclear export factor interacting directly with nuclear export signals including the Rev nuclear export signal in a RanGTP-dependent manner. We show here that NLP-1, like the previously described Rev-interacting protein hRIP/Rab and several nucleoporins, also interacts with CRM-1 both in yeast and mammalian cells.