Discovery and engineering of a 1-butanol biosensor in Saccharomyces cerevisiae.

The present study aimed to develop a universal methodology for the discovery of biosensors sensitive to particular stresses or metabolites by using a transcriptome analysis, in order to address the need for in vivo biosensors to drive the engineering of microbial cell factories. The method was successfully applied to the discovery of 1-butanol sensors. In particular, the genome-wide transcriptome profiling of S. cerevisiae exposed to three similar short-chain alcohols, 1-butanol, 1-propanol, and ethanol, identified genes that were differentially expressed only under the treatment of 1-butanol. From these candidates, two promoters that responded specifically to 1-butanol were characterized in a dose-dependent manner and were used to distinguish differences in production levels among different 1-butanol producer strains. This strategy opens up new opportunities for the discovery of promoter-based biosensors and can potentially be used to identify biosensors for any metabolite that causes cellular transcriptomic changes.

The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus.

RNA helicases of the DEAD (Asp-Glu-Ala-Asp)-box family of proteins are involved in many aspects of RNA metabolism from transcription to RNA decay, but most of them have also been shown to be multifunctional. The DEAD-box helicase DDX5 of host cells has been shown to interact with the RNA-dependent RNA polymerase (NS5B) of HCV (hepatitis C virus). In the present study, we report the presence of two independent NS5B-binding sites in DDX5, one located at the N-terminus and another at the C-terminus. The N-terminal fragment of DDX5, which consists of the first 305 amino acids and shall be referred as DDX5-N, was expressed and crystallized. The crystal structure shows that domain 1 (residues 79-303) of DDX5 contains the typical features found in the structures of other DEAD-box helicases. DDX5-N also contains the highly variable NTR (N-terminal region) of unknown function and the crystal structure reveals structural elements in part of the NTR, namely residues 52-78. This region forms an extensive loop and an α-helix. From co-immunoprecipitation experiments, the NTR of DDX5-N was observed to auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-51 residues in NTR and the NS5B-binding site in DDX5-N. Furthermore, NMR investigations reveal that there is a direct interaction between DDX5 and NS5B in vitro.

Expression, purification and preliminary crystallographic analysis of recombinant human DEAD-box polypeptide 5.

The DEAD-box RNA helicase DDX5 is involved in many aspects of RNA processing and has been implicated in a number of cellular processes involving alteration of RNA secondary structure. The N-terminal region of DDX5, which contains the conserved domain 1 of the DEAD-box helicases, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of this region is reported. X-ray diffraction data were processed to a resolution of 2.7 A. The crystals belonged to space group I222, with unit-cell parameters a = 66.18, b = 73.80, c = 104.00 A, alpha = beta = gamma = 90 degrees .

Comparing the antibody responses against recombinant hemagglutinin proteins of avian influenza A (H5N1) virus expressed in insect cells and bacteria.

The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1 isolate (A/chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus-insect cell system. As expected, full-length HA5 elicits strong neutralizing antibodies, as evaluated in micro-neutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C-terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum against the N-terminal fragment is only four times lower than the anti-serum against the full-length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities.

The human severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells.

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesis.

Proteomic approach identifies HSP27 as an interacting partner of the hepatitis C virus NS5A protein.

Chronic infection by HCV is closely correlated with liver diseases such as cirrhosis, steatosis, and hepatocellular carcinoma. To understand how long-term interaction between HCV and the host leads to pathogenesis, we identified cellular proteins that interact with NS5A and NS5B using a biochemical approach. Stable cell lines that express flag-NS5A or flag-NS5B under tetracycline induction were generated. The induced flag-tagged proteins were immunoprecipitated (IP'd) and associated proteins separated on 2D gels. Protein spots that specifically co-IP'd with NS5A or NS5B were identified by mass spectrometry. HSP27 was identified as a protein that specifically co-IP'd with NS5A but not with NS5B. The N-terminal regions of NS5A (a.a. 1-181) and HSP27 (a.a. 1-122) were defined to be the domains that interact with each other. HSP27 is generally distributed in the cytoplasm. When heat shocked, HSP27 is concentrated in the ER where NS5A is co-localized.