Oligomerization state and supramolecular structure of the HIV-1 Vpu protein transmembrane segment in phospholipid bilayers

HIV‐1 Vpu is an 81‐residue protein with a single N‐terminal transmembrane (TM) helical segment that is involved in the release of new virions from host cell membranes. Vpu and its TM segment form ion channels in phospholipid bilayers, presumably by oligomerization of TM helices into a pore‐like structure. We describe measurements that provide new constraints on the oligomerization state and supramolecular structure of residues 1–40 of Vpu (Vpu_1–40), including analytical ultracentrifugation measurements to investigate oligomerization in detergent micelles, photo‐induced crosslinking experiments to investigate oligomerization in bilayers, and solid‐state nuclear magnetic resonance measurements to obtain constraints on intermolecular contacts between and orientations of TM helices in bilayers. From these data, we develop molecular models for Vpu TM oligomers. The data indicate that a variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, since oligomers of various sizes have similar intermolecular contacts and orientations, molecular models developed from our data are most likely representative of Vpu TM oligomers that exist in host cell membranes.     

High affinity human antibody fragments to dengue virus non-structural protein 3

Background

The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein.

Methodology/Principal Findings

In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells.

Conclusions/Significance

Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.     

InnateDB: facilitating systems‐level analyses of the mammalian innate immune response

Although considerable progress has been made in dissecting the signaling pathways involved in the innate immune response, it is now apparent that this response can no longer be productively thought of in terms of simple linear pathways. InnateDB ( www.innatedb.ca ) has been developed to facilitate systems‐level analyses that will provide better insight into the complex networks of pathways and interactions that govern the innate immune response. InnateDB is a publicly available, manually curated, integrative biology database of the human and mouse molecules, experimentally verified interactions and pathways involved in innate immunity, along with centralized annotation on the broader human and mouse interactomes. To date, more than 3500 innate immunity‐relevant interactions have been contextually annotated through the review of 1000 plus publications. Integrated into InnateDB are novel bioinformatics resources, including network visualization software, pathway analysis, orthologous interaction network construction and the ability to overlay user‐supplied gene expression data in an intuitively displayed molecular interaction network and pathway context, which will enable biologists without a computational background to explore their data in a more systems‐oriented manner.     

DNA sequence representation without degeneracy

Graphical representation of DNA sequence provides a simple way of viewing, sorting and comparing various gene structures. A new two-dimensional graphical representation method using a two- quadrant Cartesian coordinates system has been derived for mathematical denotation of DNA sequence. The two-dimensional graphic representation resolves sequences’ degeneracy and is mathematically proven to eliminate circuit formation. Given x-projection and y-projection of any point on the graphical representation, the number of A, G, C and T from the beginning of the sequence to that point could be found. Compared with previous methods, this graphical representation is more in-line with the conventional recognition of linear sequences by molecular biologists, and also provides a metaphor in two dimensions for local and global DNA sequence comparison.     

The Ca-activated Cl channel and its control in rat olfactory receptor neurons

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide-gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was approximately 30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were approximately 8 and 62 micro m-2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current.     

Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass spectrometry

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1-2 compounds using the conventional methods.     

Rod sensitivity during Xenopus development

We have measured the sensitivity of rod photoreceptors from overnight-dark-adapted Xenopus laevis through developmental stages 46-66 into adulthood by using suction-pipette recording. The dark current increased gradually from approximately 5 pA at stage 46 to approximately 20 pA at stage 57, compared with an adult (metamorphosed) current of approximately 35 pA. This increase in dark current largely paralleled the progressive increase in length and diameter of the rod outer segment (ROS). Throughout stages 46-66, the dark current increased approximately linearly with ROS surface area. At stage 53, there was a steep (approximately 10-fold) increase in the rod flash sensitivity, accompanied by a steep increase in the time-to-peak of the half-saturated flash response. This covariance of sensitivity and time-to-peak suggested a change in the state of adaptation of rods at stage 53 and thereafter. When the isolated retina was preincubated with 11-cis-retinal, the flash sensitivity and the response time-to-peak of rods before stage 53 became similar to those at or after stage 53, suggesting that the presence of free opsin (i.e., visual pigment without chromophore) in rods before stage 53 was responsible for the adapted state (low sensitivity and short time-to-peak). By comparing the response sensitivity before stage 53 to the sensitivity at/after stage 53 measured from rods that had been subjected to various known bleaches, we estimated that 22-28% of rod opsin in stage 50-52 tadpoles (i.e., before stage 53) was devoid of chromophore despite overnight dark-adaptation. When continuously dark adapted for 7 d or longer, however, even tadpoles before stage 53 yielded rods with similar flash sensitivity and response time-to-peak as those of later-stage animals. In conclusion, it appears that chromophore regeneration is very slow in tadpoles before stage 53, but this regeneration becomes much more efficient at stage 53. A similar delay in the maturity of chromophore regeneration may partially underlie the low sensitivity of rods observed in newborn mammals, including human infants.     

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.