Genetic evidence for the interaction between Bacillus anthracis-encoded phage receptors and their cognate phage-encoded receptor binding proteins.

Bacteriophages such as γ and AP50c have been shown to infect strains of Bacillus anthracis with high specificity, and this feature has been exploited in the development of bacterial detection assays. To better understand the emergence of phage resistance, and thus the potential failure of such assays, it is important to identify the host and phage receptors necessary for attachment and entry. Using genetic approaches, the bacterial receptors of AP50c and γ have been identified as sap and GamR, respectively. A second AP50c-like phage, Wip1, also appears to use sap as a receptor. In parallel with this work, the cognate phage-encoded receptor binding proteins (RBPs) have also been identified (Gp14 for γ, P28 for AP50c, and P23 for Wip1); however, the strength of evidence supporting these protein-protein interactions varies, necessitating additional investigation. Here, we present genetic evidence further supporting the interaction between sap and the RBPs of AP50c and Wip1 using fluorescently tagged proteins and a panel of B. anthracis mutants. These results showed that the deletion of the sap gene, as well as the deletion of csaB, whose encoded protein anchors sap to the bacterial S-layer, resulted in the loss of RBP binding. Binding could then be rescued by expressing these genes in trans. We also found that the RBP of the γ-like prophage λBa03 relied on csaB activity for binding, possibly by a different mechanism. RBPλBa03 binding to B. anthracis cells was also unique in that it was not ablated by heat inactivation of vegetative cells, suggesting that its receptor is still functional following incubation at 98°C. These results extend our understanding of the diverse attachment and entry strategies used by B. anthracis phages, enabling future assay development.

Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop.

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

Mitogen-responsive expression of RhoB is regulated by RNA stability.

The small GTPase-encoding gene RhoB is strongly induced as part of the immediate early response of serum-stimulated fibroblasts. In this report, we have characterized the mechanism for growth factor responsiveness of RhoB in Rat-2 fibroblasts. By Northern blotting and ribonuclease protection, we observed low or barely detectable levels of RhoB mRNA in quiescent cells, but expression was transiently induced in response to serum stimulation, such that the mRNA peaked within 30 min and then declined over the next hour. Analysis of the rat promoter revealed cis-elements conserved with the mouse and human genes, including a pair of CEBP sites near the transcriptional start site. However, in contrast to the analysis of RNA, RhoB promoter fusions were constitutively expressed in quiescent cells in transient transfections, and were unaffected by serum. Similarly, stable RhoB promoter integrants were highly expressed in quiescent cells, and growth factor caused a slight decrease in activity. This indicates that growth factor-inducible RhoB expression cannot be mediated by transcriptional activation. We then examined decay of the RhoB mRNA and found that serum caused significant stabilization. Additionally, fusion of the 3' RhoB untranslated region (UTR) to a constitutively expressed reporter gene caused serum and growth factor as well as DNA damage-inducible expression. These observations are consistent with the view that RhoB mRNA is produced constitutively but its abundance is controlled in response to growth factors, and other signals including DNA damage, by stabilization through elements within the 3' UTR.

Over-expression of protein kinase Balpha enhances recombinant protein expression in transient systems.

As more genes are being identified through genomic techniques,the need to rapidly express recombinant proteins for functionalstudies has become increasingly acute. Transient expression ofrecombinant protein using COS-1, CV-1 and 293 cells is widelyused to address this need. To improve the robustness of hostcells for transient expression, the effect of over-expression ofProtein Kinase Balpha has been explored. In this report wedemonstrate that over-expression of Protein Kinase Balpha canimprove transient recombinant protein expression 40% to >200%depending on the protein being expressed and the cell line used.