Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics

The sphingosine 1-phosphate receptor 1 (S1P1) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P1 internalization is necessary for this effect. We characterize a knockin mouse (S1p1rS5A/S5A) in which the C-terminal serine-rich S1P1 motif, which is important for S1P1 internalization but dispensable for S1P1 signaling, is mutated. T cells expressing the mutant S1P1 showed delayed S1P1 internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P1 agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P1 expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P1 on T cells is a primary determinant of lymphocyte egress kinetics in vivo

Synthetic biology approaches in drug discovery and pharmaceutical biotechnology

Synthetic biology is the attempt to apply the concepts of engineering to biological systems with the aim to create organisms with new emergent properties. These organisms might have desirable novel biosynthetic capabilities, act as biosensors or help us to understand the intricacies of living systems. This approach has the potential to assist the discovery and production of pharmaceutical compounds at various stages. New sources of bioactive compounds can be created in the form of genetically encoded small molecule libraries. The recombination of individual parts has been employed to design proteins that act as biosensors, which could be used to identify and quantify molecules of interest. New biosynthetic pathways may be designed by stitching together enzymes with desired activities, and genetic code expansion can be used to introduce new functionalities into peptides and proteins to increase their chemical scope and biological stability. This review aims to give an insight into recently developed individual components and modules that might serve as parts in a synthetic biology approach to pharmaceutical biotechnology

Development and validation of a TaqMan probe-based real-time PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains.

&lt;p&gt;Lumpy skin disease (LSD) is a transboundary viral disease of cattle with severe economic impact. Immunization of cattle with homologous live attenuated vaccines poses a number of diagnostic problems, as it has been associated with adverse reactions resembling disease symptoms. The latter hampers clinical diagnosis and poses challenges in virus identification. To this end, a duplex quantitative real-time PCR method targeting the GPCR gene was developed and validated, for the concurrent detection and differentiation of wild type and vaccine Lumpy skin disease virus (LSDV) strains. The method was evaluated in three laboratories. The evaluation included a panel of 38 poxvirus isolates/strains and the analytical characteristics of the method were determined. Amplification efficiencies were 91.3% and 90.7%, for wild type and vaccine LSDV, respectively; the limit of detection was 8 DNA copies for both targets and the inter-assay CV was 0.30% for wild type and 0.73% for vaccine LSDV. The diagnostic performance was assessed using 163 LSDV-positive samples, including field specimens and samples from experimentally vaccinated/infected animals. The method is able to confirm diagnosis in suspect cases, it differentiates infected from vaccinated animals (DIVA) and can be regarded as an important tool for effective LSD surveillance and eradication during vaccination campaigns.&lt;/p&gt;</p

Ubiquitin receptor binding and signaling in primary human leukocytes

Utilizing the human monocyte/macrophage cell line THP1, we recently identified extracellular ubiquitin as an endogenous agonist of the G protein-coupled receptor CXC chemokine receptor (CXCR) 4. Because receptor binding and signaling properties of extracellular ubiquitin have not been evaluated in primary human leukocytes, we analyzed its binding characteristics and subsequent Ca2+ signaling in freshly isolated human B cells, T cells and monocytes. Ubiquitin binding shows typical receptor binding characteristics and promotes intracellular Ca2+ flux within seconds in all three cell populations. The Kd for the ubiquitin receptor interaction in freshly isolated human monocytes is consistent with the affinity of the ubiquitin CXCR4 interaction that we reported for THP1 cells. As detected in THP1 cells previously, the ubiquitin induced Ca2+ flux can be attenuated with a phospholipase C inhibitor in all primary leukocyte cultures. Our observations further support the finding that ubiquitin is a CXCR4 agonist and demonstrate that extracellular ubiquitin induces physiological relevant signaling events in primary human leukocytes. Although the exact mechanism of the ubiquitin CXCR4 interaction, its receptor selectivity and subsequent signaling events remain to be determined, our findings identify a novel and unexpected biological role of extracellular ubiquitin as an endogenous immune modulator

Allostery and Substrate Channeling in the Tryptophan Synthase Bienzyme Complex: Evidence for Two Subunit Conformations and Four Quaternary States

The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. (19)F NMR studies of bound α-site substrate analogues, N-(4’-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4’-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of -0.12 ppm. X-ray crystal structures show the F6 and F9 CF(3) groups located at the α-β subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in (19)F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and β-subunits have closed conformations wherein access of ligands into the α- and β-sites from solution is blocked. Internal and external aldimine structures show the α- and β-subunits with closed and open global conformations, respectively. These results establish that β-subunits exist in two global conformation states, designated open, where the β-sites are freely accessible to substrates, and closed, where the β-site portal into solution is blocked. Switching between these conformations is critically important for the αβ-catalytic cycle