Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Alcohol enquiry by GPs - Understanding patients' perspectives: A qualitative study.

BACKGROUND: Patients' beliefs and attitudes toward receiving alcohol enquiry from general practitioners (GPs) are unclear. These need to be understood to implement pragmatic, early detection and brief intervention strategies. METHODS: We purposively sampled 23 participants from respondents of an earlier survey conducted in a general practice clinic in Sydney, Australia. Semi-structured interviews were conducted between June and August 2014, recorded, transcribed and analysed using grounded theory method to develop an explanatory model. RESULTS: There were three factors that influenced patients' acceptability of alcohol enquiry by GPs: • perceived relevance of the alcohol enquiry dialogue to the consultation • approach and language used in the patient-doctor interaction • unease regarding the moral and stigmatising dimension of alcohol consumption. DISCUSSION: Patients are positive towards the role of GPs in health promotion, but nonetheless have reservations towards engaging in alcohol discussions. Setting the context for alcohol dialogue, linking it to patients' agendas, collaborative consultation styles and respecting patients' sensitivity may improve acceptability.

Let's listen to patients' and GPs' perspectives on alcohol-screening research.

Alcohol-screening questionnaires have been found to be effective in the early detection of risky drinking but are rarely used by clinicians in primary care. As research agenda tend not to seek the perspectives of patients and general practitioners (GPs), the best way to address the barriers to implementation is unclear. Contemporary research to explore patient beliefs and attitudes towards alcohol enquiry by GPs is needed.

Consultation contexts and the acceptability of alcohol enquiry from general practitioners - a survey experiment.

BACKGROUND: General practitioners have a crucial role in detecting risky drinking in patients. However, little is known about how the context of the consultation affect patient acceptability of these discussions. METHODS: During one week in May 2014, adult patients seen at a community general practice in Sydney were randomised to receive one of two postal questionnaires. Participants rated the acceptability of alcohol enquiry in 20 vignettes of general practice consultations, either within a SNAP (smoking, nutrition, alcohol, physical activity) framework (intervention) or alone (control). RESULTS: Of the 441 patients who received the questionnaires, 144 returned completed and returned it. The intervention group rated an additional 2.1 (95% CI = 0.38-3.7, P = 0.016) vignettes as acceptable compared to the control group. Alcohol enquiry acceptability varied greatly between individual scenarios. DISCUSSION: Alcohol-use assessment may be more acceptable to patients when it is framed within the SNAP framework, especially in certain presentations (eg diabetes management).

Fluorescent labeling of proteins in living cells using the FKBP12 (F36V) tag.

Over the past decade live cell imaging has become a key technology to monitor and understand the dynamic behavior of proteins in the physiological context of living cells. The visualization of a protein of interest is most commonly achieved by genetically fusing it to green fluorescent protein (GFP) or one of it variants. Considerable effort has been made to develop alternative methods of protein labeling to overcome the intrinsic limitations of fluorescent proteins. In this report we show the optimization of a live cell labeling technology based on the use of a mutant form of FKBP12 (FKBP12(F36V)) in combination with a synthetic high affinity ligand (SLF') that specifically binds to this mutant. It had been previously shown that the use of a fluorescein-conjugated form of SLF' (5'-fluorescein-SLF') allowed the labeling of proteins genetically fused to FKBP-F36V in living cells. Here we describe the identification of novel fluorescent SLF'dye conjugates that allow specific labeling of FKBP12(F36V) fusion proteins in living cells. To further increase the versatility of this technology we developed a number of technical improvements. We implemented the use of pluronics during the labeling process to facilitate the uptake of the SLF'-dye conjugates and the use suppression dyes to reduce background signal. Furthermore, the time and dose dependency of labeling was investigated in order to determine optimal labeling conditions. Finally, the specificity of the FKBP12(F36V) labeling technology was extensively validated by morphological analysis using a diverse set of FKBP12(F36V) fusions proteins. In addition we show a number of different application examples, such as translocation assays, the generation of biosensors, and multiplex labeling in combination with different labeling technologies, such as FlAsH or GFP. In summary we show that the FKBP12(F36V)/SLF' labeling technology has a broad range of applications and should prove useful for the study of protein function in living cells.

Simultaneous sequence transfer into two independent locations of a reporter vector using MultiSite Gateway technology.

The bacteriophage lambda recombination system is increasingly used for recombinant DNA applications that involve the frequent transfer of sequences into and between shuttle and reporter vectors. This approach bypasses the need for restriction endonucleases or ligases and, as such, is easily scalable and automated. However this system has not yet been tested for the ability to support the simultaneous introduction of donor fragments into two separate target sites of a single reporter plasmid. This attribute would greatly facilitate studies of cis-regulatory elements that only function in specific combinations, such as a class of regulatory elements known as chromatin insulators. With the goal of facilitating a screen for chromatin insulators, we sought to determine whether the commercially available MultiSite Gateway Technology recombination system could be used to simultaneously insert candidate insulator elements into two separate locations of a functional reporter plasmid. We show that this application is both highly efficient and specific, generating the desired recombination products nearly three quarters of the time without disrupting the specificity of the reporter system. As such, these studies establish a novel application of the MultiSite Gateway Technology for the generation of recombinant reporter plasmids where the constituent elements function in a combinatorial fashion.

Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA.

The approximately 1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream approximately 1.0 kb and the downstream approximately 0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete (and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most approximately 0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Psi and Omega) in the presence and absence of the upstream approximately 1.0 kb. Although the systematic deletion of sequences within the 3'-most approximately 0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.