Thirty-five thousand people die as a result of more than 2.8 million antibiotic-resistant infections in the United States of America per year. Pseudomonas aeruginosa (P. aeruginosa) is classified a serious threat, the second-highest threat category of the U.S. Department of Health and Human Services. Among others, the World Health Organization (WHO) encourages the discovery and development of novel antibiotic classes with new targets and mechanisms of action without cross-resistance to existing classes. To find potential new target sites in pathogenic bacteria, such as P. aeruginosa, it is inevitable to fully understand the molecular mechanism of homeostasis, metabolism, regulation, growth, and resistances thereof. P. aeruginosa maintains a sophisticated copper defense cascade comprising three stages, resembling those of public safety organizations. These stages include copper scavenging, first responder, and second responder. Similar mechanisms are found in numerous pathogens. Here we compare the copper-dependent transcription regulators cueR and copRS of Escherichia coli (E. coli) and P. aeruginosa. Further, phylogenetic analysis and structural modelling of mexPQ-opmE reveal that this efflux pump is unlikely to be involved in the copper export of P. aeruginosa. Altogether, we present current understandings of the copper homeostasis in P. aeruginosa and potential new target sites for antimicrobial agents or a combinatorial drug regimen in the fight against multidrug resistant pathogens.

Pruritoceptive (dermal) itch was long considered an accompanying symptom of diseases, a side effect of drug applications, or a temporary sensation induced by invading pruritogens, as produced by the stinging nettle. Due to extensive research in recent years, it was possible to provide detailed insights into the mechanism of itch mediation and modulation. Hence, it became apparent that pruritus is a complex symptom or disease in itself, which requires particular attention to improve patients’ health. Here, we summarize recent findings in pruritoceptive itch, including how this sensation is triggered and modulated by diverse endogenous and exogenous pruritogens and their receptors. A differentiation between mediating pruritogen and modulating pruritogen seems to be of great advantage to understand and decipher the molecular mechanism of itch perception. Only a comprehensive view on itch sensation will provide a solid basis for targeting this long-neglected adverse sensation accompanying numerous diseases and many drug side effects. Finally, we identify critical aspects of itch perception that require future investigation.

Protease-activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling, and signaling studies, we determined the molecular mechanism for tethered ligand–mediated PAR4 activation. H/D exchange identified that the LBS is composed of transmembrane 3 (TM3) domain and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and 2 experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the International Network Against Venous Thrombosis (INVENT) consortium multi-ancestry genome-wide association study (GWAS) meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% reduction in relative risk for VTE (odds ratio, 0.85; 95% confidence interval, 0.77-0.94) compared with the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE.