Mcnallyabdi1752

Of the ARBs tested (including losartan and fimasartan), only telmisartan increased p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19. GW9662 had no effects on telmisartan-induced changes. Similar to the in vitro results, telmisartan enhanced p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19 in endothelium-deprived aortas. Furthermore, the telmisartan-inhibited vessel contraction in the aortas was significantly reversed by MG-132 or compound C. In conclusion, we demonstrated that telmisartan inhibits VSMC contractility and vessel contraction by activating AMPK/proteasome/MLCK degradation signaling axis. These results suggest that telmisartan can be used to treat pathological vasospasms. Enzymes are pliable systems and core cellular components allowing the performance of several processes. They can also be utilized as "green" synthetic factories to generate bioactive therapeutic, diagnostic or theranostic compounds. Methods to enable the mapping of enzyme substrates as well as the understanding of the interactions of the formed products with target proteins could be of importance. This chapter describes the utilization of the "NMR tube bioreactor" method. This method, carried out inside an NMR tube, can allow for the prediction of compounds that are able to serve as potential enzyme substrates, and also exploit the regioselectivity of the enzymatic reactions. Furthermore, it enables the real time monitoring of multiple-biotransformation products in the NMR tube without the need of fractionation or isolation of each individual component. Finally, it allows for the screening of the resulting biotransformation products as ligands for protein targets. © 2020 Elsevier Inc. All rights reserved.Enzyme inhibitors are central tools for chemical biology. In this chapter we will discuss the application of chemical probes for competitive profiling of inhibitors of the quinolone biosynthesis enzyme PqsD of Pseudomonas aeruginosa. The human pathogen P. aeruginosa produces a large diversity of 2-alkyl-4(1H)-quinolones and their derivatives as metabolites with major roles in quorum sensing, virulence, and interspecies competition. PqsD is a central enzyme in the biosynthesis of all of these quinolones and hence an interesting target for inhibitor discovery. Activity-based probes with an electrophilic warhead bind covalently to active site nucleophiles like cysteine or serine. An α-chloroacetamide probe with terminal alkyne tag allowed to selectively label the active site cysteine of PqsD and was demonstrated to be a useful tool for inhibitor discovery using competition experiments. Potent inhibitors bind to the active site and thereby prevent labeling of the enzyme by the probe. Labeling intensity is quantified on polyacrylamide gels by the fluorescence of a reporter tag appended by bioorthogonal click chemistry. The competitive inhibitor profiling strategy has many advantages over traditional screening approaches and is applicable in vitro as well as in live cells. Here we describe the synthesis of an activity-based probe and provide our detailed protocols for target enzyme labeling as well as its application for the screening for potent enzyme inhibitors of PqsD by a competitive profiling strategy. © 2020 Elsevier Inc. All rights reserved.Siderophores have important functions for bacteria in iron acquisition and as virulence factors. In this chapter we will discuss the engineering of cyclic hydroxamate siderophores by various biochemical approaches based on the example of Shewanella algae. The marine gamma-proteobacterium S. algae produces three different cyclic hydroxamate siderophores as metabolites via a single biosynthetic gene cluster and one of them is an important key player in interspecies competition blocking swarming of Vibrio alginolyticus. AvbD is the key metabolic enzyme assembling the precursors into three different core structures and hence an interesting target for metabolic and biochemical engineering. Synthetic natural and unnatural precursors can be converted in vitro with purified AvbD to generate siderophores with various ring sizes ranging from analytical to milligram scale. These engineered siderophores can be applied, for example, as swarming inhibitors against V. alginolyticus. Here, we describe the synthesis of the natural and unnatural siderophore precursors HS[X]A and provide our detailed protocols for protein expression of AvbD, conversion of HS[X]A with the enzyme to produce ring-size engineered siderophores and secondly for a biosynthetic feeding strategy that allows to extract engineered siderophores in the milligram scale. © 2020 Elsevier Inc. check details All rights reserved.Measuring protein-protein interactions using purified proteins in vitro is one of the most frequently used approach to understand the biochemical and mechanistic details of cellular signaling pathways. Typically, affinity tags are genetically fused to proteins of interest, and they are used to capture and detect them. However, in some cases, fusion of bulky affinity tags might present a significant limitation in these experiments, especially if the regions in close proximity of tags are involved in protein-protein interactions. Here, we present a step-by-step protocol for an alternative approach that involves reversible biotinylation of purified proteins using a simple chemical-conjugation of cleavable biotin moiety. Biotinylated proteins can be directly used as bait for selective immobilization on solid support for measuring protein-protein interactions. Furthermore, biotinylation of protein of interest also allows specific detection in standard biochemical assays. This simple, straightforward and modular protocol can be directly adapted and applied to facilitate the detection of novel protein-protein interactions as well as measuring apparent affinities of such interactions. © 2020 Elsevier Inc. All rights reserved.β-arrestins (βarrs) are multifunctional proteins that interact with activated and phosphorylated G protein-coupled receptors (GPCRs) to regulate their signaling and trafficking. Understanding the intricate details of GPCR-βarr interaction continues to be a key research area in the field of GPCR biology. Bimane fluorescence spectroscopy has been one of the key approaches among a broad range of methods employed to study GPCR-βarr interaction using purified and reconstituted system. Here, we present a step-by-step protocol for labeling βarrs with monobromobimane (mBBr) in a site-directed fashion for measuring their interaction with GPCRs and the resulting conformational changes. This simple protocol can be directly applied to other protein-protein interaction modules as well for measuring interactions and conformational changes in reconstituted systems in vitro. © 2020 Elsevier Inc. All rights reserved.