A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE)

The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction. Keywords: AGEs and ALEs, RAGE, Pull-down assay, VC1 domain, Reactive carbonyl species, MD

Practical Considerations for the Use of DREADD and Other Chemogenetic Receptors to Regulate Neuronal Activity in the Mammalian Brain

Chemogenetics is the process of genetically expressing a macromolecule receptor capable of modulating the activity of the cell in response to selective chemical ligand. This chapter will cover the chemogenetic technologies that are available to date, focusing on the commonly available engineered or otherwise modified ligand-gated ion channels and G-protein-coupled receptors in the context of neuromodulation. First, we will give a brief overview of each chemogenetic approach as well as in vitro/in vivo applications, then we will list their strengths and weaknesses. Finally, we will provide tips for ligand application in each case.Each technology has specific limitations that make them more or less suitable for different applications in neuroscience although we will focus mainly on the most commonly used and versatile family named designer receptors exclusively activated by designer drugs or DREADDs. We here describe the most common cases where these can be implemented and provide tips on how and where these technologies can be applied in the field of neuroscience