Molecular Determinants for Ligand Selectivity of the Cell-Free Synthesized Human Endothelin B Receptor

Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms.     

DehydroalanylGly,a new post translational modification resulting from the breakdown of glutathione

Background

The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues.

Characterization of Plasmodium falciparum 6-Phosphogluconate Dehydrogenase as an Antimalarial Drug Target

The enzyme 6-phosphogluconate dehydrogenase (6PGD) of the malaria parasite Plasmodium falciparum catalyzes the third step of the pentose phosphate pathway converting 6-phosphogluconate (6PG) to ribulose 5-phosphate. The NADPH produced by 6PGD is crucial for antioxidant defense and redox regulation, and ribose 5-phosphate is essential for DNA and RNA synthesis in the rapidly growing parasite. Thus, 6PGD represents an attractive antimalarial drug target. In this study, we present the X-ray structures of Pf6PGD in native form as well as in complex with 6PG or nicotinamide adenine dinucleotide phosphate (NADP⁺) at resolutions of 2.8, 1.9, and 2.9?Å, respectively. The overall structure of the protein is similar to structures of 6PGDs from other species; however, a flexible loop close to the active site rearranges upon binding of 6PG and likely regulates the conformation of the cofactor NADP⁺. Upon binding of 6PG, the active site loop adopts a closed conformation. In the absence of 6PG, the loop opens and NADP⁺ is bound in a waiting position, indicating that the cofactor and 6PG bind independently from each other. This sequential binding mechanism was supported by kinetic studies on the homodimeric wild-type Pf6PGD. Furthermore, the function of the Plasmodium-specific residue W104L mutant was characterized by site-directed mutagenesis. Notably, the activity of Pf6PGD was found to be post-translationally redox regulated via S-nitrosylation, and screening the Medicines for Malaria Venture Malaria Box identified several compounds with IC₅₀s in the low micromolar range. Together with the three-dimensional structure of the protein, this is a promising starting point for further drug discovery approaches.     

Cellular thiols in rat liver cell lines possessing different growth characteristics

Thiol levels were measured in three cell lines derived from rat hepatocytes with different growth rates and degrees of tumorigenicity: IAR20 having normal epithelial morphology and no tumour forming ability; IAR6.1 being a chemically-transformed malignant cell line; and IAR6.1RT7 derived from an epithelial tumour obtained after injection of IAR6.1 cells into a syngenic animal. The mean levels of GSH, GSSG, low molecular weight thiols (LMWT), macromolecular thiols (MT) and total reactive protein sulphur (TRPS), expressed as nmoles-SH mg-1 protein, were found to be 25.5, 7.5, 50.1, 114.5 and 143.6 respectively for IAR20; 37.6, 3.9, 65.4, 126.8 and 148.4 for IAR6.1; 17.2, 4.4, 52.3, 141.0 and 168.2 for IAR6.1RT7. Cultures were treated with D,L-buthionine-S,R-sulphoximine (BSO) to cause greater than 70 per cent depletion of GSH and the measurements of cellular thiols repeated. Although treatment with BSO caused a substantive decrease in the LMWT fraction, there were no major changes in macromolecular thiols or in total reactive protein sulphur. The respective mean values for LMWT, MT and TRPS (expressed as nmoles-SH mg-1 protein) were 19.4, 109.8, 136.3 for IAR20; 17.2, 119.3, 143.6 for IAR6.1; 21.6, 150.7 and 163.5 for IAR6.1RT7. It is concluded that significant differences in thiol levels exist between the three rat liver cell lines studied. However, severe acute depletion of GSH is not reflected by changes in the levels of macromolecular thiols which suggests that there is only a slow equilibrium between these two major thiol pools.