The neuropeptide Y monomer in solution is not folded in the pancreatic‐polypeptide fold

Bettio, Andrea; Dinger, Michaela C.; Beck‐Sickinger, Annette G.

doi:10.1110/ps.0204902

Cited by 34 publications

(15 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is in agreement with previous results on ET‐1 folding, which report on the induction of secondary structures by cell membranes 27 . Similar effects were demonstrated for other peptides hormones like the neuropeptide Y, which displays different folding patterns depending on the absence or presence of hydrophobic environments 28 . Contrary to the wt peptide, the linearized ET‐1 (peptide 1 ) showed only random coil structures in aqueous solution, although addition of the membrane mimetic induced an ET‐1‐like helical fold.…”

Section: Discussionsupporting

confidence: 92%

The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

Wolf

Beck‐Sickinger

2021

Journal of Peptide Science

View full text Add to dashboard Cite

Cardiovascular diseases (CVDs) like hypertension are a major cause for death worldwide. In the cardiovascular tissue, the endothelin system—consisting of the receptor subtypes A (ETAR) and B (ETBR) and the mixed agonist endothelin 1 (ET‐1)—is a major key player in the regulation of vascular tone and blood pressure. Tight control of this system is required to maintain homeostasis; otherwise, the endothelin system can cause severe CVDs like pulmonary artery hypertension. The high sequence homology between both receptor subtypes limits the development of novel and selective ligands. Identification of small differences in receptor–ligand interactions and determination of selectivity constraints are crucial to fine‐tune ligand properties and subsequent signaling events. Here, we report on novel ET‐1 analogs and their detailed pharmacological characterization. We generated simplified ET‐1‐derived monocyclic peptides to provide an accessible synthesis route. By detailed in vitro characterization, we demonstrated that both G protein signaling and the subsequent arrestin recruitment of activated ETBR remain intact, whereas activation of the ETAR depends on the intramolecular ring size. Increasing of the intramolecular ring structure reduces activity at the ETAR and shifts the peptide toward ETBR selectivity. All ET‐1 analogs displayed efficient ETBR‐mediated signaling by G protein activation and arrestin 3 recruitment. Our study provides in‐depth characterization of the ET‐1/ETAR and ET‐1/ETBR interactions, which has the potential for future development of endothelin‐based drugs for CVD treatment. By identification of Lys9 for selective labeling, novel analogs for peptide‐mediated shuttling by ET‐1 are proposed.

show abstract

Section: Discussionsupporting

confidence: 92%

The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

Wolf

Beck‐Sickinger

2021

Journal of Peptide Science

View full text Add to dashboard Cite

show abstract

“…Ligands carrying two fluorophores with spectral characteristics, that are well-suited for FRET measurements, can provide further insights into the bioactive conformation versus the conformation in solution of the ligand by changes in intramolecular FRET due to distance change between the fluorescently labeled residues [ 168 ].…”

Section: Reviewmentioning

confidence: 99%

Illuminating the life of GPCRs

Böhme

Beck‐Sickinger

2009

Cell Commun Signal

View full text Add to dashboard Cite

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. Gprotein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, autofluorescent proteins as well as the evolving technologies for chemical labeling with peptide-and protein-tags are described and their major applications concerning the GPCR life cycle are presented.

show abstract

“…In the first step monomeric NPY associates with the micellar/lipid surrounding, which forms hydrophobic contacts with the α-helix (Bader et al, 2001; Lerch et al, 2002; Thomas et al, 2005, 2009). The second step involves the entrance of NPY through the helices into the binding pocket (Bettio et al, 2002; Lerch et al, 2004; Thomas et al, 2005). The N terminus of Y 1 R seems to be involved in this binding mechanism of NPY and important for guidance of the ligand into the binding pocket.…”

Section: Discussionmentioning

confidence: 99%

Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide Y2 Receptor

et al. 2019

Self Cite

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y 2 receptor (Y 2 R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y 2 R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y 2 R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p -benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y 1 R, NPY position 27 remains flexible when bound to Y 2 R. These results are in agreement with the suggested binding mode of NPY at Y 2 R.

show abstract

The neuropeptide Y monomer in solution is not folded in the pancreatic‐polypeptide fold

Cited by 34 publications

References 34 publications

The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

Illuminating the life of GPCRs

Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide Y2 Receptor

Contact Info

Product

Resources

About