Acylated and unacylated ghrelin binding to membranes : towards a better understanding of the underlying mechanisms
(2010)
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- Préat, Véronique
- Abstract
- (en) In 1999, Kojima and co-workers isolated ghrelin from stomach extracts as the endogenous ligand of the orphan growth hormone secretagogue receptor 1a (GHS-R1a), now called the ghrelin receptor. Ghrelin is a 28-aa peptide hormone which is O-octanoylated at its Ser3 residue. Ghrelin is a pleiotropic hormone. In particular, besides its growth hormone releasing activity, it plays a key role in energetic homeostasis. Ghrelin is unique in four ways: (i) it is the only hormone peptide to be modified by a fatty acid on one of its lateral chains; (ii) O-octanoylation is a medium chain length fatty acid (C8) modification whereas other acylated peptides are usually acylated with either much shorter (C2) or much longer fatty acids (C14 or C16); (iii) other acylated peptides are usually N- or S-linked; (iv) its unacylated form is also endogenous and also seems to play physiological roles, and the charge of both forms is identical. In the nanomolar range, the acylation of ghrelin is required for efficient binding and activation of the ghrelin receptor that mediates most of its endocrinological activities. However, some other effects of ghrelin have been attributed to the activation of a yet unidentified receptor that could bind acylated and/or unacylated ghrelin. Unacylated ghrelin is also a full agonist of the ghrelin receptor but in the micromolar range. As ghrelin effects seem to be receptor mediated, the interaction of ghrelin with cell membranes is potentially important for all its biological activities. Moreover, membrane lipids and lipidation of peptides have both been shown to modulate the activity of peptides. Therefore, the aims of this thesis were to determine the importance of this unique post-translational modification of ghrelin as well as of its basic residues (i) in the extent of ghrelin binding to membranes; (ii) on the mechanisms that drive acylated and unacylated ghrelin to the membrane and hence to the ghrelin receptor; (iii) on ghrelin transmembrane and transepithelial transport. The originality of this work is that we gained some insights into the mechanisms of action of a unique hormone peptide modification, O-octanoylation, and the concomitant role of its basic residues, especially at the level of peptide-membrane interactions and their possible biological implications. Moreover, whenever possible, we worked with natural lipids mixtures and this is quite unusual in the field of peptide-lipid interactions. In this work we have shown differential behaviours of acylated and unacylated ghrelin towards the membrane. We have also shown by PAMPA and intestinal monolayer experiments, that O-octanoylation of ghrelin does not seem to help its transport across membranes and epithelia. After having shown that ghrelin could bind to model plasma membranes (liposomes) as well as to biological membranes and that the acylation significantly enhanced ghrelin binding to negatively charged membranes, we explored the underlying mechanisms of ghrelin binding. The electrostatic and/or hydrophobic interactions of ghrelin with the membrane were investigated using various liposome and buffer compositions in binding, zeta potential and isothermal titration calorimetry studies. On the basis of the results of these experiments it appears that unacylated ghrelin only seems to interact with the lipids by Coulombic interactions, which are long range electrostatic interactions. Acylated ghrelin, however, is not only driven to the membrane by electrostatic attraction between the positively charged residues of ghrelin and the negatively charged lipids, but acylated ghrelin binding to the membrane is also accompanied by: (i) partitioning into the lipid headgroup and into the lipid backbone regions; (ii) a 120-fold increase in peptide concentration in the membrane. However, as shown by ATR-FTIR, DPH and TMA-DPH fluorescence polarization studies as well as by the use of a binding model, acylated ghrelin did neither deeply penetrate the membrane nor did it perturb its organization. Conformational studies by circular dichroism and attenuated total reflection Fourier transform infrared as well as in silico modelling revealed that both forms of ghrelin mainly adopted the same structure in aqueous, micellar and bilayer environments even though acylated ghrelin structure is slightly more -helical in a lipid bilayer environment. Besides, based on our results along with the fact that the ghrelin binding pocket is thought to be located in the transmembrane domains of the ghrelin receptor. I hypothesized that the membrane probably acts as a “catalyst” in acylated ghrelin-ghrelin receptor interactions, as it does for other peptides. Physiologically our findings also imply that unacylated ghrelin is less likely to encounter and thus to bind to the ghrelin receptor than acylated ghrelin. Moreover, ghrelin conformational flexibility may be associated with its various roles in different tissues.
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Staes, E. (2010). Acylated and unacylated ghrelin binding to membranes : towards a better understanding of the underlying mechanisms. https://hdl.handle.net/2078.5/148315