Half-Life Extending Modifications of Peptide YY_3–36 Direct Receptor-Mediated Internalization

Abstract

Peptide YY3–36 (PYY3–36) is an endogenous ligand of the neuropeptide Y2 receptor (Y2R), on which it acts to reduce food intake. Chemically modified PYY3–36 analogues with extended half-lives are potential therapeutics for the treatment of obesity. Here we show that the common half-life extending strategies PEGylation and lipidation not only control PYY3–36’s pharmacokinetics but also affect central aspects of its pharmacodynamics. PEGylation of PYY3–36 inhibited endocytosis by increasing receptor dissociation rates (k off), which reduced arrestin-3 (Arr3) activity. This is the first link between Arr3 recruitment and Y2R residence time. C16-lipidation of PYY3–36 had a negligible impact on Y2R signaling, binding, and endocytosis. In contrast, C18acid-lipidation minimized endocytosis, which indicated a decreased internalization through non-arrestin-related mechanisms. We propose a temporal model that connects the properties and position of the half-life extender with receptor Gi versus Arr3 signaling bias. We believe that this will be important for future design of peptide therapeutics.

Direct Observation of Sophorolipid Micelle Docking in Model Membranes and Cells by Single Particle Studies Reveals Optimal Fusion Conditions

“…Prolonged imaging will bleach the DiO chromophore, as shown by the mono-exponential decay of intensity, also confirming that each docking event is an SL assembly rather than individual DiO molecules (see Figure 1 B, red trace, and Figure S7 ). Quantification of interaction events and their separation from kiss-and-run type of events was done using custom made script in Python, based on earlier publications [ 31 , 33 , 37 ]. Briefly, events were detected using a method similar to earlier published [ 31 , 37 , 40 , 41 ], where each intensity trace is scanned for changes larger than five standard deviations.…”

“…Upon docking and membrane fusion, the resulting mixing of lipid species and rapid diffusion away from the docking spot will result in fluorescent signal dilution and loss [ 45 , 46 ] (see Figure S9 for time lapse of particle docking and subsequent signal loss). Analysis was performed in python using a custom routine adapted from [ 33 , 47 ]. Shortly, a binary mask was used to integrate the intensity of fluorescence signal localized with cell membrane and internalized or outside of the cell membrane ( Figure 3 ).…”

“…Similar sets of experiments were performed over all three different pH buffer liposomes. All the data were collected and analyzed by a homemade python script adapted from an earlier publications by our group [ 31 , 32 , 33 ]. The interactions of SL micelles were recorded as docking and kiss-and-run type events on the liposomes.…”

“…For cytotoxicity, Cellmask red marker was applied to the counting of live cells, while the NucGreen marker (ThermoFisher, membrane disruption dye) was used for the number of dead cells. All cells were outlined using a custom made routine in python [ 33 ] and afterwards counted manually. All the experiments were performed in triplicates and repeated twice, and the data are presented as mean ± SD.…”

“…To quantify the combined uptake and membrane interacting SL micelles on live cells, we used a slightly modified version of earlier published software [ 31 , 33 , 37 ]. Briefly, based on the membrane signal, the software will create a binary mask for all cells and use this to integrate fluorescent intensity from the cell interior (including the membrane) and exterior, respectively.…”