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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/137497
Understanding the transport mechanism of BBB peptide shuttles: THRre and MiniAp-4 as case studies
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[eng] The research work presented in this thesis aims to understand the transport mechanism of two described BBB-shuttles, THRre and MiniAp-4. To achieve these objectives, different techniques have been used as solid phase peptide synthesis (SPPS), blood-brain barrier (BBB) cellular models, biophysical techniques, nuclear magnetic resonance (NMR), photocrosslinking techniques and microscopy. The work was performed at the Institute for Research in Biomedicine (IRB) supervised by Dr. Meritxell Teixidó and Dr. Macarena Sánchez in the group of Prof. Ernest Giralt.
The introduction presents an overview of the main topics discussed in this thesis, how the presence of the BBB difficult the pass of most of potential therapeutics and the need to find tools to overcome this problem. Also, a thorough introduction on the main transport mechanism across the barrier is given.
The thesis is divided in two chapters. The first one consists of THRre peptide. This peptide was developed by Dr. Roger Prades and corresponds to the retroenantio version of THR, which was discovered by phage display. This two peptides present a capacity to transport a wide variety of cargoes in vitro and in vivo. It was already known that they use the transferrin receptor (TfR) to cross the BBB. Our first objective was to prove it by a saturated transfer difference (STD) NMR experiment. We were able to confirm the interaction between the two peptides and by measuring the STD effect, we could hypothesize the part of the sequence which is responsible for the interaction. Then, we tried to characterize this interaction by measuring the constant of dissociation (KD) by isothermal titration calorimetry (ITC) and sensor acoustic wavelength (SAW). A KD value around 1 mM was obtained for THR, however it was not possible to measure it for THRre, suggesting that the interaction is weak. We also wanted to elucidate the binding site of THR and THRre to the receptor by photocrosslinking. To this end, two photoreactive version were synthesised for each peptide, substituting one of the tryptophan for a non-natural commercial amino acid bearing a benzophenone moiety, a 4-benzoylphenylalanine (Bpa).
The second chapter is based on MiniAp-4, a short non-toxic cyclic version of apamin venom developed by Dr. Benjamí Oller in our group. The good transport properties of this peptides were attributed at its high stability against proteases and its remarkable 1:1 cis/trans proline conformation. The first objective was to synthesize seven different analogues changing the length of the cycle and swapping the proline for a dimethylproline residue in order to see if differences in the cis/trans ratio lead to differences in transport capacity. After, by NMR techniques cis/trans ratios were measured and peptides were assessed in a human BBB model, however no significant differences were observed between analogues. In addition, the candidate that presented an 85% cis conformation showed better transport capacity for a small cargo. The second objective was to study the uptake of MiniAp-4 in brain endothelial cell line using quantum dots (QDs) nanoparticles. Nevertheless, we could conclude that even though the peptide was able to carry QDs
inside the cell, these type of nanoparticles were not the ideal ones. Finally, we aimed to understand the transport mechanism of this peptide by confocal microscopy and photocrosslinking. A conjugated cyanine_MiniAp-4 was co-incubated with transferrin and cholera toxin, which are described to internalize via clathrin dependent and clathrin independent mechanism respectively. A co-localization with both controls was observed however, we suggest that the main route of entry is through caveolae. Also, different antibody staining performed in order to understand the route that MiniAp-4 takes once inside the cell suggested that maybe a degradation pathway is followed.
For the photocrosslinking experiments, we developed a photo-crosslinking strategy in which we designed and synthesized a biotinylated photo-reactive analogue that contains a photo-proline and a cleavable linker. After evaluation by top down mass spectrometry, we obtained different potential membrane proteins, however further experiments need to be done in order to confirm it.
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FUSTER JUNCÀ, Cristina. Understanding the transport mechanism of BBB peptide shuttles: THRre and MiniAp-4 as case studies. [consulta: 6 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/137497]