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Author
Soler Palazón, Adrià
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Abstract
The Hedgehog (Hh) pathway is a crucial signaling cascade during embryogenesis. The Sonic Hedgehog gene (SHH) was identified in vertebrates, which encodes for Sonic Hedgehog protein (Shh), acting as a ligand in Hh pathway. The Hh signaling pathway is activated when this ligand is present, allowing the transcription of diverse genes. The fatty acid present in the N-terminal domain of Shh (ShhN) regulates its affinity by the receptor Patched (Ptch), while the absence of cholesterol at its C-terminal part does not have a similar effect on the binding.
In the las years it was discovered that the Hh pathway can be modulated through some agents such as Robotnikinin and the antibody 5E1, acting as inhibitors. These antagonist could have therapeutic utility, since an inappropriate activation of Hh signaling pathway has been shown in some cancers. These two are one of the few reported molecules capable to bind ShhN, so new potential peptide-based ShhN inhibitors were synthesized in this work. The sequences of the new inhibitors were identified by phage display studies performed against Palm-ShhN-Biotin in the previous thesis of the group.
The unique clones obtained from the corresponding library were synthesized by Solid Phase Peptide Synthesis (SPPS), making on it some modifications. A peptide fragment of 25 amino acids containing a thioester in its C-terminal domain has been ligated with different peptides of 37 amino acids having a cysteine in its N-terminal domain by means of Native Chemical Ligation (NCL), obtaining yields from 37 % to 70 %. The resulting peptides of 62 amino acids have been desulfurized and purified obtaining yields between 59 % and 75 %.
These reactions have been monitored through different techniques such as Reverse Phase – High Performance Liquid Chromatography (RP-HPLC) and Matrix Assisted Laser Desorption Ionization – Time of Flight (MALDI-TOF). The synthesized peptides have also been characterized by mass spectrometry employing Electrospray Ionization (ESI). Finally, the folding of these potential ShhN inhibitors has been characterized by Circular Dichroism (CD) and their percentage of helicity was calculated. Their binding with the protein was studied using native Polyacrylamide Gel Electrophoresis (PAGE) and Immobilized Metal Affinity Chromatography (IMAC) combined with SDS-PAGE as a detection method. The results were not conclusive for His6-SUMO-ShhN. Future work is focused to the analysis of the binding using Surface Plasmon Resonance (SPR).
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