Studies On Posttranslational Modification On Function Of Peptide

Post-translational modifications is a very important step after proteins and peptides translated by ribosomes. To become mature functionally proteins, the proteins and peptides have to go through a series of biochemical reactions to add the functional group covalently. The most common forms of post-translational modifications including phosphorylation, ubiquitination, lipid. The modifications determine the structure and function of the proteins and peptides. The core work of this thesis is to study the influence of post-translational modifications on the function of peptides.It is reported that the modifications of the substrate, inclding phosphorylation and glycosylation, interferes with the recognition of the protease to the substrate and cleavage process. The modifications of the substrate can be regulated in dynamic manner. TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins and peptides. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated.Here, we developed peptide-based protease sensor, using 5(6)-Carboxyfluorescein and pyrene as a high-efficiency mutual fluorophore-quencher pair. We demonstrated that phosphorylation at P3 tyrosine severely hindered the recognition of TEV protease to the substrate by using the peptide-based protease sensor. And based on phosphorylation in the substrate, we managed to dynamically regulate the cleavage capability of TEV protease in a facile and reversible method.The influence of lipidation of the peptide on transmembrane ability was also discussed here. In the previous work, we discovered the partial native sequence of cancer-associated protein Kras, Pep-OMe 4, which consisted with 12 amino acids, had good penetrating ability. The penetrating efficiency of Pep-OMe 4 was comparable to that of(Arginine)7, which is frequently used as cell penetrating peptides. Pep-OMe 4has the potential to be the native sequence of CPP. Kras plays a very important role in organism in lipid-based modification, especially in farnesyl-modified way. In this thesis,we also studied the influence of farnesyl modification of Pep-OMe 4 on its transmembrane efficiency. The results showed, the farnesyl modification significantly increased the transmembrane ability of Pep-OMe 4, which provided a potential way toimprove its penetrating ability in modified way.In this thesis, the effects of two different post-translational modifications on two respective peptides were studied: The phosphorylation of the substrate improved their stability to TEV protease and we managed to dynamically regulate the cleavage capability of TEV protease based on phosphorylation in the substrate; The farnesyl modification significantly improve the penetrating efficiency of Pep-OMe 4, the short sequence from cancer-associated protein Kras. These findings provided important references for the next investigation, which is about in vitro regulating the function of peptides in modified ways.