The Mechanism And Application Of Phosphorylation Modification To Regulate TDP-43 And Melittin

Phosphorylation is the most common post-translational modification of proteins,with approximately two-third of proteins in mammalian cells presenting with one or more phosphorylation modifications.The nature often regulate the structure and function of peptides and proteins through phosphorylation,so phosphorylation is closely related to numerous physiological and pathological processes.In the nerve cells of patients with ALS and FTLD,there are a large number of aggregates of TDP-43 and TDP-43 C-terminal fragments.Such aggregates are often modified by hyperphosphorylation and ubiquitination.Studies have confirmed that the level of phosphorylation of TDP-43-related proteins is correlated with disease progression.Specifically,the level of phosphorylation of TDP-43-related proteins significantly increases in patients with late stage of diseases.However,the current researches on the regulation of the pathological functions of TDP-43 related proteins by phosphorylation are still confusing.The major reason is lacking TDP-43 related proteins with site-specific post-translational modifications.In order to accurately address above problems,we selected TDP-43 protein C-terminal domain,TDP PLD protein as model.We combined the protein expression with chemical modification and synthsis to design a novel stratrgy.Using this strategy,we successfully synthesized w TDP PLD protein and serine404 phosphorylated p TDP PLD protein without any purification tag for the first time.It was confirmed that the p TDP PLD protein had a faster aggregation process,more cytotoxicity and more infectious ability than w TDP PLD protein.This suggested that pathologically related p S404 modification could mediate TDP PLD protein to obtain stronger pathological functions.Thus inhibition of Ser404 phosphorylation of TDP-43 protein will be a potential target for the treatment of ALS and FTLD diseases.Based on the fact that phosphorylation could regulate TDP PLD functions,phosphorylation modification was used to regulate the function of melittin and its derivatives.And an alkaline phosphatase(ALP)emzyme-induced gain of function(EIGF)strategy was developed to enhance the cell selectivity of melittin and its derivatives.Specifically,by screening for phosphorylation modification sites,we obtained phosphorylated melittin derivative with loss of function.The phosphorylated peptide underwent a dephosphorization process in response to ALP activity in vitro and on the cell surface,thereby restoring its membrane lysis ability.Thus,the phosphorylated peptide could recognize and selectively kill cancer cells that highly expressed ALP,while greatly reduce the toxic side effects on normal mammalian cells.This targeting strategy based on phosphorylation modification and ALP enzyme activity can not only enhance the cell selectivity of melittin and its derivatives,but also can act on other bioactive molecules to improve their targeting ability.Studying the mechanism of phosphorylation for regulating the structure and function of peptides and proteins,is an important view for understanding the dynamics of life activities.Similarly,learning from the subtle manipulations of nature,we can also use phosphorylation to regulate functions of biologically active molecules.