engleski

THERMALLY RESPONSIVE ELP-dnMAML PROTEIN AND IT'S EFFECT ON U251 CELLS IN VITRO

Mastermind-like (MAML) family of proteins has 3 members in mammalian organisms and has gotten its name because of the similarities between them and the Drosophilla Mastermind protein both in structure and in function. MAML proteins function as transcriptional co-activators in Notch signaling and several other pathways. MAML is essential for the assembly of transcription activation complex of Notch target genes. MAML proteins are structurally simple and can be the base for binding more complex proteins and getting them in close contact necessary for proper function. dnMAML1 mutant is a truncation of MAML1 protein consisting of 62 amino acids (13-74) from the N-terminal basic domain of MAML1. Since N-terminus is the most conserved part of all MAML proteins it can inhibit all four receptors and MAML proteins. Depending on the part of MAML involved dnMAML is presumed to be able to mimic MAML in Notch independent functions as well. That is true for p53 where binding is accomplished between N-terminal part of MAML and the DNA binding domain of p53. MAML family of co-activators makes an excellent candidate for targeting since they modulate a wide number of signaling pathways. N-terminal fragment of MAML1 protein further named dnMAML was cloned into a vector carrying ELP and SynB1 cell penetrating peptide. SynB1-ELP-dnMAML inhibition potential was tested in U251 glioblastoma derived cell line and confirmed by appropriate controls. Precise mechanism of inhibition was explored by testing levels of apoptosis induction and cell cycle distribution. SynB1-ELP-dnMAML show cytoplasmic accumulation in the cells. Protein uptake in U251 cells doubles when heat is applied. Cells were treated with growing concentrations of SynB1-ELP-dnMAML for 1h at different temperatures in two 72h cycles. Same was done with SynB1-ELP to show that the vehicle protein itself is not cytotoxic. Treatment with dnMAML inhibits cell growth significantly up to 60%. The effect can partially be explained by increased apoptosis (up to 20% in heated samples). There is no significant cell cycle arrest. dnMAML also negatively effects expression levels of key regulatory protein MAPK, pAKT and p53, which is mutated in the tested cell line. Further investigation is necessary but SynB1-ELP-dnMAML holds great potential for targeted molecular therapy.

Notch pathway ; dnMAML ; elastin-like polypeptide

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