engleski

Thermally responsive ELP-dnMAML protein and its effect on U251 cells in vitro

Introduction: The Mastermind-like (MAML) family of proteins has gotten its name because of the similarities with the Drosophila Mastermind protein in both structure and function. MAML proteins are 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 the 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 the N-terminal part of MAML and the DNA binding domain of p53. MAML family of co-activators make an excellent candidate for targeting since they modulate a wide number of signaling pathways. Materials and Methods: The N- terminal fragment of MAML1 protein, further named dnMAML, was cloned into a vector carrying elastin like polypeptide (ELP) and SynB1 cell penetrating peptide. SynB1-ELP-dnMAML inhibition potential was tested in U251 glioblastoma derived cell line. Cells were treated with increasing concentrations of SynB1-ELP-dnMAML for 1 h at different temperatures in two 72 h cycles. The same was done with SynB1-ELP to show that the vehicle protein itself is not cytotoxic. Precise mechanism of inhibition was explored by testing levels of apoptosis induction and cell cycle distribution. Results and Discussion: SynB1-ELP-dnMAML shows cytoplasmic accumulation in the cells. Protein uptake in U251 cells doubles when heat is applied. 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 effect on cell cycle or expression levels of Notch canonical targets Hes and Hey. dnMAML also negatively affects expression levels of key regulatory proteins MAPK, pAKT and p53 (mutated in the tested cell line). Conclusion: The effect of SynB1-ELP-dnMAML on cell growth and signaling is evident. It affects cytoplasmic targets due to the lack of nuclear penetration. The ability of this peptide to be modified and tailored more specifically further increases its potential for development.

NOTCH, MAML

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