Homeoproteins, transcription factors involved in physiological development, can be secreted and internalized by eukaryotic cells, thus acting as paracrine cellular messengers. The third helix of the DNA-binding homeodomain, penetratin, has been characterized as a cell-penetrating peptide and is primarily responsible for the cellular internalization of homeoproteins. Engrailed-2, a homeoprotein involved in neurological development, has been chosen as a model to study how different regions of the protein affect the quantity of protein internalized and the mechanism of internalization. We have used an isotopic labeling strategy in conjunction with MALDI-TOF MS to quantify the entry into CHO K1 cells of the isolated Engrailed-2 homeodomain and a fragment of Engrailed-2 that includes the homeodomain and the nuclear localization sequence. We also present preliminary experiments studying the internalization of these proteins in CHO PgsA-745 cells, which lack glycosaminoglycans essential for endocytosis.

Quantitative analysis of the cellular internalization of Engrailed-2 homeoprotein

249th National Meeting (2015)

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