Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Maria Morales - University of Puerto Rico at Cayey
Co-Author(s): Kuangyu Chen and Dehua Pei, Ohio State University, Columbus, OH
Bacterial toxins can enter the cytosol of mammalian cells by endocytosis and endosomal escape. However, the mechanism of their endosomal escape remains poorly defined in most cases. In this study, in attempt to gain a better understanding of the mechanism of entry of the Pseudomonas exotoxin A (PE) to cells, we synthesized a peptide corresponding to a region of PE (peptide 1), which we hypothesized was responsible for the endosomal escape of the toxin into the cytosol, and a mutant of that sequence (peptide 2) and evaluated their cellular entry efficiency. In addition, we attached those two peptides to the HIV Tat peptide sequence to test their ability of enhancing the cytosolic entry efficiency of the Tat peptide. The peptides were chemically synthesized, purified by reversed-phase high pressure liquid chromatography (HPLC), and authenticated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Peptides 1 and 2 were fluorescently labeled with fluorescein isothiocyanate (FITC) and the Tat peptide conjugates were fluorescently labeled with naphtofluorescein. Flow cytometry was used to measure the HeLa cell’s uptake of the labeled peptides. The results showed that peptides 1 and 2 were able to enter HeLa cells, but their uptake efficiency was lower than that of a cyclic cell-penetrating peptide (which was used as a positive control). In addition, the results suggest that the Tat peptide conjugates enter the cytosol of HeLa cells 7-8 times better that the Tat peptide. These are promising results, suggesting that the peptide studied from PE could be the sequence responsible for the endosomal escape of PE into the cytosol and that this sequence can be used to enhance the cytosolic delivery efficiency of other cell-penetrating peptides.
Funder Acknowledgement(s): This work was supported by The Ohio State University Graduate School.
Faculty Advisor: Dehua Pei, firstname.lastname@example.org
Role: For this research project, I was fully responsible of chemically synthetizing the peptide corresponding to a region of PE and the mutant peptide. Also, I chemically attached the HIV Tat peptide to the previous mentioned peptides. In addition, I was responsible of fluorescently labeling, purifying by high performance liquid chromatography (HPLC), and authenticating by MALDI-TOF mass spectrometry all of the peptides. Furthermore, I helped with the preparation of the HeLa cells treated with the labeled peptides for their analysis by flow cytometry.