Delivery of biomolecules into mammalian cells using anthrax toxin

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2015.Cataloged from PDF version of thesis.Includes bibliographical references.The intracellular delivery of biomolecules into mammalian cells is a major challenge due to the plasma membrane, which acts as a barrier between the extracellular environment and intracellular components. Recently, a non-toxic delivery platform derived from anthrax lethal toxin has been developed to overcome this challenge for the delivery of biomolecules into the cytosol of mammalian cells. The PA/LFN delivery platform has been used to deliver over 30 known biomolecules of diverse sequences, structures, and functionalities. Collectively, these translocation studies have helped to elucidate the translocation mechanism and to probe intracellular biological processes. In this thesis, the PA/LFN delivery platform was used to analyze the delivery of assorted biomolecules through the PA pore. A facile, modular ligation strategy using sortase A was developed for the conjugation of biomolecules to LFN. The biomolecules for this analysis included antibody mimic proteins with defined sizes and secondary structures, mirror image peptides and proteins, polypeptides containing non-canonical amino acids or small molecule drugs, and cyclic peptides. Our translocation analyses have led to guidelines for translocation as well as insight into design parameters for the efficient delivery of new cargos. The PA/LFN delivery platform has also been used to translocate bioactive cargos for the disruption of intracellular protein-protein interactions (PPI). The translocation efficiency and bioactivity of a tandem monobody to Bcr-Abl, an affibody to hRaf- 1, and a mirror image peptide to MDM2 were analyzed. Efficient translocation and disruption of the intended PPI in each case indicated that the delivery platform could be used to deliver bioactive cargos into cells for therapeutic utility. As an application of this technology, the PA/LFN delivery platform was employed to analyze the intracellular stability of mixed chirality proteins. One major factor that governs a protein's stability is the N-end rule, which states that the N-terminal residue of a protein impacts its intracellular stability through the ubiquitin (Ub)/proteasome system. Utilizing the PA/LFN delivery platform, the stability of proteins containing one N-terminal D-amino acid was analyzed. In contrast to N-terminal L-amino acids, each N-terminal D-amino acid abrogates protein degradation by the N-end rule pathway.by Amy Ellen Rabideau.Ph. D