This dissertation describes the design and evaluation of cell-permeable miniature proteins containing arginines displayed on an alpha-helix. Protein therapeutics are garnering great interest recently but still face challenges, particularly in delivery for intracellular targets. Even prior to use in the clinic, cell impermeability of proteins is a significant limitation to examination of their functions in the laboratory. The use of cell-penetrating peptides (CPPs) as tags is one of the more promising tools to overcome this challenge. However, particularly for small proteins, where a tag may be nearly as large as the protein it is appended to, CPPs can have negative effects on function, structure, and toxicity. The first chapter describes our use of a technique known as protein grafting to incorporate arginines into a folded alpha-helix of the miniature protein avian pancreatic polypeptide (aPP). We successfully generated a variant of aPP displaying five arginines on the alpha-helix, and only seven arginines in total, that had significantly higher uptake at 1 microM than five commonly used CPPs, including octaarginine. This miniature protein also retained the PP-fold structure of the wild type aPP at physiological temperature.;In the second chapter, we used the arginine display technique to impart cell permeability to a different miniature protein, YY2. YY2 itself is based on PYY, a miniature protein with a similar PP-fold but somewhat different primary sequence from aPP. YY2 has been modified from PYY to present an SH3-binding epitope on the PPII helix, and it has been shown in vitro to activate Src family kinases possibly by disruption of an intramolecular SH3-binding autoinhibitory interaction. YY2 itself, which contains six total arginines---two on each of the PPII helix, the alpha-helix, and the unstructured tail---showed cell-permeability only at higher concentrations. We were able to impart cell permeability to YY2 at low concentrations by use of our arginine grafting technique, but only with loss of thermostability as a price.;We returned to aPP in the third chapter to further explore the technique of arginine display on the alpha-helix. We generated a series of nine miniature proteins to examine the effects on cell uptake of arginine clustering on two or three faces of the helix. We discovered a threshold number of arginines on this helix; variants with four or more arginines possessed cell-penetrating ability, and those with three or fewer did not. Miniature proteins with exactly the threshold number of arginines had uptake that was strongly dependent on the position of those arginines, with the variant with closer clustering showing much higher uptake. Interestingly, both of these variants, compared to the variants with five arginines on the alpha-helix, possessed higher levels of cytosolic, versus endosomal, localization.;Due to the dramatic difference in localization among the miniature protein series, the fourth chapter describes an analysis of the mechanism of entry, specifically for two variants with punctate localization in addition to the one with high uptake and diffuse cytosolic staining. The uptake of all of these miniature proteins was energy-dependent, but the similarities ended there. The derivative with cytosolic localization was significantly more dependent on the presence of sulfated glycosaminoglycans, and was also found to utilize a different endocytotic pathway to enter the cell. Entry by lipid-raft-mediated endocytosis, rather than the clathrin-mediated endocytosis used by the other two variants, may help explain the greater escape of this miniature protein to the cytosol. In addition, this result further underscores the importance of evaluating the mechanism of entry for every known CPP, rather than trying to find a common mechanism used by all.
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