Cytolysin A (ClyA) is a member of a class of proteins called pore-forming toxins (PFTs). ClyA is secreted by Gram-negative bacteria, and it attacks a number of mammalian cells by inserting into and forming channels within the cell membrane (Oscarsson J et al., 1999). It has been suggested that ClyA binds to cholesterol (Oscarsson J et al., 1999) and thus can insert into the membranes of many different cell types of eukaryotic origin. In our studies we propose to engineer a ClyA protein that can only attack a small subset of cell types. We propose to engineer ClyA that can be only activated when exposed to specific cell-surface proteases produced by a specific cell type. We ultimately want to target breast cancer cells that differentially secrete or express proteases such as matrix-metalloproteases (Stautz D et al., 2012; Zhang, M et al. 2013). To engineer this protein we took advantage of the N-terminus of ClyA. The N-terminus of ClyA, which is highly hydrophobic (Oscarsson J et al), undergoes a conformational change to insert into the target cell membrane (Oscarsson J et al). This conformational change allows ClyA to penetrate the target membrane to form a transmembrane domain of ClyA. The hydrophobic nature of lipid membranes makes it highly unfavorable for any charged residues to cross the membrane (Hunt J 1997). With this in mind, we hypothesize that negative charges inserted into the N-terminus of ClyA will inhibit it from inserting into the membrane. Thus, we mutated the N-terminus of the ClyA protein by inserting an inactivation site composed of negatively charged amino acids that we hypothesize would prevent insertion into the plasma membrane of the target cell. Once we confirmed that this construct was an inactive ClyA mutant, we inserted a thrombin cleavage site right after the inserted negative charges. This site should allow us to remove the negative charges once the protein is exposed to thrombin. Once the negative charges are removed, the protein should recover its activity. This approach will allow us to create a version of ClyA that is protease-switchable.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-1135 |
| Date | 07 November 2014 |
| Creators | Avelino, Alzira Rocheteau |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
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