Investigation of perforin pore function and formation

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells. Perforin contains a membrane attack complex / perforin (MACPF) domain and oligomerises to form an aqueous pore in the plasma membrane. Therefore, the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. However, the mechanics of pore assembly and granzyme delivery remain unclear. Perforin has been compared to Streptolysin O (SLO), a structurally similar bacterial pore-forming protein that is part of the cholesterol dependent cytolysin (CDC) family. The assembly of CDC pores has been extensively studied, and sets the paradigm for assembly of MACPF protein pores. This thesis comprehensively compares SLO and perforin in terms of cargo delivery and pore-forming mechanisms. Granzymes contain two cationic sites that have been previously shown to be important for delivery of human granzyme B by perforin. Human granzyme B contains the highest proportion of positive residues at these sites, while granzymes A and K both have fewer. It was shown that human and mouse granzyme A and K are delivered more efficiently by SLO than perforin. Human granzyme B, on the other hand, was delivered equally well with both perforin and SLO. These results reinforce the idea that positive residues are important for efficient delivery of granzymes by perforin, but not by SLO. Supporting this notion, it was found that perforin effectively delivered molecules other than granzymes, provided the cargo was cationic. Anionic and neutral molecules were inefficiently delivered by perforin. This suggests that perforin delivers cationic cargoes via “facilitated diffusion”, which may involve conserved features of oligomerised MACPF proteins, such as an anionic lumen. In a different approach, quartz crystal microbalance with dissipation monitoring (QCM-D) showed that the mechanism of pore formation of SLO is different to perforin. Perforin binding to membranes is cooperative whereas SLO binding follows first order kinetics. Cholesterol contributes to binding and activity of both SLO and perforin on intact cells and on model membranes. Finally, QCM-D revealed that perforin is able to efficiently bind membranes in the absence of calcium. When calcium was subsequently introduced the QCM-D traces changed significantly, indicating that perforin had bound calcium and then became active on the membrane. Taken together, these results demonstrate that there are fundamental differences between the cargo delivery and pore-forming mechanisms between MACPFs proteins and CDCs