Alpha-synuclein is a natively unfolded protein widely expressed in neurons at the presynaptic level. It is linked to Parkinson's disease by two lines of evidence: amyloid fibrils of the protein accumulate in patients' brains and three genetic mutants cause autosomal dominant forms of the disease. The biological role of the protein and the mechanisms involved in the etiopathogenesis of Parkinson's disease are still unknown. Membrane binding causes the formation of an amphipathic alpha-helix, which lies on the surface without crossing the bilayer. Recent observations however reported that the application of a voltage induces a pore-like activity of alpha-synuclein. This study aims to characterize the pore forming activity of the protein starting from its monomeric form. In particular, experiments with planar lipid membranes allowed recording of conductance activity bursts with a defined and reproducible fingerprint. Additional experiments with deletion mutants and covalently bound alpha-synuclein dimers were performed to understand both pore assembly and stoichiometry. The information acquired allowed formulation of a model for pore formation at different conductance levels.

The cytotoxic cell granule secretory pathway is essential for immune defence. How the pore-forming protein perforin (PFN) facilitates the cytosolic delivery of granule-associated proteases (granzymes) remains enigmatic. Here we show that PFN is able to induce invaginations and formation of complete internal vesicles in giant unilamellar vesicles. Formation of internal vesicles depends on native PFN and calcium and antibody labeling shows the localization of PFN at the invaginations. This vesiculation is recapitulated in large unilamellar vesicles and in this case PFN oligomers can be seen associated with the necks of the invaginations. Capacitance measurements show PFN is able to increase a planar lipid membrane surface area in the absence of pore formation, in agreement with the ability to induce invaginations. Finally, addition of PFN to Jurkat cells causes the formation of internal vesicles prior to pore formation. PFN is capable of triggering an endocytosis-like event in addition to pore formation, suggesting a new paradigm for its role in delivering apoptosis-inducing granzymes into target cells. P athogen-infected and tumor cells are eliminated through granule-mediated apoptosis. Perforin (PFN), which is secreted together with proteases (granzymes) from cytotoxic T lymphocytes or natural killer cells, has a central role in this process, aiding the intracellular delivery of granzymes to initiate apoptosis of target cells (1, 2). Two models are currently available for the function of PFN. The first involves formation of stable plasma membrane pores that allow the direct transfer of granzymes into the target cell, which are later removed by membrane repair (1,3,4). This model is based primarily on visualization of pore-like structures on the membrane of the target cells (5-7). The other model, originally postulated by Podack and coworkers, proposes that the delivery process requires endocytosis of both PFN and granzyme, with the protease subsequently being released to the cytosol (4, 8).Mature PFN is a 533 residue protein consisting of three domains: an N-terminal membrane attack complex/perforin (MACPF) domain, an intervening EGF-like domain, and a Cterminal calcium-binding C2 domain, which is responsible for the initial Ca 2þ -dependent binding of PFN to membrane surfaces (9-11). The structures of PFN and other MACPF domain proteins (12-15) reveal they have folds related to those of the cholesteroldependent cytolysins (CDCs) of Gram-positive bacteria, such as pneumolysin from Streptococcus pneumoniae (16-19). These observations suggest that the effects of PFN and other MACPF proteins on membranes derive from a mechanism related to the CDCs, which involves membrane binding and oligomerization to large arc or ring structures. MACPF/CDC domains refold into membrane-inserted pore forms via an oligomeric prepore state; both prepore and pore states have been observed for PFN (15,20,21). It has been argued CDCs form pores as full ring-shaped oligomers and as arcs (17, 22); a recent study by us supports this view f...