Final Activity Report Summary - APOPTOSIS MECHANISMS (Structural and biophysics studies of programmed cell death) Programmed cell death, also known as apoptosis, consists of a series of cellular mechanisms that lead to cell suicide as a response to external and internal stimuli. This form of death is an essential process in the development of organisms by eliminating damaged or unwanted cells. As a key component of the cell life cycle, the dysfunction of apoptosis results in several human pathologies including neurodegenerative and autoimmune disorders, and it is also a relevant cause in the onset of cancer. The molecular mechanisms of programmed cell death are intricate, involving numerous protein-protein interaction events, the formation of large supramolecular assemblies through protein oligomerisation, as well as protein structural changes resulting in translocation and insertion in cellular membranes. The project aimed to understand the molecular basis of these mechanisms as a key step toward the foundation of clinic strategies to treat disease. In particular, the project focuses on protein-protein interactions and oligomerisation in the Death Domain superfamily, heterodimer formation within members of the Bcl-2 family and their translocation and interaction with membranes. Protein-protein interaction and oligomerisation were studied using as model the protein ASC, a Death Domain superfamily member that mediates in apoptosis and inflammation. ASC interacts with cell death executioners and oligomerises into functional supramolecular assemblies through its two Death Domains. Nuclear Magnetic Resonance (NMR) and Atomic Force Microscopy (AFM) were used to study the structure, dynamics and oligomerisation properties of ASC. Based on the obtained results a model is proposed to illustrate how ASC oligomerises via homotypic Death Domain interactions. This study shows that the molecular architecture of ASC facilitates self-association and multiple binding to several proteins, which in turn can result in the assembly of supramolecular platforms. The overall dimension and shape of the ASC oligomers were analysed by AFM showing a predominant species of disk-like particles whose size agrees with the formation of a ~7-member ring that is proposed based on the model. Taken together, the structural and dynamic features of ASC shed light into the function of this protein as an adapter molecule and its capability to form supramolecular complexes (Journal of Biological Chemistry (2009), vol. 284: 32932-32941). Protein heterodimerization within prosurvival and proapoptotic Bcl-2 members, as well as membrane translocation and insertion are mechanisms that control apoptosis. To better understand these processes the project centers at studying the structure, dynamics and interactions of two Bcl-2 proteins: Harakiri and Diva. Harakiri is a cell death-inducing protein that localizes in membranes and binds to prosurvival Bcl-2 proteins. The obtained NMR and Circular Dichroism data show that the N-terminal region of harakiri is highly unstructured, although it populates the a-helical conformation. The three-dimensional structure of this fragment determined by enhancing its population resembles other BH3 domains bound to prosurvival partners, which suggests that intrinsic structure propensity is very relevant in the binding mechanism. In addition, harakiri contains a C-terminal transmembrane domain that folds into a monomeric a-helix in micelles. The resulting structure reveals features explaining its function as membrane anchor. The data on the N-terminal and transmembrane regions are used to propose a tentative model of how harakiri works. Diva is a peculiar Bcl-2 member found to promote or inhibit cell death depending on the cellular context. Preliminary data (J. Biomol. NMR Assign. (2009), in press) show that Diva consists of 8 a-helices with a particularly long loop connecting helices 5 and 6. Further structural studies will help to understand Diva's dual behaviour in apoptosis.