Figure 1. Pro-apoptotic and antiapoptotic signalling through death receptor family. Pro-apoptotic function: Ligation of the TNF receptor (TNFR), death receptor 3 (DR3), Fas (CD95) or death receptors 4 and 5 (DR5/DR4) by their respective ligands TNF, TL1A, FasL (CD95L) or TRAIL results in receptor trimerization. Consequently, the oligomerized death domains of the receptor bind cognate domains of TNFR-associated death domain (TRADD) and Fas-associated death domain (FADD). The death effector domain (DED) of FADD in turn binds a related domain in the propeptide region of caspase-8, facilitating transactivation of the enzyme. The active enzyme is able to trigger two different apoptotic pathways depending on the cell type. In type I cells, active caspase-8 cleaves procaspase-3 which is fully activated by autocleaving. Active caspase 3 is able to degrade and/or activate several proteins inducing cell death by apoptosis. In type II cells caspase-8 cleaves the Bcl-2 family member Bid generating the active truncated form of the protein (tBid) which translocates to the mitochondria where it activates Bak and Bax. Active Bak/Bax induces cytochrome c release, which in the presence of dATP form a complex with Apaf-1 and recruit and activates caspase 9 generating a structure known as apoptosome. Active caspase-9 induces caspase 3 activation and apoptosis. In addition, other pro-apoptotic proteins such as AIF, endonuclease G (EndoG) or the serineprotease HtrA2 are released from the mitochondria by this pathway. Antiapoptotic function: In the case of DR3 and TNFR, TRADD also binds RIP, which then interacts with TNFR-associated factor 2 (TRAF2). This latter interactions activate the NFB offering antiapoptotic regulation (survival) of the death receptors. DcRs with either a truncated or an absent death domain (DD) act as a ‘sink’ for TRAIL minimizing activation of DR4 and DR5. FLIP (FLICE inhibitor protein) is able to block pro-apoptotic signals by blocking procaspase-8 recruitment by FADD.

Figure 2. Proposed model for perforin-mediated intracellular granzymes delivery. Perforin pore model: Perforin (perf) is able to form pores in the membrane of target cells and granzymes (gzms) would enter the cytosol via those pores. Granzyme endocytosis model: granzymes would be endocyted via specific receptors such as mannose-6-phosphate receptor (M6PR) or by membrane charge-dependent interactions and pinocytosis. At the same time perforin insertion in the cell membrane would induce a cell repairment response that would induce perforin endocytosis. It is not clear if perforin and granzymes would be endocytosed in the same endosome or if granzyme endosomes fuse with perforin endosomes during intracellular traffic. Finally, perforin would disrupt the endosome allowing granzymes to release in the cytosol where they would induce cell death.

Figure 3. Antitumoural and antibacterial action mechanism of granulysin. (a) Granulysin-induced apoptosis does not require perforin. The first studies showed that granulysin-induced plasma membrane-associated sphingomyelinase (neutral SMase) activation, degradation of sphingomyelin and generation of ceramide, a lipid responsible of the activation of some pro-apoptotic pathways. However, ceramide generation was only detected after 8–10 h and granulysin was able to induce apoptosis faster. Later on it was found that granulysin induced a fast caspase-independent apoptosis pathway mediated by the release of AIF from the mitochondria. This release was Bcl-2-dependent and mediated by the opening of the mitochondrial transition pore by a Ca²⁺ increase in the cytosol. Cytosolic Ca²⁺ increase is a consequence of granulysin-induced disruption of plasma membrane. This mechanism is already evident after 3–4 h. (b) Although granulysin is able to directly lyse pathogens, it needs perforin to reach intracellular bacteria.

Figure 4. Pro-apoptotic pathways activated by granzymes. (a) Intracellular granzyme B is able to induce cell death by at least three different pathways. Granzyme B cleaves and activates the effector caspases-3 and -7, which are mandatory for phosphatidylserine translocation and ROS production. On the other side Bid/Bak/Bax pathway is activated and crucial for cytochrome c (cyt c) release from the mitochondria and apoptosome formation. Both pathways independently contribute to depolarization of the mitochondria. Finally, granzyme B induces cell death without apoptotic phenotype by a caspase- and Bid-independent pathway. Extracellular granzyme B is able to cleave proteins from the extracellular matrix (ECM) such as filamin, vitronectin or laminin, inducing cell detachment and death by anoikis. Cells die because they need integrin-mediated prosurvival signalling, which is disrupted when cells are not attached to ECM. (b) Intracellular granzyme A is able to induce mitochondrial depolarization and ROS production by a not known pathway. ROS generation is crucial for PS translocation. In addition, ROS production would induce DNA damage and the subsequent activation of DNA-repairing mechanisms. Among them, the translocation of the SET complex from the ER to the nucleus. In the nucleus granzyme A would degrade some proteins of that complex such as SET, pp32 and Ape1 releasing from the nuclease NM23H1 that would induce DNA damage and cell death. This mechanism has been tested with purified proteins and is not clear. Extracellular granzyme A is able to induce ECM degradation and cell detachment.