2.1 The intrinsic pathway
The occurrence of the intrinsic apoptosis signal pathway of apoptosis mainly involves various non-receptor-mediated stimuli, generating intracellular signals, and directly acting on intracellular target (mitochondria), leading to apoptosis. Stimulants that cause endogenous apoptosis act in an active or passive manner. Passive signals include the loss of specific growth factors, hormones and cytokines, leading to an unlimited death program, thereby triggering cell apoptosis(Papaianni et al., 2015). Other stimuli that act in an active manner include but are not limited to radiation, toxins, hypoxia, high fever, viral infections and free radicals.
The mitochondrial pathway in apoptosis is mainly regulated by members of the Bcl-2 family proteins(Ashkenazi & Salvesen, 2014; Papaianni et al., 2015). Bcl-2 family proteins have pro-apoptotic or anti-apoptotic effects. So far, 25 genes have been identified in the Bcl-2 family. Anti-apoptotic proteins include Bcl-2, Bcl-XL, Bcl-x, Bcl-XS, Bcl-w and BAG, pro-apoptotic proteins include Bax, Bak, Bid, Bad, Bim, Bcl-10, Bik and Blk(Borner & Andrews, 2014; Kilbride & Prehn, 2013; Wong & Puthalakath, 2008). These proteins all play a role in determining whether a cell undergoes apoptosis. Studies have found that the main mechanism of Bcl-2 family protein function is to modulate the mitochondrial membrane permeability to regulate the release of cytochrome c (Cyt-c).
The stimulus causes changes in the inner mitochondrial membrane, leading to the opening of the permeability transition pore (PTP), the loss of mitochondrial membrane potential (MMP), and the release of two groups of pro-apoptotic proteins from the membrane space into the cytoplasm(Tait & Green, 2010) (Figure 3). The first group contains Cyt-c, DIABLO/Smac and serine protease HtraA2/Omi(Garrido et al., 2006; van Loo et al., 2002). These proteins activate the caspases-dependent mitochondrial pathway, cyt-c binds and activates apaf-1 and procaspase-9 to form “apoptosome”(Yuan & Akey, 2013). The accumulation of procaspase-9 leads to the activation of caspase-9, and caspase-9 triggers the caspase cascade, then the activated caspase-3/6/7 executes apoptosis. DIABLO/Smac and serine protease HtrA2/Omi have been reported to inhibit IAPs (inhibitors of apoptotic protein) and promote apoptosis(Schimmer, 2004). The second group of pro-apoptotic proteins includes AIF (apoptosis-inducing factor) and EndoG (endonuclease G), and the functions of AIF and EndoG are independent of caspases. These two proteins are released from the mitochondria when apoptosis occurs, and as a late event, it occurs in the dying cells. AIF translocates into the nucleus, causing DNA fragmentation and condensation of peripheral nuclear chromatin(Joza et al., 2001). EndoG can also be translocated into the nucleus to cut off nuclear chromatin and produce oligomeric nucleosome DNA fragments(Jang et al., 2015). As a site for protein synthesis and processing, the ER is imbalanced due to physical and chemical stimulation or infection of pathogenic microorganisms, resulting in the inability of protein folding or misfolding. When these conformationally dissimilated proteins accumulate in the ER and cannot be effectively eliminated, the cells will initiate an unfolded protein response (UPR). Long-term activation of UPR can trigger cell apoptosis through IRE1 (inositol requiring protein-1), PERK (protein kinase RNA-like ER kinase) and ATF6 (activating transcription factor-6) pathways, which are connected with mitochondria-mediated apoptosis pathway(Verma & Datta, 2012). First, phosphorylated IRE1 recruits TRAF2 (TNF receptor-associated factor 2) and triggers a cascade of phosphorylation events, such as the activation of ASK1 (apoptosis signaling kinase 1), and ultimately phosphorylates and activates JNK. Then, JNK activates pro-apoptotic genes Bax, Bak, Bim, PUMA and NOXA, these genes are transferred to the mitochondrial to initiate apoptosis(Upton et al., 2012). The homomultimerization and autophosphorylation of PERK lead to phosphorylation of eIF2α (eukaryotic translation initiation factor 2α), which increases the translation of ATF4 (activating transcription factor 4). Then, ATF4 upregulates the expression of CHOP (C/EBP-homologous protein) and activates apoptosis(Tabas & Ron, 2011). CHOP can reduce the expression of anti-apoptotic genes Bcl-2 and Bcl-XL(Puthalakath et al., 2007). ATF6 binds to the ER membrane, but when protein homeostasis is disrupted, ATF6 then migrates to the Golgi apparatus to undergo cleavage, first by Site 1 Protease (S1P) and then by Site 2 Protease (S2P)(Ye et al., 2000), cleaved ATF6 can also promote cell apoptosis via upregulating of CHOP(Hirsch, Weiwad, Prell, & Ferrari, 2014; Morishima, Nakanishi, & Nakano, 2011).