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Core Apoptosis Pathways
Apoptosis is a biochemically well-defined programmed cell death that is essential for normal development and cellular homeostasis; dysregulation of apoptosis is involved in several diseases. The classical morphological features of apoptosis include cell shrinkage, membrane blebbing, nuclear condensation, and deoxyribonucleic acid (DNA) fragmentation. The biochemical hallmarks of apoptosis include permeabilization of the outer mitochondrial membrane (MOMP), activation of caspases, and the externalization of phosphatidylserine, leading to phagocytosis of the apoptotic remnants without inducing inflammation.
In mammals, apoptosis is initiated by either internal or external stimuli and is governed by two molecular programs that terminate in caspase activation: the intrinsic (mitochondria-mediated) pathway and the extrinsic (death receptor-mediated) pathway. The intrinsic pathway is regulated by B-cell lymphoma (BCL-2) family proteins that determine whether MOMP occurs. Upon MOMP, cytochrome C is released into the cytosol, where it binds apoptotic protease activating factor 1 (APAF-1) and caspase-9 to form the “apoptosome,” which in turn cleaves and activates the downstream effectors caspase-3 and caspase-7. Caspases (cysteinyl aspartate–specific protease) are a family of cysteine proteases that function as the central effectors of apoptosis. These executioner caspases cleave multiple cellular substrates, resulting in the nuclear condensation, DNA fragmentation, and membrane blebbing that are characteristic of apoptosis. Other apoptogenic factors released from mitochondria, including apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspase (SMAC)/Diablo, Omi/HtrA2, and endonuclease G, augment intrinsic apoptosis. The extrinsic pathway is initiated upon ligation of the death receptors of the tumor necrosis factor (TNF) receptor family, which recruit the adapter molecule FAS-associated death domain protein (FADD) and then caspases-8 and -10, leading to activation of other downstream caspases. Conventional, nonconventional, and other mechanisms of apoptosis and other modes of cell death are described in detail below ( Table 18.1 ).
Table 18.1
Mechanisms of Apoptosis and Cell Death
| Conventional Mechanisms |
| Mitochondrial (intrinsic) pathway |
| Death receptor (extrinsic) pathway |
| Nonconventional Mechanisms |
| Necroptosis |
| Ferroptosis |
| Pyroptosis |
| Other Mechanisms |
| Autophagy |
| Unfolded protein response |
The Intrinsic Apoptotic Pathway
The intrinsic apoptotic pathway relies on the BCL-2 family of proteins, which include multiple proteins that share BCL-2-like homology domains 1-4 (BH1-BH4). The BCL-2 family proteins can be classified by their functions: suppressors (BCL2, BCL-XL, BCL-W, BCL2-A1, and myeloid cell leukemia [MCL-1]), activators (BCL2-interacting mediator of cell death [BIM] and p53 upregulated modulator of apoptosis [PUMA]), effectors (BCL2 associated X [BAX] and Bcl-2 homologous antagonist/killer [BAK]), and sensitizers (NOXA). Activators and effectors oligomerize, creating pores in the mitochondrial outer membrane. Subsequently, cytochrome C is released through the membrane and caspase-9 activated, ultimately leading to proteolytic cell death. Suppressors bind to and counteract activators and effector, while sensitizers bind directly to suppressors, which causes their disassociation from effector and activator proteins ( Fig. 18.1 ).
The proapoptotic proteins (including activators and effectors) share a homology only in their BH3 domain and have specific interaction with the five anti-apoptotic proteins (or suppressors) outlined above. By exposing mitochondria to known concentrations of BH3 peptides and measuring the resulting MOMP, it is possible to understand the specific antiapoptotic proteins on which a cell depends for its survival. This technique, called BH3 profiling, is considered the most accurate assessment of BCL-2 family member dependence.
Nonapoptotic Roles of Bcl-2 Family Proteins
Biochemical evidence indicates that select BCL-2 proteins interact with several non-BCL-2 protein partners that endow them with homeostatic roles beyond regulation of apoptosis. These functions include, but are not limited to, mitochondrial energy and nutrient metabolism, calcium signaling, cell cycle checkpoints, and DNA damage response. In addition, several BCL-2 family proteins have been implicated in the regulation of mitochondrial electron transport chain activity in healthy cells that have not been stressed with any apoptotic signals. For example, BCL-XL interacts with the voltage-dependent anion channel protein that mediates ADP/ATP exchange across the mitochondrial membrane and facilitates mitochondrial respiration. BCL2 was reported to be upregulated in quiescent leukemia stem cells (LSCs), where it maintains oxidative phosphorylation; in another report, the combination of BCL2 inhibitor venetoclax and the hypomethylating agent azacitidine disrupted the tricarboxylic acid cycle in LSCs by targeting glutathionylation of mitochondrial complex II of the electron transport chain. Proteolytic processing of MCL-1 produces an N-truncated MCL-1 isoform that is localized to the mitochondrial matrix, where it maintains normal inner mitochondrial membrane structure, regulates mitochondrial fusion, and supports mitochondrial bioenergetic functions. This MCL-1 isoform does not have antiapoptotic activity. Furthermore, this inner mitochondrial membrane form of MCL-1 interacts with very-long-chain acyl-CoA dehydrogenase, a key enzyme in the mitochondrial fatty acid oxidation pathway. Understanding the precise molecular mechanisms underlying these nonapoptotic functions is important for effective targeting of these proteins in disease settings.
The Death Receptor (Extrinsic) Pathway of Apoptosis
The death receptor or extrinsic apoptotic pathway is initiated upon activation of the TNF death receptors ( Fig. 18.2 ). TNF is the canonical member of the TNF superfamily; additional closely related family members include the TNF-related apoptosis-inducing ligand (TRAIL) and the CD95 ligand (CD95L, also known as FASLG and APO-1L). Upon ligation, signals from the death receptor CD95 (FAS) recruit the adapter FADD and, subsequently, the initiator caspase-8. This brings additional caspase-8 molecules that dimerize and are then activated and cleaved, inducing downstream cleavage of the executioners caspase-3 and caspase-7. This process can be inhibited by c-FLIP, which blocks the dimerization of caspase-8. The protein XIAP, an inhibitor of apoptosis, inhibits the executioner caspases. However, caspase-8 can also cleave the proapoptotic BH3-only protein BID which interferes with antiapoptotic BCL-2 family proteins and activates BAX and BAK, causing MOMP and release of Smac and Omi, which antagonize XIAP, facilitating execution of cell death.
Nonapoptotic Forms of Cell Death
Necroptosis
Necroptosis (necrosis-like cell death) is a form of caspase-independent programmed cell death that is important in inflammation and viral infection. Necroptosis can be initiated by death receptors, such as FAS and TNFR1, TLR3 and TLR4, which recognize pathogens (double-stranded RNA viruses or lipopolysaccharides, respectively), or by the intracellular nucleic acid sensor ZBP1. Necroptosis is dependent on the activation of RIPK1 and RIPK3; the latter phosphorylates and activates the effector molecule mixed-lineage kinase domain-like protein (MLKL). Phosphorylated MLKL oligomerizes and disrupts the plasma membrane through exposing phospholipids to trigger cell membrane permeabilization and cellular destruction. The cell then dies by necroptosis, a process characterized by necrosis-like features, which include cell swelling, membrane disruption, and the release of the intracellular contents. Death-receptor triggered necroptosis can only proceed when the caspase-8–c-FLIP complex is inhibited because caspase-8 can cleave RIPK1 and RIPK3, preventing necroptosis. Inhibition of caspases is observed during certain viral infections, in which case necroptosis proceeds as a dominant cell death pathway. Importantly, necroptotic cells release damage-associated molecular patterns (DAMPs) that can induce inflammatory responses. They activate caspase-1, which in turn cleaves proinflammatory interleukin (IL)-1β and IL-18 into mature inflammatory forms. Release of IL-1β is dependent on RIPK3 through caspase-8 or an MLKL-dependent NLRP3 inflammasome. Among the three NLR inflammasomes, NLRP1 and NLRC4 inflammasomes recognize bacterial muramyl dipeptide and flagellins, respectively, while NLRP3 recognizes multiple stimuli, including saturated fatty acids, bacterial RNA, and urate crystals. An inflammatory cell death termed pyroptosis (see below) is initiated by inflammasome activation. Recent findings suggest that dysregulated necroptosis during hematopoiesis promotes bone marrow progenitor cell death that induces inflammation, impairs hematopoietic stem cells, and recapitulates the features of the bone marrow failure disorder myelodysplastic syndrome (MDS). Genetic loss of Ripk1 in hematopoietic cells can be responsible for this bone marrow failure. In cancer, necroptosis may facilitate metastasis through damaging endothelial cells.
Ferroptosis
Ferroptosis is an iron-dependent form of regulated necrosis. In ferroptosis, lipid peroxidation occurs in the presence of free iron, which reacts with hydrogen peroxide produced by reactive oxygen species (ROS), a reaction that is further fueled by lipid oxidation in the presence of oxygen and the disruption of cellular membranes. The physiological role of ferroptosis in development and homeostasis remains to be elucidated. Ferroptotic cells exhibit a necrotic-like phenotype, including mitochondrial shrinkage, disrupted cristae, and a ruptured outer mitochondrial membrane, yet these features occur independently of caspase activation or necrosis. Ferroptosis is inhibited by the lipid peroxidase GPX4, which in turn requires glutathione and NADPH for recycling. Accordingly, ferroptosis can be observed under conditions of cysteine/cystine deprivation or inhibition of the cystine/glutamate antiporter Xc- (i.e., by sorafenib) because these interfere with glutathione synthesis. Reduction in NAPDH or inhibition of GPX4 (i.e., by the alkylator altretamine) can also induce ferroptosis. Ferroptosis was first discovered in cancer cells treated with the VDAC2/3 inhibitor erastin, which causes ferroptosis by promoting iron-dependent ROS accumulation. Ferroptosis can be blocked by depletion of free iron (i.e., iron chelation), inhibition of synthesis of polyunsaturated fatty acids, or ROS scavenging using lipophilic antioxidants such as ferrostatin-1. Identification of agents that can selectively induce ferroptosis is actively being pursued in cancer research.
Pyroptosis
Pyroptosis is a cell-intrinsic inflammatory form of regulated cell death in response to bacterial, viral, fungal, and protozoan infections. It occurs upon cleavage and oligomerization of the effector molecule gasdermin D (GSDMD), which executes pyroptosis via forming large pores in the membranes. Pyroptosis requires activation of caspases distinct from those involved in apoptosis, including caspase-1 and caspases-4 and -5 in humans and their rodent homolog caspase-11. Caspases-4, -5, and -11 are directly activated by cytosolic lipopolysaccharides from invading gram-negative bacteria and cleave GSDMD in monocytes and other cell types, triggering pyroptosis. In turn, activation of caspase-1 by pathogen-associated pattern recognition receptors like the NLRs and the cytosolic DNA sensor AIM2—or in some instances indirectly through an adapter protein, apoptosis-associated speck-like (ASC) protein—leads to formation of inflammasomes, caspase-1–mediated cleavage of GSDMD, and processing of the cytokines IL-1β and IL-18, which can be released through GSDMD pores. Additional members of the gasdermin family were recently identified as mediators of pyroptosis, one of which, GSDME (also known as DFNA5), is cleaved by caspase-3 that has been activated by TNF or chemotherapy drugs and could be responsible for normal tissue toxicity from chemotherapy. Further characterization of caspase-3 as an activator of GSDME/DFNA5 broadened the role of this type of cell death, which has been recently shown to augment mitochondrial apoptosis.
Cell Survival and Cell Death Pathways and Adaptive Responses to Stress
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