Apoptosis and Autophagy PDF

MDC 811 Dr. Green Apoptosis and Autophagy The goal of this lecture is to describe the process of apoptosis and its role in disease. By the end of this lecture, the student should be able to: 1) Describe the roles of the Bcl-2 family proteins in apoptosis....

MDC 811 Dr. Green Apoptosis and Autophagy The goal of this lecture is to describe the process of apoptosis and its role in disease. By the end of this lecture, the student should be able to: 1) Describe the roles of the Bcl-2 family proteins in apoptosis. 2) Describe the components of the intrinsic pathway of apoptosis and how they cause cell death. 3) Describe the components of the extrinsic pathway of apoptosis and how they cause cell death. 4) Describe how apoptosis is regulated. 5) Describe the function of lysosomes in autophagy. 6) Describe the types of autophagy. Glossary Apaf-1: apoptotic protease-activating factor; complexes with cytochrome c and ATP to activate caspase-9 apoptosis: programmed cell death apoptosome: Apaf-1/ATP/caspase-9/cytochrome c complex autophagosome: vesicle that delivers cytosolic material to lysosome in macroautophagy autophagy: mechanism to degrade unnecessary or dysfunctional cellular organelles and components and recycle the by-products Bcl-2: family of genes involved in apoptosis; some inhibit apoptosis and others promote apoptosis blebbing: formation of irregular bulges in the plasma membrane of a cell during apoptosis death domain: amino acid sequence present on C-terminus of both Fas and FADD DED: death effector domain; amino acid sequence present on N-terminus of both FADD and pro-caspase-8 FADD: Fas-associated death domain protein; complexes to Fas and pro-caspase-8 when Fas is stimulated Fas: cell surface receptor in immune cells that binds Fas ligand leading to apoptosis FLIP: negative regulator of apoptosis IAP: inhibitor of apoptosis protein TRAIL: TNF-related apoptosis-inducing ligand 1 I. What occurs during apoptosis? A. Apoptosis irreversible once started B. Cell breaks down from inside 2 C. Chromatin and DNA 1. chromosomal DNA fragments due to cleavage between nucleosomes 2. chromatin condenses and nucleus breaks up into small pieces Gel electrophoresis of DNA from apoptotic cells DNA degrades to fragments corresponding to multiples of 200 base pairs D. Cytoskeleton breaks up E. Cell shrinks and breaks up into membrane-enclosed fragments (blebbing) 3 F. Apoptotic cell recognized and phagocytosed by macrophages and neighboring cells 1. one signal phosphatidylserine a. normally expressed on inner leaflet of plasma membrane b. during apoptosis expressed on outer leaflet Phagocytosis of apoptotic cells 4 Events in apoptosis 5 II. What are caspases and what is their role in apoptosis? A. Function 1. normally present as inactive precursors, which are activated by cleavage by other caspases 2. cleave proteins at only a few residues 3. initiators and executioners a. initiator caspases activate executioner caspases, which can then activate other executioner caspases 4. cleave many different cell proteins a. inhibitor of DNase that cleaves genomic DNA between nucleosomes b. nuclear lamins c. cytoskeletal proteins d. inhibitors of apoptosis (discussed later) 1) Bcl-2 2) Bcl-xL 3) FLIP 4) IAP 5) XIAP 5. can both inactivate and activate proteins during apoptosis Caspase targets 6 III. How does apoptosis occur in vertebrates? A. Two main pathways 1. Intrinsic/Stress 2. Extrinsic/Death receptors B. Mitochondria source and amplifier of apoptotic signals induced by stress C. Bcl-2 family of proteins determine if apoptosis occurs 1. both inhibitors and activators of apoptosis The Bcl-2 family 2. antiapoptotic proteins a. inhibit apoptosis by sequestering pro-apoptotic proteins 1) Bcl-2 2) Bcl-xL 3. proapoptotic proteins a. block Bcl-2 and Bcl-xL interaction with pro-apoptotic proteins 1) Bad 2) Bid b. translocate to mitochondria, where they insert in membrane and disrupt its integrity, allowing cytochrome c release 1) Bak 2) Bax 4. family members can either heterodimerize or homodimerize 5. current model of interactions of Bcl-2 proteins a. one group of pro-apoptotic proteins (Bid, Bad) inactivates anti-apoptotic Bcl-2- like proteins b. this allows other pro-apoptotic proteins (Bax, Bak) to activate apoptosis 7  Model for interactions of Bcl-2 proteins Regulatory interactions between Bcl-2 family members 8 D. Mitochondrial/Intrinsic pathway 1. predominantly release of proteins that trigger activation of apoptosis, but could also involve disruption of electron transport or production of reactive oxygen species 2. if damage to DNA or mitochondria occurs, Apaf-1 and cytochrome c from mitochondria complex with ATP in cytoplasm 3. this multi subunit complex called apoptosome 4. caspase-9 binds and activated by autocleavage 5. downstream caspases activated The mitochondrial pathway of apoptosis 9 E. Death receptor/Extrinsic pathway 1. in immune system, Fas receptor controls apoptosis 2. ligand for Fas, FasL, is trimer of identical polypeptides F. Pathway 1. membrane-bound FasL on surface of one cell binds to Fas on surface of another cell 2. this induces trimerization of Fas 3. trimerized Fas binds to adaptor, FADD, via death domains (DDs) at C-termini of both proteins 4. FADD has death effector domain (DED) at its N-terminus, which binds to inactive caspase-8 via DEDs at N-termini 5. this results in autocleavage of caspase-8 to remove N-terminal DED 6. activated caspase-8 translocates from cytoplasmic tail of Fas-FADD complex to cytoplasm 7. downstream caspases activated Death receptor pathway 10  Death receptor complex (CD95L = Fas ligand; CD95/Apo1 = Fas) FADD = Fas-associated death domain protein (DD = death domain; DED = death effector domain) These are two good videos I can recommend that describe the apoptotic pathways. They focus on the death receptor pathway but the first includes the mitochondrial pathway. http://www.dnatube.com/video/1188/Apoptosis-animation http://www.dnatube.com/video/2463/Apoptosis-animation 11 G. TRAIL pathway (TNF-related apoptosis-inducing ligand) 1. similar to Fas/FasL system 2. TRAIL expressed on immune cells 3. TRAIL binds to DR 4/5 receptor on surface of tumor cell 4. specific for tumor cells 5. selectively induces cancer cell apoptosis without causing normal cell toxicity FADD = Fas-associated death domain protein (DD = death domain; DED = death effector domain) H. interaction of extrinsic and intrinsic pathways 1. in some cells, caspase-8 also cleaves Bid, which is member of Bcl-2 family a. Bid normally inactive in cytosol, but when cleaved, translocates to mitochondria and disrupts membrane b. this releases cytochrome c, which leads to activation of caspase-9, amplifying caspase cascade 12 13  Summary of apoptosis pathways (From Igney and Krammer, Nature Reviews Cancer, 2:277-288, 2002. Figure 1) 14 IV. How is apoptosis regulated? A. Positive feedback and amplification loops 1. caspases activate downstream caspases 2. cleave anti-apoptotic proteins B. Buffers/dampeners 1. Bcl-2 family of anti-apoptotic proteins 2. inhibitors-of-apoptosis proteins (IAPs) a. bind to procaspases to prevent their activation b. bind to caspases to inhibit their activity c. these in turn inhibited by proteins such as Smac/DIABLO, Reaper, Hid, Grim Regulators of apoptosis 15  Regulation of caspases by IAPs 3. FLIP a. similar in structure to caspase-8, but lack catalytic residues b. competes with caspase-8 for binding to FADD c. inhibits aopotosis 16 4. Decoy receptors a. bind to apoptosis-inducing ligand, but cannot transduce apoptotic signal Decoy receptors 17 5. p53 a. tumor suppressor protein b. mediates cell cycle arrest and apoptosis c. DNA damage activates ATM and Chk2 protein kinases d. these kinases phosphorylate and stabilize p53 e. p53 initiates transcription of pro-apoptotic genes PUMA and Noxa Role of p53 in DNA damage-induced apoptosis 18 V. Apoptosis and disease A. Excessive/inappropriate apoptosis 1. neurodegenerative diseases 2. immune deficiency diseases 3. cardiovascular disease 4. emphysema 5. AIDS B. Too little apoptosis 1. cancer 2. autoimmune diseases C. Apoptosis and cancer 1. ability to escape apoptosis characteristic of most cancer cells a. IAPs expression increased b. anti-apoptotic Bcl-2 protein levels increased 2. reactivating apoptosis target of cancer treatment strategies a. BH3 mimetics b. XIAP antagonists c. FasL mimetics and Fas activators 19  Apoptosis-based therapies Top figure - stimulating intrinsic pathway with BH3 analog and blocking XIAP with SMAC analog Bottom figure - cancer cell on left; blocking XIAP with antagonist on right 20 VI. Autophagy A. Definition 1. regulated process by which cytosolic components and organelles are delivered to lysosome, degraded, and recycled 2. usually doesn’t kill cell but cell can die if process deregulated B. Inducers 1. environmental cues a. starvation b. high temperature c. low oxygen d. hormonal stimulation 2. intracellular stress a. damaged organelles b. accumulation of misfolded or aggregated proteins c. microbial and viral infection C. Lysosome 1. degrades cytoplasmic material 2. contains enzymes that digest nucleic acids, proteins, carbohydrates, and lipids 3. constituent macromolecules released back into cytosol to be reused D. Types 1. microautophagy a. cytosolic components taken up directly by lysosome b. lysosomal membrane invaginates 2. macroautophagy a. cytosolic components delivered to lysosome by vesicle called autophagosome b. fuses with lysosome to form autolysosome 3. chaperone-mediated autophagy a. targeted proteins translocated across lysosomal membrane complexed with chaperone protein b. proteins have specific amino acid sequence recognized by chaperone c. complex recognized by protein receptor (LAMP-2A) in lysosomal membrane 21 E. Regulation 1. macroautophagy regulated at autophagosome formation 2. chaperone-mediated autophagy mediated by levels of LAMP-2A protein in lysosomal membrane F. Autophagy in disease 1. neurodegenerative diseases a. Parkinson’s b. Alzheimer’s c. Huntington’s d. characterized by accumulation of aberrant or misfolded proteins that are not removed by autophagy 2. cancer a. depends on level of autophagy b. autophagy can help tumors survive stresses such as hypoxia and nutrient deprivation c. some cancer drugs promote lethal autophagy 22  Review questions 1. You are working on a drug that will inhibit the extrinsic pathway of apoptosis as a therapy for immune deficiency. Which of the following proteins is the best target? A. Apaf-1 B. Bax C. Caspase 9 D. FADD 2. After a heart attack, active caspase-9 levels are increased in damaged cardiac cells. Caspase-9 has to bind to which of the following to be activated? A. Apoptosome B. Bid C. Caspase-8 D. FADD E. Fas 23  Answers 1. The correct answer is D. The other proteins are components of the mitochondrial/intrinsic pathway. 2. The correct answer is A. The intrinsic/mitochondrial activates caspase-9 through the formation of apoptosomes. FADD and Fas are in the death receptor/extrinsic pathway, which activates caspase-8. Caspase-8 cleaves Bid, which can activate caspase-9. 24