GMS 6644: Apoptosis. Introduction

Transcription

1 GMS 6644: Apoptosis Introduction (Feb. 15, 2006) Lei Xiao, Ph.D. Department of Anatomy & Cell Biology UF Shands Cancer Center ARB Rm R4-250, ,

2 Outline of the Lecture Different types of cell death Apoptosis versus Necrosis Other types of cell death Molecular mechanisms of apoptosis Apoptotic signaling Caspases Bcl-2 family proteins Physiological roles and significance of apoptosis Embryonic development Viral and microbial infections ER stress response Cancer

3 Two distinct modes of cell death Apoptosis 1. Energy-dependentorganized 2. Plasma membrane integrity maintained 3. Ordered DNA degradation 4. Immuno-suppressive 5. Cell elimination via phagocytosis 6. Cell: shrinking and nuclear condensed; PS exposure Necrosis 1. Bioenergetic catastrophedisordered 2. Plasma membrane integrity lost 3. Random DNA degradation 4. Immuno-stimulating 5. Cell demise; Initiation of cell growth and tissue repair 6. Cell: swell and burst

4 Apoptosis vs. Necrosis The fundamental feature that distinguishes most forms of necrosis from apoptosis is the rapid loss of cellular membrane potentials.

5 Apoptosis and necrosis may be inter-connected. Many insults induce apoptosis at lower doses and necrosis at higher doses. Apoptosis and necrosis may coexist in the same cell. Apoptosis-induced dysfunction of mitochondria may ultimately lead to cellular energy depletion, therefore necrosis. (delayed necrosis or slow cell death) In the absence of phagocytosis, dead cells in the late stages of apoptosis may present necrotic features. (apoptotic necrosis or secondary necrosis)

6 Cell death can take many forms Classic apoptosis: caspase-dependent Caspase-independent cell death Apoptosis-like PCD: induced by AIF Necrosis-like PCD: absence of chromatin condensation; induced by ROS and Ca ++ Autophagy: formation of large, lysosomederived cytosolic vacuoles Mitotic catastrophe: an intermediate phase of apoptosis and necrosis

7 Nuclear alternation in different forms of programmed cell death

8 Outline of the Lecture Different types of cell death Apoptosis versus Necrosis Other types of cell death Molecular mechanisms of apoptosis Apoptotic signaling Caspases Bcl-2 family proteins Physiological roles and significance of apoptosis Embryonic development Viral and microbial infections ER stress response Cancer

9 Induction Phase Effector/Commitment Phase Execution Phase

10 Apoptotic Pathways The extrinsic death-receptor pathway TNF receptor superfamily The intrinsic mitochondrial pathway Stress/DNA damage

11 Mitochondrial Death Pathways Leist & Jaattela (2001)

12 Function-based Caspase Subfamilies All known active caspases family members are cysteine proteases with an absolute specificity for Asp in the substrate P1 position.

13 Caspases: the central executioners All caspases are zymogens, composed of three domains. Activation of a procaspase by proteolytic cleavage yields a large and a small subunits. Two large and two small subunits form a tetrameric, active caspase.

14 Mechanisms of Caspase Activation Effector caspases are activated via proteolytic cleavage by an upstream caspase. (a) Initiator caspases are activated through regulated proteinprotein interaction. Induced proximity (b) (e.g. procaspase-8) Holoenzyme formation (c) (e.g. procaspase-9)

15 Caspase Activation Pathways

16 Inhibitors of Apoptosis Proteins (IAPs) Consisting of NAIP, XIAP, ciap1, ciap2, and survivn Suppress apoptosis by preventing procaspase activation and inhibiting the activity of mature caspases (caspase-3, -7, and -9) by directly binding to caspases. Expression of ciap1/2 is stimulated by NF-κBmediated survival signals. Negative regulators of IAPs: Smac/DIABLO, XAF1, and OMI/HTRA2

17 The Bcl-2 family of Proteins Pro- and anti-apoptotic members localize to separate subcellular compartments. Post-translational modifications determine active/inactive confirmations. Activation of pre-apoptotic members may involve translocation and/or oligomerization. Pro- and anti-apoptotic genes are transcriptional responsive.

18 Mitochondrial Integrity Model (Bcl-2 survival activity) Cory & Adams (2002) Members of the Bcl-2 family control permeability of the OMM. Baxand Bakprovoke the permeabilization of the OMM by forming channels or via interacting with the permeability transition pore (PTP), allowing efflux of apoptotic molecules.

19 Mechanisms of restraining pro-apoptotic proteins

20 Modulating mitochondria by Bcl-2 family proteins

21 Apoptotic and Survival Pathways Involving Bcl-2 Members Gross et al, Genes Dev. (1999)

22 Outline of the Lecture Different types of cell death Apoptosis versus Necrosis Other types of cell death Molecular mechanisms of apoptosis Apoptotic signaling Caspases Bcl-2 family proteins Physiological roles and significance of apoptosis Embryonic development Viral and microbial infections ER stress response Cancer

23 Apoptosis: essential for a health life A major form of cell death to remove excess, damaged or infected cells throughout life. A fundamental process in the development of an organism and in control of self-renewing tissues A balance of cell division (e.g. regulating cell numbers in the developing nervous system) A self-defense mechanism against viral and pathogen infection Loss of control of the apoptotic program contributes to many diseases Accumulation of unwanted cells through inefficient apoptosis (e.g. cancer) Cell loss as a result of excessive apoptosis (e.g. neurodegeneration, stroke and heart failure)

24 Interdigital cell death by apoptosis in the developing mouse paw Mouse paws are sculpted by cell death during embryonic development: they start out as spadelike structures, and the individual digits separate only as the cells between them die.

25 Impact of the disruption of apoptotic genes in the development of the nervous system Gene Lethality Apoptotic phenotype Neural phenotype Cellular phenotype Caspase 3 -/- 1-3 weeks Decreased Brain hyperplasia Increase cell numbers in cortex, retina cerebellum, etc. Caspase 8 -/- E12.5 No change in nervous system Neural tube defects Decreased neurogenic gene expression Apaf-1 -/- E16.5 Decreased Brain overgrowth Reduced cell death of progenitors Bax -/- Bak -/- Perinatal Decreased Brain hyperplasia Increase cell numbers in regions rich in stem cells Boya & de la Rosa: Birth Defects Res. (2005)

26 Significance of apoptosis in Developing Neural Cells Early neural cell death is a phase of physiological cell death that controls neural cell populations. Apoptosis affects the proliferation and neurogenesis in early neural development. Prevention of cell death results in expanded nervous tissue, frequently, embryonic lethality.

27 Deregulation of apoptosis leads to embryonic malformations Hernandez-Sanchez et al. EMBO J (2003)

28 Apoptosis: a natural defense mechanism against viral infection Protein CrmA/SPI-2 IAPs TNF Receptor Decoys Mitochondrial Inhibitors Gene VVWR-B13R CPXV-B12R AMV-021 MSV-242, -248 CrmB, C, D, E Myx-MT-2 M11L, F1L, FPV- Bcl-2 Function Inhibits caspases 1, 8 Caspase inhibitors Sequester TNF Inhibit cyto c release Taylor & Barry: Virology (2005)

29 ER Stress Response (ESR) ER function is essential for cell physiology Vesicle trafficking Lipid and membrane biogenesis Protein targeting and secretion ERS: three major pathways UPR: the unfolded protein pathway (transcription-dependent) ERAD: proteasome-dependent ER-associated degradation Control protein translation The ERS acts both to increase the capacity of the ER to fold and process client proteins, and to alleviate the burden on the organelles by reducing the amount of proteins inside the ER.

30 ER Stress induced apoptotic pathways Boyce & Yuan: Cell Death Differ. (2006)

31 Apoptotic pathway from the mitochondria and ER M. Germain et al., Sci. STKE, pe10 (2003)

32 Essential alterations in cell physiology for human cancer Self-sufficiency in growth signals (activation of Ras oncogene) Insensitivity to antigrowth signals (loss of the retinoblastoma suppressor, Rb) Evasion of apoptosis (activation of survival pathways) Limitless replicative potential immortalization (telomere maintenance) The capacity to initiate and sustain angiogenesis (induction of VEGF) Tissue invasion and metastasis (inactivation of E-cadherin) Hanahan & Weinberg: The hallmarks of cancer (Cell 100:57, 2000)

33 Damages/Stress p53 Cell cycle arrest Apoptosis DNA Repair Angiogenesis inhibition Tumor Inhibition Chemo- and radio-sensitivity Loss of p53 pathway function can contribute not only to aggressive tumor behavior but also to therapeutic resistance.

34 p53-mediated Apoptosis Models of p53 action: Transcriptional upregulation of proapoptotic genes Pro-apoptotic Bcl-2 members Death receptors (e.g. CD95 & DR5) Transcriptionindependent activation of Bax (BH3-like activity), initiating cyto c release. Bratton & Cohen (2001)

35 Dysregulation of the Intrinsic Apoptotic Pathway in Cancer Cells Upstream from the mitochondria Mutations on those targeting upstream components of the apoptotic program (p53, PTEN, Akt, Ras) At the Mitochondria Bcl-2 family members (pro- and anti-apoptotic) Downstream from the mitochondria Inhibitors of apoptosis proteins (IAPs) and heat shock proteins (Hsp70/90) Epigenetic silencing of Apaf-1, caspase-3 deletion, etc. Caspase-independent mechanisms AIF (apoptosis inducing factor)

36 The mitochondrial pathway plays the central role in chemotherapy-induced apoptosis Chemotherapeutic agents induce mitochondrial membrane disruption and mitochondrial release of cytochrome c that is inhabitable by Bcl-2 and BclxL. Apaf-1 overexpression sensitizes cancer cells to chemotherapeutic agents, accompanied with increased caspase-9 and -3 activation. Cells deficient in Apaf-1 or caspase-9 are protected from apoptosis induced by anticancer drugs, whereas cells deficient in caspase-8 and -2 show no protecting effect against anticancer drugs.

37 Hanahan and Weinberg (Cell 100:57)