Molecular Mechanisms of Programmed Cell Death Xuejun Jiang

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Molecular Mechanisms of Programmed Cell Death Xuejun Jiang, MSKCC • Programmed cell death and

Molecular Mechanisms of Programmed Cell Death Xuejun Jiang, MSKCC • Programmed cell death and autophagy • Regulation of the tumor suppressor PTEN • Mechanism-based cancer therapy

Programmed Cell Death

Programmed Cell Death

What shall we discuss? • An overview of the field • In-depth discussion of

What shall we discuss? • An overview of the field • In-depth discussion of certain topics • Methods for measuring apoptosis • Cell death and cancer biology

Cancer, a genetic disease/diseases

Cancer, a genetic disease/diseases

Chromosomal Translocations in Follicular Lymphoma Janet Rowley found 18: 14 translocation in 1970 s

Chromosomal Translocations in Follicular Lymphoma Janet Rowley found 18: 14 translocation in 1970 s Normal Chromosomes 18 Follicular Lymphoma 14 ? Follicular lymphoma

Stanley Korsmeyer Normal Chromosomes 18 Follicular Lymphoma 14 Ig Gene Ig Promoter Overexpression of

Stanley Korsmeyer Normal Chromosomes 18 Follicular Lymphoma 14 Ig Gene Ig Promoter Overexpression of Gene X driven by strong Ig enhancer Gene X 1984: Stan Korsmeyer et al and Carlo Croce et al

Stanley Korsmeyer IL-3 Normal B-cells FL B-cells IL-3 David Vaux/Susan Cory et al (1988)

Stanley Korsmeyer IL-3 Normal B-cells FL B-cells IL-3 David Vaux/Susan Cory et al (1988) and Korsmeyer et al (1989) discovered the pro-survival function of Bcl-2. A new class of oncogenes Proper cell death is crucial for life!

Cell Death • Programmed cell death • Physiological cellular suicide • Apoptosis

Cell Death • Programmed cell death • Physiological cellular suicide • Apoptosis

Why Programmed Cell Death? Genomic integrity --- Cancer Immune function --- Immune disorders Roles

Why Programmed Cell Death? Genomic integrity --- Cancer Immune function --- Immune disorders Roles in development

Kerr, J. F. , Wyllie, A. H. , and Currie, A. R. (1972). Apoptosis:

Kerr, J. F. , Wyllie, A. H. , and Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239 -257

Morphological Markers of Programmed Cell Death v v v Cell shrinkage Membrane blebbing Nuclear

Morphological Markers of Programmed Cell Death v v v Cell shrinkage Membrane blebbing Nuclear membrane breakdown Chromatin condensation Formation of dead bodies Phagocytosis

Kerr, J. F. , Wyllie, A. H. , and Currie, A. R. (1972). Apoptosis:

Kerr, J. F. , Wyllie, A. H. , and Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239 -257

Sydney Brenner Lower organism development C. elegans Hermaphrodite Cell lineage study Bob Horvitz, John

Sydney Brenner Lower organism development C. elegans Hermaphrodite Cell lineage study Bob Horvitz, John Sulston

Bob Horvitz, 1990 s Death of 131 specific neuronal cells out of 1090 cells

Bob Horvitz, 1990 s Death of 131 specific neuronal cells out of 1090 cells in defined developmental stages Egl-1 Ced-9 Ced-4 Ced-3 Bcl-2

The 2002 Nobel Price Sydney Brenner, H. Robert Horvitz and John E. Sulston for

The 2002 Nobel Price Sydney Brenner, H. Robert Horvitz and John E. Sulston for their discoveries concerning "genetic regulation of organ development and programmed cell death"

Early stage: morphological observations, hypothesis raised Medicine: pathology and molecular basis of diseases (Bcl-2)

Early stage: morphological observations, hypothesis raised Medicine: pathology and molecular basis of diseases (Bcl-2) Basic research: the fundamental mechanisms (C. elegans genetics)

When it’s too low When it’s too high Cancers Neural degenerative diseases Alzheimer’s Parkinson’s

When it’s too low When it’s too high Cancers Neural degenerative diseases Alzheimer’s Parkinson’s ALS Autoimmune diseases

Apoptosis, A Conserved Pathway Apoptosis of 131 neuronal cells out of 1090 cells Egl-1

Apoptosis, A Conserved Pathway Apoptosis of 131 neuronal cells out of 1090 cells Egl-1 Ced-9 (Bcl-2) Ced-4 Ced-3 (Caspases)

Caspases, a Family of Cysteine Proteases N. A. Thornberry, Y. Lazebnik. Science 281: 1312

Caspases, a Family of Cysteine Proteases N. A. Thornberry, Y. Lazebnik. Science 281: 1312 -6

Bao Q, Shi Y, Cell Death and Differentiation 2006

Bao Q, Shi Y, Cell Death and Differentiation 2006

How to study apoptosis in mammals? Strategically … triggers Caspase activation What happens after

How to study apoptosis in mammals? Strategically … triggers Caspase activation What happens after the trigger? What are the caspase substrates? What are the binding partners of known components? Cloning based on sequence homology Confirmation/mechanism (molecular biology, pharmacology…)

Xiaodong Wang Biochemical mechanisms of apoptosis in mammalian cells

Xiaodong Wang Biochemical mechanisms of apoptosis in mammalian cells

Induction of Caspase-3 Activation by d. ATP Procaspase-3 Caspase-3 Liu et al. Cell 86:

Induction of Caspase-3 Activation by d. ATP Procaspase-3 Caspase-3 Liu et al. Cell 86: 147 -157

Cytochrome c-Mediated Caspase Activation He. La S 100 PC-FT PC-B Cytochrome c Apaf-1 Procaspase-9

Cytochrome c-Mediated Caspase Activation He. La S 100 PC-FT PC-B Cytochrome c Apaf-1 Procaspase-9

A Conserved Apoptotic Pathway from Worms to Mammals Worms: Egl-1 Ced-9 Ced-4 Ced-3 BH

A Conserved Apoptotic Pathway from Worms to Mammals Worms: Egl-1 Ced-9 Ced-4 Ced-3 BH 3 Bcl-2 Apaf-1 Caspase-9 Mammals: Cytochrome C

The Apoptotic Role of the Apoptosome in Brain Development Nijhawan et al. Annu. Rev.

The Apoptotic Role of the Apoptosome in Brain Development Nijhawan et al. Annu. Rev. Neurosci. 23: 73 -87. Courtesy of Razqallah Hakem and Tak Mak. Cytochrome c knock-in Tak Mak and colleagues, Cell 121: 579

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion d. ATP/ATP Procaspase-3, 7 Procaspase-9 Apoptosome

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion d. ATP/ATP Procaspase-3, 7 Procaspase-9 Apoptosome Active caspase-3, 7

Scorrano L, Korsmeyer SJ. Biochem Biophys Res Commun. 2003 May 9; 304(3): 437 -44

Scorrano L, Korsmeyer SJ. Biochem Biophys Res Commun. 2003 May 9; 304(3): 437 -44

The Binding Pocket of Bcl-XL Bound to the Bak BH 3 Peptide Sattler et

The Binding Pocket of Bcl-XL Bound to the Bak BH 3 Peptide Sattler et al. , Science, 275, 983

Regulation by the Bcl-2 Family Opferman JT, Korsmeyer SJ. Nat Immunol. 2003 May; 4(5):

Regulation by the Bcl-2 Family Opferman JT, Korsmeyer SJ. Nat Immunol. 2003 May; 4(5): 410 -5.

The Active Apoptosome-caspase-9 Holoenzyme Apaf-1 BSA Procaspase-9 (D 315 A) Cytochrome C * 1.

The Active Apoptosome-caspase-9 Holoenzyme Apaf-1 BSA Procaspase-9 (D 315 A) Cytochrome C * 1. 5 m. M Apaf-1, 3 m. M PC 9, 5 m. M Cyt. C, 1 m. M d. ATP, 0. 1 mg/ml BSA, 30 o. C 1 hr, then Superdex 200

Model of Apoptosome Assembling Acehan et al, Mol. Cell 2002, 9: 423

Model of Apoptosome Assembling Acehan et al, Mol. Cell 2002, 9: 423

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion d. ATP/ATP Procaspase-3, 7 Procaspase-9 Apoptosome

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion d. ATP/ATP Procaspase-3, 7 Procaspase-9 Apoptosome Active caspase-3, 7

Endogenous Caspase Inhibitors --- IAP (Inhibitor of Apoptosis) Verhagen et al, Genome Biology, 2:

Endogenous Caspase Inhibitors --- IAP (Inhibitor of Apoptosis) Verhagen et al, Genome Biology, 2: reviews 3009

Verhagen et al, Genome Biology, 2: reviews 3009

Verhagen et al, Genome Biology, 2: reviews 3009

SMAC/Diablo, the Second Mitochondria-Derived Activator of Caspases, Functions to Antagonize IAP Du et al,

SMAC/Diablo, the Second Mitochondria-Derived Activator of Caspases, Functions to Antagonize IAP Du et al, Cell 2000, 102: 33

Smac Interacts with the XIAP-BIR 3 Through the First Four Amino Acid Residues, AVPI

Smac Interacts with the XIAP-BIR 3 Through the First Four Amino Acid Residues, AVPI Yigong Shi

Interaction of IAPs with IAP Antagonists Alanine as the N-terminal residue, how so? Shi

Interaction of IAPs with IAP Antagonists Alanine as the N-terminal residue, how so? Shi Y, NCB 2001, 8: 394

A Conserved Apoptotic Pathway from Worms to Mammals ? Modified from Shi Y, NCB

A Conserved Apoptotic Pathway from Worms to Mammals ? Modified from Shi Y, NCB 2001, 8: 394

Regulation of Apaf-1 -Mediated Caspase Activation in Different Organisms Worms: suppression and de-suppression of

Regulation of Apaf-1 -Mediated Caspase Activation in Different Organisms Worms: suppression and de-suppression of Ced-4 (Ced-9 directly inhibits Ced-4, and Egl-1 can release the inhibition; also have IAP-like proteins, role unclear) Flies: suppression and de-suppression of caspases (DApaf-1 is thought to be constitutively active) (Bcl-2 function and mechanism unclear) (IAPs as the brake, released by Reaper/Grim/Hid) Mammals: additional mechanism for Apaf-1 activation (with almost all other regulatory mechanisms; but no direct inhibitor of Apaf-1, as Ced-9 for Ced-4, identified yet)

Ced-4 and Its Homologs Nucleotide exchange/binding versus nucleotide hydrolysis

Ced-4 and Its Homologs Nucleotide exchange/binding versus nucleotide hydrolysis

A Novel Apoptosome Regulatory Pathway PETCM Pro. T PHAP CAS HSP 70 Apoptosome Jiang

A Novel Apoptosome Regulatory Pathway PETCM Pro. T PHAP CAS HSP 70 Apoptosome Jiang et al, 2003, Science 299: 223 Kim et al, 2008, Molecular Cell 30: 239 Pan et al, 2009, JBC 284: 6946

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Endo G

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Endo G & AIF Nuclear DNA degradation Procaspase-3, 7 Procaspase-9 Apoptosome Active caspase-3, 7

What is the point of no-return of the mitochondrial pathway? After cytochrome c release,

What is the point of no-return of the mitochondrial pathway? After cytochrome c release, will cells die even without caspase activation? Do regulatory events after cytochrome c release and before caspase activation really matter? All-or-none release model (MOMP vs. i. MOMP) Death after cytochrome c release but in the absence of caspase activation Mouse knockout data Mechanisms underlying apoptotic resistence of specific tissues Second hit model Non-death functions of the mitochondrial caspase activation pathway

Death Receptor-Mediated Caspase Activation DISC

Death Receptor-Mediated Caspase Activation DISC

Crosstalk between the receptor pathway and the intrinsic pathway 1. Type I cells vs

Crosstalk between the receptor pathway and the intrinsic pathway 1. Type I cells vs Type II cells (the crucial role of Bid in initiation and amplification of cytochrome c release) 2. Smac and c. IAP 1/2 (physiological relevance? ) 3. Determine which pathway is responsible for a death process (amplification; specificity of caspases, caspase substrates, and chemical inhibitors)

Life after Death What events follow caspase activation?

Life after Death What events follow caspase activation?

What is needed for proper phagocytosis? • “Eat-me” signal and its receptor • “Find-me”

What is needed for proper phagocytosis? • “Eat-me” signal and its receptor • “Find-me” signal and its receptor • The mechanic force/molecules for engulfment What happens to the corpse after engulfment? The biological functions of apoptosis-coupled phagocytosis • Immune • Organ development (heart), etc…. The function of DNA fragmentation (Nagata) (CAD/DFF 40 and DNase II)

How to measure apoptosis reliably and quantitatively? • Caspase activity (enzymatically) • Other specific

How to measure apoptosis reliably and quantitatively? • Caspase activity (enzymatically) • Other specific morphological and molecular events

Features of Apoptosis versus Necrosis APOPTOSIS NECROSIS • Chromatin condensation • Nuclear Swelling •

Features of Apoptosis versus Necrosis APOPTOSIS NECROSIS • Chromatin condensation • Nuclear Swelling • Cell shrinkage • Cell Swelling • Preservation of cell membranes • Disruption of organelles • Rapid engulfment of apoptotic bodies by neighboring cells to prevent inflammation • Rupture of cells and release of cellular contents • Inflammatory response

Detection of Apoptosis --- Chromosomal DNA Fragmentation Two types of DNA fragmentation

Detection of Apoptosis --- Chromosomal DNA Fragmentation Two types of DNA fragmentation

Detection of Apoptosis --- Chromosomal DNA Fragmentation and Condensation Sub-G 1/PI staining The low

Detection of Apoptosis --- Chromosomal DNA Fragmentation and Condensation Sub-G 1/PI staining The low molecular weight fragments are readily extracted from ethanol-fixed cells by treatment with aqueous buffers, DNA-content in apoptotic cells is low compared to normal cells after staining with DNA-binding dyes such as propidium iodide (PI) and detected by flow cytometry. Morphological detection: Hoechst 33342 dye staining for condensed chromatin

Detection of Apoptosis --- TUNEL Staining Terminal Deoxynucleotidyl Transferase (Td. T)-Mediated d. UTP Nick

Detection of Apoptosis --- TUNEL Staining Terminal Deoxynucleotidyl Transferase (Td. T)-Mediated d. UTP Nick End-Labeling Analysis (TUNEL). Enzymatic labeling of DNA double strand breaks (blunt, or with overhang) induced by apoptotic stimuli.

Detection of Apoptosis --- Annexin V labeling of externalized PS Early detection of apoptosis;

Detection of Apoptosis --- Annexin V labeling of externalized PS Early detection of apoptosis; PS, phosphatidylserine, is normally confined to the inner leaflet of the plasma membrane. During apoptosis, PS translocates to the cell surface. This externalization of PS marks the apoptotic cells for phagocytosis and removal. Once on the cell surface, PS can be labeled by binding of fluorescent-labeled annexin V, followed by flow cytometry detection or fluorescent microscope observation. Annexin V: a 35 -36 k. Da, Ca 2+-dependent, phospholipid binding protein with a high affinity for PS.

Detection of Apoptosis --- Annexin V labeling of externalized PS PI Annexin V Dose-Dependent

Detection of Apoptosis --- Annexin V labeling of externalized PS PI Annexin V Dose-Dependent Induction of Apoptosis in Lung Cancer PC-9 Cells by EGFR Inhibitor Tarceva (Gong et al, 2007 PLo. S Medicine 4: 1655)

Detection of Apoptosis • Caspase activity of cell extracts • IF or IHC of

Detection of Apoptosis • Caspase activity of cell extracts • IF or IHC of activated caspase-3 *** • Cytochrome c release (Western or IF) • Mitochondrial membrane potential • • •

TRAIL-Induced Apoptosis in MCF 10 A Cells as Measured by Caspase Activation Caspase-3 activity

TRAIL-Induced Apoptosis in MCF 10 A Cells as Measured by Caspase Activation Caspase-3 activity (RFU) Time (min)

TRAIL-Induce Apoptosis in MCF 10 A Cells DIC m. Cherry-Smac

TRAIL-Induce Apoptosis in MCF 10 A Cells DIC m. Cherry-Smac

Cell Death and Cancer Therapeutically, cytostasis vs cytotoxicity

Cell Death and Cancer Therapeutically, cytostasis vs cytotoxicity

Conventional Therapies Chemotherapies (genotoxic) Radiation therapies p 53 The mitochondrial apoptotic pathway

Conventional Therapies Chemotherapies (genotoxic) Radiation therapies p 53 The mitochondrial apoptotic pathway

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Endo G

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Endo G & AIF Nuclear DNA degradation Procaspase-3, 7 Procaspase-9 Apoptosome Active caspase-3, 7

Oncogene-Induced Apoptosis c-myc (Gerald Evan) Ras … How and why?

Oncogene-Induced Apoptosis c-myc (Gerald Evan) Ras … How and why?

Tumor Initiation versus Tumor Maintenance • Cancer: a multistep process in which tumor cells

Tumor Initiation versus Tumor Maintenance • Cancer: a multistep process in which tumor cells acquire activating mutations in proto-oncogenes and inactivating mutations in tumor suppressor genes • Is a mutation needed for tumor initiation or progression also required for tumor maintenance? • ‘Oncogene addiction’ has therapeutic implications – BCR-ABL – CML – C-KIT/PDGFRa – GIST – PDGFRa – HES All targeted by imatinib (Gleevec)

Oncogene Addiction The mechanisms of oncogene addiction are unknown. Some hypothesis have been proposed:

Oncogene Addiction The mechanisms of oncogene addiction are unknown. Some hypothesis have been proposed: “Life-in balance”: in order to maintain homeostasis, the proliferation-enhancing effects of a specific oncogene in cancer cells might be partially buffered through negative feedback mechanisms, through increased expression of proliferation-inhibitory factors. If this oncogene is inactivated, the cancer cells might suffer a relative excess of the latter inhibitory factors and undergo apoptosis. “Differential decay”: the apoptotic response observed in tumor cells upon acute disruption of an oncogene product results from differential decay rates of the various multiple pro-survival and pro-apoptotic signals emanating from the oncoprotein following its inactivation. The downstream pro-survival signals are relatively short-lived following oncogene inactivation when compared with the longer-lived apoptotic signals, which persist for a sufficient period of time to drive the tumor cell down an irreversible pathway of apoptotic death. Sharma et al, Cancer Cell 2006, 10: 425

Clinically Effective Signal Transduction Inhibitors Disease Oncogene Target Drug CML BCR-ABL Imatinib/Dasatinib GIST Mutant

Clinically Effective Signal Transduction Inhibitors Disease Oncogene Target Drug CML BCR-ABL Imatinib/Dasatinib GIST Mutant CKIT/PDGFRa Imatinib/Sunitinib HES FIP 1 L 1 -PDGFRa Imatinib NSCLC Mutant EGFR Gefitinib/Erlotinib RCC AML Multiple kinases – mutant? Sorafenib/Sunitinib FLT 3 -ITD PKC 412

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Endo G

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Endo G & AIF Nuclear DNA degradation Procaspase-3, 7 Procaspase-9 Apoptosome Active caspase-3, 7

Smac Interacts with the XIAP-BIR 3 Through the First Four Amino Acid Residues, AVPI

Smac Interacts with the XIAP-BIR 3 Through the First Four Amino Acid Residues, AVPI

1996 -1998: Discovery of the cytochrome c-mediated caspase activation pathway (X. Wang) 1995 -1998

1996 -1998: Discovery of the cytochrome c-mediated caspase activation pathway (X. Wang) 1995 -1998 s: Discovery of IAP proteins as caspase inhibitors (Miller, Vaux, Reed, Goeddel, et al). 2000: Identification of SMAC/DIABLO as IAP antagonist (X. Wang; D. Vaux) 2000: Structure of SMAC and SMAC-IAP complex (Y. Shi, Fesik) 2004: X. Wang et al. , a cancer drug lead that mimics AVPI peptide (N-terminus of SMAC)

SMAC AVPI Peptide Mimic Synthesis Li et al, Science 2004, 305: 1471

SMAC AVPI Peptide Mimic Synthesis Li et al, Science 2004, 305: 1471

The Binding Pocket of Bcl-x. L Bound to the Bak BH 3 Peptide Sattler

The Binding Pocket of Bcl-x. L Bound to the Bak BH 3 Peptide Sattler et al. , Science, 275, 983

1970 s: Janet Rowley found 18: 14 translocation 1984: Carlo Croce and Stan Korsmeyer

1970 s: Janet Rowley found 18: 14 translocation 1984: Carlo Croce and Stan Korsmeyer cloned Bcl-2 David Vaux (1988) and Korsmeyer (1989) discovered the pro-survival function of Bcl-2. 1990 s: Cloning of multiple Bcl-2 family members, both pro- and anti-death; interaction between members (Korsmeyer, Thompson, et al) 1990 s: Multiple structural studies (Fesik et al) 1997: Bcl-2 can inhibit cytochrome c release from mitochondria (Xiaodong Wang; Newmeyer/Green) 1990 s-2000 s: Differential regulation of individual Bcl-2 members (Korsmeyer, Thompson, Wang …) 2004: Korsmeyer et al. , a cancer drug lead mimicking the BH 3 motif of Bid

Mechanism-based drug design Functionality Stability Cell-permeability Pharmacokinetics Side effects Molecular targeting vs cell targeting

Mechanism-based drug design Functionality Stability Cell-permeability Pharmacokinetics Side effects Molecular targeting vs cell targeting

Cyto c/d. ATP 7 Apaf-1 (Apaf-1)7 Apaf-1 monomer Apoptosome [Apoptosome] ~ [Apaf-1]7 Caspase-3 Activity

Cyto c/d. ATP 7 Apaf-1 (Apaf-1)7 Apaf-1 monomer Apoptosome [Apoptosome] ~ [Apaf-1]7 Caspase-3 Activity (RFU) 6000 0 n. M 2 n. M 5000 4000 3000 2000 1000 0 0 10 20 30 Time (min) 40 50 60

Apoptosis --- Mechanisms • Two families of key players --- the executioners: caspases; and

Apoptosis --- Mechanisms • Two families of key players --- the executioners: caspases; and the upstream regulators: the Bcl-2 family proteins • Two caspase activation pathways --- the mitochondrial pathway and the death receptor-mediated pathway • Mitochondria as the center of apoptosis in mammals • Other important components --- Apaf-1, SMAC/Diablo, IAP, death receptors and their agonists … • Post-death event --- phagocytosis; ‘find-me’ signal, ‘eat-me signal’, and the corresponding receptors …

Apoptosis --- Physiological Functions • In mammals, many cells undergo apoptosis during development. –

Apoptosis --- Physiological Functions • In mammals, many cells undergo apoptosis during development. – hand fingers – brain – heart • Immune cells • Tissue homeostasis and metabolism • The average adult eliminates 50 -70 billion cells daily. Over the course of a year, each of us will produce and destroy a mass of cells almost equal to our entire body weight.

Apoptosis/Programmed Cell Death --- Pathology Too low: • Cancers • Autoimmune disorders, • Persistent

Apoptosis/Programmed Cell Death --- Pathology Too low: • Cancers • Autoimmune disorders, • Persistent viral infections. . . Too high: • Neurodegenerative disorders • Heart disease (ischemic injury from stroke) • Post-menopausal osteoporosis…

Induction ? Developmental cues Immunological Hormonal Stressful Pathological Execution Termination ? Caspase activation Cell

Induction ? Developmental cues Immunological Hormonal Stressful Pathological Execution Termination ? Caspase activation Cell death Morphology: ? Cell shrinkage Membrane blebbing Nuclear breakdown Chromatin condensation Formation of dead bodies DNA fragmentation PS flipping Apoptosis malfunction and human diseases When it’s too low Cancers Autoimmune diseases When it’s too high ? Neural degenerative diseases Alzheimer’s Parkinson’s ALS

Beyond apoptosis …

Beyond apoptosis …

The Potential Non-apoptotic Functions of Canonical Apoptotic Proteins Caspases • Spermatogenesis in flies •

The Potential Non-apoptotic Functions of Canonical Apoptotic Proteins Caspases • Spermatogenesis in flies • Erythrocyte differentiation and maturation • Capase-8 and wound healing • Caspase-6 in neuronal axonal degeneration • Cytochrome c pathway in long-term neuronal depression • Casp-8 and -3/7 in microglia activation and neurotoxicity Bcl-2 members • Cellular calcium homeostasis • Glucose metabolism • Mitochondrial dynamics (Bak/Bax, fusion) IAP proteins • Cell cycle (survivin for kinetochore function) • NF-k. B signaling

Non-apoptotic, programmed cell death • Caspase-independent • Distinctive morphology • Programmed (what does that

Non-apoptotic, programmed cell death • Caspase-independent • Distinctive morphology • Programmed (what does that mean? )

TNF receptor pathway: From necrosis to apoptosis and back to necrosis What is necrosis?

TNF receptor pathway: From necrosis to apoptosis and back to necrosis What is necrosis?

Necrosis, originally a morphological term Apoptosis Necrosis • Chromatin condensation • Nuclear Swelling •

Necrosis, originally a morphological term Apoptosis Necrosis • Chromatin condensation • Nuclear Swelling • Cell shrinkage • Cell Swelling • Preservation of cell membranes • Rapid engulfment of apoptotic bodies by neighboring cells to prevent inflammation • Disruption of organelles • Rupture of cells and release of cellular contents • Inflammatory response • Thought to be always passive and harmful He et al, 2009, Cell 137: 1100

Can necrosis ever be programmed? Definition of “programmed” Molecular basis (What is the molecular

Can necrosis ever be programmed? Definition of “programmed” Molecular basis (What is the molecular executioner? ) Biological function Necroptosis, or programmed necrosis Degterev et al, 2005 Nat. Chem. Biol. 1: 112 (TNF pathway, requirement of RIPK 1 and RIPK 3) Activation methods all appear to quite artificial

Overview of TNF induced apoptosis and necroptosis Berghe et al. , Nat Rev Mol

Overview of TNF induced apoptosis and necroptosis Berghe et al. , Nat Rev Mol Biol Cell (2014)

Abstract Receptor-interacting protein kinase 1 (RIPK 1) and RIPK 3 trigger pro-inflammatory cell death

Abstract Receptor-interacting protein kinase 1 (RIPK 1) and RIPK 3 trigger pro-inflammatory cell death termed "necroptosis. " Studies with RIPK 3 -deficient mice or the RIPK 1 inhibitor necrostatin-1 suggest that necroptosis exacerbates pathology in many disease models. We engineered mice expressing catalytically inactive RIPK 3 D 161 N or RIPK 1 D 138 N to determine the need for the active kinase in the whole animal. Unexpectedly, RIPK 3 D 161 N promoted lethal RIPK 1 - and caspase-8 -dependent apoptosis. In contrast, mice expressing RIPK 1 D 138 N were viable and, like RIPK 3 -deficient mice, resistant to tumor necrosis factor (TNF)-induced hypothermia. Cells expressing RIPK 1 D 138 N were resistant to TNFinduced necroptosis, whereas TNF-induced signaling pathways promoting gene transcription were unperturbed. Our data indicate that the kinase activity of RIPK 3 is essential for necroptosis but also governs whether a cell activates caspase-8 and dies by apoptosis.

Journal Club 11/19/14

Journal Club 11/19/14

Cells Die by Different Mechanisms • Necrotic cell death (pathological or not) Cell swelling

Cells Die by Different Mechanisms • Necrotic cell death (pathological or not) Cell swelling and breakage • Apoptotic cell death (programmed type I) Caspase activation Nuclear condensation and DNA fragmentation • Autophagic cell death (programmed type II) Autophagy morphology

Autophagy (Self-eating) Christian de Duve George Palade Albert Claude

Autophagy (Self-eating) Christian de Duve George Palade Albert Claude

Histone Deacetylase (HDAC) Inhibitors Suberoylanilide Hydroxamic Acid (SAHA)

Histone Deacetylase (HDAC) Inhibitors Suberoylanilide Hydroxamic Acid (SAHA)

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Procaspase-3, 7

Apoptotic Stimuli Bcl-2 members Apaf-1 Cytochrome c Mitochondrion IAP d. ATP/ATP Smac/Diablo Procaspase-3, 7 Procaspase-9 Apoptosome Active caspase-3, 7

SAHA Can Induce Caspase-Independent Cell Death

SAHA Can Induce Caspase-Independent Cell Death

Autophagy Detected by Electron Microscopy Control SAHA treatment Yufang Shao

Autophagy Detected by Electron Microscopy Control SAHA treatment Yufang Shao

Correlation, consequence, versus causality

Correlation, consequence, versus causality

Basis of Yoshinori Ohsumi’s yeast genetic screening Takeshige et al, JCB 1992 (119) 301

Basis of Yoshinori Ohsumi’s yeast genetic screening Takeshige et al, JCB 1992 (119) 301

 • Dan Klionsky’s Cvt screening Harding et al, JCB 1995 (131): 591 •

• Dan Klionsky’s Cvt screening Harding et al, JCB 1995 (131): 591 • M. Thumm’s screening (monitoring fatty acid synthase amount) Thumm et al, FEBS Letters, 1994 (349) 275

Autophagy, A Highly Conserved Cellular Process Klionsky et al, Developmental Cell, 2003 (5) 539–

Autophagy, A Highly Conserved Cellular Process Klionsky et al, Developmental Cell, 2003 (5) 539– 545

Metabolism, Motility, Proliferation, Survival, and Autophagy PTEN ? Small GTPases Modified from Nature, vol

Metabolism, Motility, Proliferation, Survival, and Autophagy PTEN ? Small GTPases Modified from Nature, vol 451, 28, 1069 -1073, 2008

Physiological and Pathological Functions of Autophagy • Survival and also debatable suicidal roles in

Physiological and Pathological Functions of Autophagy • Survival and also debatable suicidal roles in biology • Interplay/overlap with other cellular processes • Pathology and diseases (neurodegeneration, cancer, metabolic diseases…) • Cargo-specific degradation Example 1: mitophagy (pathological and physiological) Example 2: cellular protein aggregates Example 3: xenophagy

Molecular Pathway of Autophagy Eric H. Baehrecke Nature Reviews Molecular Cell Biology 2005

Molecular Pathway of Autophagy Eric H. Baehrecke Nature Reviews Molecular Cell Biology 2005

ATG 8/LC 3, a ubiquitin-like protein essential for autophagy Ubiquitin LC 3 Sugawara et

ATG 8/LC 3, a ubiquitin-like protein essential for autophagy Ubiquitin LC 3 Sugawara et al, Genes to Cells, 2004, 9: 611

Autophagy (self-eating) LC 3 -PE conjugation

Autophagy (self-eating) LC 3 -PE conjugation

GFP-LC 3/ATG 8 as a cellular marker for autophagy Ian Ganley

GFP-LC 3/ATG 8 as a cellular marker for autophagy Ian Ganley

LC 3 -PE conjugation detected by Western blot Starvation. SAHA Ctrl 12 18 (hours)

LC 3 -PE conjugation detected by Western blot Starvation. SAHA Ctrl 12 18 (hours) LC 3 (LC 3 -I) LC 3 -PE (LC 3 -II)

Autophagy (self-eating) LC 3 -PE conjugation

Autophagy (self-eating) LC 3 -PE conjugation

Starvation-induced GFP-m. Cherry-LC 3 formation and fusion with lysosomes

Starvation-induced GFP-m. Cherry-LC 3 formation and fusion with lysosomes

Sensing and Relaying m. TOR signal by the ULK 1 Complex

Sensing and Relaying m. TOR signal by the ULK 1 Complex

ULK 1 co-localizes with ATG 5 (isolation membrane) upon starvation in MEF cells

ULK 1 co-localizes with ATG 5 (isolation membrane) upon starvation in MEF cells

Both ATG 13 and FIP 200 are required for maximal ULK 1 kinase activity

Both ATG 13 and FIP 200 are required for maximal ULK 1 kinase activity in vitro Ganley et al, JBC 2009

How does the ULK 1 complex mediate m. TOR signaling? • Phosphorylation-dephosphorylation of ULK

How does the ULK 1 complex mediate m. TOR signaling? • Phosphorylation-dephosphorylation of ULK 1 and ATG 13 P P P P • Autophagy addiction How about in cancer cells with intact m. TOR activity and high autophagy? • Regulated protein phosphatase for ULK 1 and ATG 13

How does the ULK 1 complex relay the signal to the downstream? • Epistatic

How does the ULK 1 complex relay the signal to the downstream? • Epistatic relationship with other upstream complexes (ATG 5 complex and VPS 34 complex) The transient nature of interaction, if any • Protein substrate(s) of the ULK 1 kinase complex (how to identify? )

Shokat Gate-keeping Kinase Mutation (Manning and Cantley, 2002)

Shokat Gate-keeping Kinase Mutation (Manning and Cantley, 2002)

The Function of Shokat ULK 1 in Test Tubes and in Cells GFP-LC 3

The Function of Shokat ULK 1 in Test Tubes and in Cells GFP-LC 3

Autophagy, ever a cause of programmed cell death? • Survival argument • Developmental observation

Autophagy, ever a cause of programmed cell death? • Survival argument • Developmental observation • Therapeutic implication

Cancer Therapeutic Implication • Autophagy is frequently activated during cancer development, shall we inhibit

Cancer Therapeutic Implication • Autophagy is frequently activated during cancer development, shall we inhibit it? • Autophagy can be activated by many cancer therapeutic treatments (m. TOR as an example), shall we inhibit it (combination therapy)? • Currently, there is not autophagy-specific inhibitor available

Targeting Autophagy:

Targeting Autophagy:

Molecular Basis of Autophagy in Mammals Induction Nutrient starvation Other stress ? Other triggers

Molecular Basis of Autophagy in Mammals Induction Nutrient starvation Other stress ? Other triggers ? Class. III PI 3 K, m. TOR, Atg 1 ? Conjugation reactions (Atg 12 -Atg 5, LC 3 -PE) ? Execution Termination ? Intracellular membrane organization and trafficking, lysosomal activity Regulation/induction of the conjugation reactions Translate biochemical reactions to membrane events Origin of autophagosomal membrane Basis of cargo specificity Mechanism of recycling after lysosomal degradation To be or not to be (what is the switch for death? ) … … Clearance mechanism to avoid inflammation? (autophagic cell death)

Any other form of programmed cell death?

Any other form of programmed cell death?

Brent Stockwell Erastin GSH Inhibitors (iron chelators, etc. ) Unique mode of death Ferroptosis,

Brent Stockwell Erastin GSH Inhibitors (iron chelators, etc. ) Unique mode of death Ferroptosis, 2012

Metabolism O 2/Fe, Fe homeostasis “Harmful” consequence: ROS/lip. ROS – death Protection systems Transsulfuration

Metabolism O 2/Fe, Fe homeostasis “Harmful” consequence: ROS/lip. ROS – death Protection systems Transsulfuration GSH, GPX 4 RSL 3 bypasses upstream - Glutamine - Autophagy - Cystine starvation Gao & Jiang, Current Opinion in Cell Biology 2018, 51: 58– 6

Clinical relevance of ferroptosis • Ischemic organ damage Ischemic heart disease; Stroke; Kidney failure;

Clinical relevance of ferroptosis • Ischemic organ damage Ischemic heart disease; Stroke; Kidney failure; Liver damage, … • Cancer Glutamine-addicted cancers; p 53 (Gu); Therapy-resistant cancers (Schreiber; Mc. Cormick)