The Death Receptor Signaling Pathway

Transcription

1 The Death Receptor Signaling Pathway Richard M. Siegel M.D., Ph.D. National Institutes of Health Bethesda, MD USA 1 The death receptor signaling pathway 1. Structure and function 2. Early signaling events and regulation 3. Review of individual receptors and physiological functions 4. Diseases associated with altered death receptor function 2 Apoptosis signaling pathways Extrinsic pathway Caspase-8 Procaspase-8 L Type II Procaspase- 3 Bid tbid IAP s Intrinsic DNA damage Cellular stress pathway Deprivation of growth factors Bcl-2 Bcl-xl Bax, Bak Smac, Omi Cytochrome C Apaf-1 Type I Caspase-3 Apoptosome Procapase-9 Apoptosis 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 A spectrum of responses triggered by TNF-family receptors Death-domai n containi ng TNFRSF s Death receptors (6 human) Truncated and soluble Decoy receptors (4 human) Other TNFRSF receptors (22 human) Apoptosis (, TRAIL-Receptors) Mixed apoptosis and inflammation (TNFR1, DR3) Block apoptosis (DcR1-3) Block other signals (OPG) Costimulation (CD40, OX40, 4-1BB, CD27 & others) Osteoclast activation (RANK) Organ formation (TNFR2, LT-receptors, EDAR) 4 TNF-SF structure-function TNF ligands Type II transmembrane proteins B-jellyroll structure Trimeric, stabilized by internal residues Receptor binding regions highly variable Cleaved from the membrane by metallo-proteases 5 TNF-SF structure-function TNF receptors: extracellular domains Cysteine repeat domains (CRD s) define TNFR-superfamily 1-6 repeats/receptor Scaffold of internal disulfide bonds stabilize structure Typical CRD contains two subdomains Variants can be classified into subgroups Naismith and Sprang, TIBS, Feb The screen versions of these slides have full details of copyright and acknowledgements 2

3 TNF-SF structure-function ligand: receptor binding Ligands crystallize in a 3:3 complex with receptors Ligand interdigitates between receptor subunits Ligand contact residues usually not at N-terminal Unliganded receptors may have a different quaternary arrangement TNF/TNFR and TRAIL/DR5 structures remarkabl y similar 7 TNF-SF structure-function death domain 6 alpha-helix knot - unique fold Shared by death receptors (, TNFR1, TRAIL-receptor s 1, 2, DR6, NGFR, EDAR) Bind other death-domain containing proteins such as and TRADD Structurally related to downstream intracellular signaling proteins in the apoptosis pathway: DED and CARD 8 Apoptosis triggered by death domain-containing TNF-SF receptors through recruitment of a death-inducing signaling complex (DISC) 1. Unbound receptors 2. Ligand-bound receptors (receptor clustering) 3. Activated receptor signaling complex Death domains (DD) 4. Release of activ e caspase into cytoplasm APOPTOSIS Pro-caspase-8 Active caspase-8 DISC 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Regulation of early events in death receptor signaling Extracellular: soluble and DcR3 decoy receptor Plasma membrane & early endosomes: Non-covalent receptor preassociation Siegel, et al., Science 288:2354 (2000) Lipid raft localization Muppidi & Siegel, Nature Immunology 5:182 (2004) Receptor superclustering Siegel, et al., Journal of Cell Biology 167: (2004) Receptor internalization Lee et al., EMBO journal. 25 (5), (2006) homotypic interactions Muppidi et al., Cell Death and Differentiation, 2006 (in press, online) 10 Preassociation of and other TNFR family members PLAD mediated preassociation found for (human), TNFR1, TNFR2, TRAILR1, TRAILR2 Discovered because of dominant negative mutants of which do not bind L May explain inhibition of and other TNFRSF receptors by soluble splice variants and decoy receptors CRD1 CRD2 CRD3 PLAD CRD4 TM domain Ligand binding domains Death domain Unbound receptors Preassociation may be regulated in different cell types; This could modulate sensitivity to receptor signaling Blocking pre-association of TNF-R family members may be a novel way to disrupt function More receptor-specific Blocking TNFR1 PLAD improves collagen-induced arthritis (Deng et al., Nature Medicine 11, , 2005) 11 TNF-SF receptors without a death domains trigger diverse gene expression programs through TNF-receptor associated adapter proteins: TRAF s 1. Unbound receptor 2. Bound receptor with signaling complex TRAF s (TNF-receptor associated proteins) TRAF-binding sequences KINASES NIK, RIP, TBK, IKK Regulators IAP1,2, A20 NFkB JNK MAP-K Differentiation Inflammation Organogenesis 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Spectrum of receptors in the TNF-R superfamily Activating TRAF Dual signaling TRADD Professional death receptors Co-stimulation Osteoclast activation Lymphoid organ formation Mixed apoptosis and inflammation Apoptosis Negative regulation of innate immunity? (TRAIL) CD30, CD40, 4-1BB RANK, RANK-L etc. TNFR1, DR3 FAS, TRAIL-Rs 13 Dual signaling by TNF receptors: the two complex model TNF Micheau and Tschopp, Cell 2003 TNFR1 Caspase-8 Complex I ciap1 TRADD RIP1 TRAF2 > 2hrs RIP1 Complex II NF-KB New gene synthesis Cell survival inflammati on c-flip Caspase-8 Cell death 14 Alternative caspase-independent receptor induced cell death TNF TNFR1 Caspase-8 ciap1 TRADD RIP1 TRAF2 > 2hrs RIP1 Complex II Caspase inhibition RIP-dependent necrosis/autophagic cell death Caspase-8 Apoptosis 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Elimination of restimulated T cells is clonotypic Antigen/MHC Activated T cell Restimulated T cell 16 T-cell receptor stimulation sensitizes cells to die via P MA ( ng/ ml) Sensitization by TCR does not depend on RNA or protein synthesis Specific cell death (%) Muppidi & Siegel, Nature Immunology 5:182 (2004) Vector Fa sl 17 Lipid rafts in antigen receptor signal transduction GFI-linked protein Coreceptor MIRR CD45 Src-family kinase From Cherukuri et al., The screen versions of these slides have full details of copyright and acknowledgements 6

7 TCR restimulation results in the accumulation of in lipid rafts % Protein 60 Activated human CD4 T cells % Protein anti-cd3 (90 min) Lck Raft fractions Caspase-8 Raft fractions Nature Immunology 5:182 (2004) 19 APC Lipid raft relocalization increases sensitivity to -induced apoptosis TCR T cell? Lipid raft What parts of the TCR signal lead to sensitization? What types of cells are pre-sensitized to? What other signals might relocalize? 20 Cholesterol depletion TNFR1 L L TCR L TNF TNFR1 Cholesterol depletion Non-raft Lipid raft Non-raft Lipid raft Monomeric Preassociated TRADD RIP1 Complex I TRADD RIP1 Caspase-8 ciap 1,2 TRAF2 Complex II TRAF2 TRAF2 Poor caspase activation No cell death Caspase activation Cell death poor NF-κB activation RIP1 TRADD cflip et al. NF-κB activation Muppidi & Siegel, Nature Immunology 5:182 (2004) Legler, et al., Immunity, (5): p Micheau, and Tschopp. Cell 114: (2003) Cell death Anti-apoptosis, inflammation 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Regulation of early events in death receptor signaling Extracellular: soluble and DcR3 decoy receptor Plasma membr ane & early endosomes: Non-coval ent receptor preassoci ati on Siegel, et al., Science 288:2354 (2000) Lipid raft localization Muppidi & Siegel, Nature Immunology 5:182 (2004) Receptor superclusteri ng Siegel, et al., Journal of Cell Biology 167: (2004) Receptor internalizati on Lee et al., EMBO journal. 25 (5), (2006) homotypi c interactions Muppidi et al., Cell Death and Differentiation, 2006 (in press, online) 22 engagement induces rapid receptor clustering in living cells Cos-7: FL-GFP Cos-7: FL-GFP Pre-tx Anti- (15 min) Receptor clustering requires the death domain Receptor clustering is upstream of caspase activation Siegel et al., J. Cell Biol 167: (2004) 23 Formation of SPOTS is dependent on the death domain and recruitment but not caspase activity WT -YFP Del DD Pre-tx Anti- Anti- + zvad Anti- D244V (ALPS Type I) Anti- (10 %) 52 % 46 % 4.9% 2.5 % % of cells with SPOTS SPOTS: Surface Protein Oligomeric Transduction Structures 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Two distinct phases of receptor redistribution in lymphocytes endogenous - SKW 6.4 cells Pre-treatment diffuse 15 SPOTS 30 caps 30 + zvad SPOTS 25 SPOTS are on the cell surface and are enhanced with caspase blockade Control Anti- 60 min + zvad Anti- 60 min Surface stain Intracellular stain Combined Relative surface anti- 20 anti- + zvad Time (min) SPOTS: platforms for signaling Formation of SPOTS requires an intact death domain and recruitment, but not caspase enzymatic activity SPOTS likely contain hundreds of individual receptors: higher-order oligomers than preassociated receptors defined by FRET or crosslinking studies SPOTS may be a molecular platform required for caspase-8 activation in the DISC What interactions underlie formation of SPOTS? 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 1. Unbound 2. Ligand binding 4. RXDLLmediated self-association SPOTS DD 5. Caspase-8 recruitment 3. recruitment RXDLLmutations 6. Caspase-8 activation and release Apoptosis DD DED DED DED Caspase-8 28 The death receptor signaling pathway 1. Structure and function 2. Early signaling events and regulation 3. Review of individual receptors and physiological functions 4. Diseases associated with altered death receptor function 29 Death receptors in T cell homeostasis Effector Post activation cell death - not known to be dependent on death receptors Memory Naive Activation HVEM Clonal expansion CD27 Survival OX40, 4-1BB, CD30 CD4+ T cells restimulation induced death FASL Helpless CD8 T cell death: TRAIL 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 (TNFRSF6): negative regulation through apoptosis induction in multiple immune cell types B cell Ag/MHC Ag/MHC L APC Resting T cell Activated T cell 31 TNF: inflammation and enhancement of immunity Two receptors for TNF TNFR1 (TNFSF1A): mediates cell death and inflammation TNFR2 (TNFSF1B): can synergize with TNFR1 but mainly a T cell costimulator Systemic release of TNF mediates septic shock and chronic wasting syndromes Direct injection of TNF produces acute inflammation Overproduction of TNF in transgenic mice produces a chronic wasting phenotype with cachexia, arthritis, and autoantibodies that is dependent on TNFR1 TNF or TNFR -/- mice are deficient in innate immune responses to bacteria but resistant to LPS-mediated endotoxic shock Pathology mediated by TNF overexpression is independent of T and B cells The same pathways that mediate innate immune protection also mediate immunopathology 32 DR3: a T cell co-stimulator? DR3 specifically expressed on T cells Can activate apoptosis as well as NF-kappaB, JNK and ERK signaling pathways Ligand is the TNF family member TL1A TL1A can enhance cytokine production by T cells DR3 knockout mice have partial defect in negative antigenic selection and no reported peripheral T cell phenotype to date 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 TRAIL: cancer cell death and innate immune cell regulation 2 receptors in human genome TRAIL-R1 (DR4, TNFRSF10A) and TRAIL-R2 (DR5, TNFRSF10B) as well as two truncated decoy receptors ; One receptor identified in mouse so far: TRAIL-R Although TRAIL induces apoptosis efficiently in many tumor cell lines and in tumors in vivo, range of normal cell types killed by TRAIL is small: dendritic cells, helpless CD8 T cells TRAIL KO mice found to have defective tumor surveillance TRAIL-R KO mice have increased activati on of myeloid cells and hyperactive responses to innate immune stimuli 34 Other TNF family receptors with a death domain DR6 (TNFRSF21) may be a negative regulator of T and B cell activation, but no direct evidence for physiological cell death induction; Ligand not identified EDAR: tooth, hair, sweat gland formation; Mutated in ectodermal dysplasia; Associates with unique adapter protein ERADD 35 ALPS - autoimmune lympho proliferative syndrome genetic defects in the pathway Clinical: lymphadenopathy, splenomegaly and accumulation Extracellular Domain Intracellular Domain of CD4 - CD8 T cells; Initial presentation: Infancy to 5 yrs Lab: hypergammaglobulinemia and autoantibodies; Primarily hematopoietic Cysteine Repeat Domains antibody-mediated Death autoimmune Domain disease; 15-fold CD95/ Apo-1/ increased incidence of lymphomas, years after initial ALPS symptoms 85% of patients harbor heterozygous mutations in the gene coding for /CD95 Exon Exon 9 α1 α2 α3 α4 α5 α Pt. 20 (C57X) Pt. 7 Pt. 2 Pt. 34 (D62fs) (P49del46, P49 fs) (T131fs) Pt. 3 (T225P) Pt. 31 (R234P) Pt. 26 (D244V) 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 ALPS mutants interfere with WT signaling through two distinct mechanisms Intracellular mutations Extracellular mutations Formation of the signaling complex is cooper ative Pre-association of receptors Martin, D. A., Zheng, L., Siegel, R. M., et al., (1999). PNAS 96, Siegel, et al., Science 288:2354 (2000) 37 Relationship between mutations and disease? Functional Haploinsufficiency variant Extracellular dominant negative Intracellular dominant negative Homozygous deficiency Contribution of other genetic loci Disease penetrance Disease severity 38 TRAPS Autosomal-domi nant inherited autoinflammator y disorder with relapsing and remitting nature Symptoms include recurrent fevers, abdomi nal pain, migratory rash, fascial and synovial inflammation Amyloidosis is a life-threatening complication Associated with heterozyg ous mutations in TNFR1 39 The screen versions of these slides have full details of copyright and acknowledgements 13

14 TRAPS-associated TNFR1 mutations PLAD Ligand binding domain Cleavage site Death domain CRD1 CRD2 CRD3 CRD4 > 46 pathogenic mutations identified in TNFR1 in TRAPS - All are extracellular (most are in CRD1 or CRD2) - Both cysteine and non-cysteine residues are equally affected - Many predicted to disrupt protein folding - No truncation or nonsense mutations - R92Q & P46L rare polymorphism occurs in 1-2.5% of the population and at increased frequency in TRAPS 40 Sources for further reading 1. Bodmer, J. L., P. Schneider, et al., (2002). "The molecular architecture of the TNF superfamily." Trends Biochem Sci 27: Siegel, R. M. Caspases at the crossorads of immune cell life and death (2006) Nature Reviews Immunology, April issue 3. Locksley, R, Killeen, N, and Lenardo M.J. (2001) The TNF and TNF Receptor Superfamilies: Integrating Mammalian Biology. Cell 104: Siegel, R.M., Chan, F.C., Chun, H., and Lenardo, M.J (2000) The multifaceted role of signaling in immune cell homeostasis and autoimmunity Nature Immunology, 1: Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998 Aug 28; 281(5381): Jagan R. Muppidi, Jürg Tschopp and Richard M. Siegel Life And Death Decisions: Secondary Complexes and Lipid Rafts in TNF Receptor Family Signal TransductionI mmunity, (2004) 21,: Peter, M.E. and Krammer, P.H. (2003) The CD95 death-inducing signaling complex and beyond. Cell Death Differ, 10, Online Table of TNF Family Ligands and receptors: The screen versions of these slides have full details of copyright and acknowledgements 14