Role of NF-  B in the Regulation of Immunity and Apoptosis

1. Role of NF-B in the Regulation of Immunity and Apoptosis Kavitha Bharatham

2. Abstract • Introduction • Structural Description • Role as regulator of Immune responses • Role as regulator of apoptosis • Regulation Mechanism • As target for Therapy • Conclusions

3. Introduction

4. Nuclear Factor kappa B (NF- B) • Multifunctional transcription factor • Play central role in the cellular response to a variety of stress signal by regulating genes • Exists as homo or hetero-dimers found inactive in the cytoplasm • Upon stimulation, active NF-kB rapidly translocates to the nucleus where it binds B-sites and activates target genes

5. Role of NF- B/Rel family • Involved in proinflammatory response: a first line of defense against infectious diseases and cellular stress • Signal Activated NF- B  immune defence activated • Immune response, inflammatory response, accute phase response • NF-B also a major anti-apoptopic factor • aberrant activation of NF- B causes Inflammatory diseases, Rheumatoid arthritis, Asthma, Atherosclerosis, Alzheimer • helps keeping cancer cells alive • NF-B also promoting growth • Activated NF- B  cyclin D expression enhanced  growth

6. Factors that induce NF-kB Reactive Oxygen Species (ROS) Leukemia16:1053-1068, 2002

7. Genes Regulated by NF-kB Nat.Rev.Cancer 2:301-310, 2002

8. Disorders associated with aberrant NF-kB activation • Rheumatoid arthritis • Atherosclerosis • Vascular dysfunction • Multiple sclerosis • Neurodegenerative disorders • Inflammatory bowel disease • H. pylori-associated gastritis • Systemic inflammatory response syndrome • And the list is growing… • Autoimmune thyroid disease • Cystic fibrosis • Diabetes • Aging • Macular degeneration • HIV/AIDS • Cancer • Septic shock

9. Structural Description

10. The NF- B/Rel family NF-B belongs to the Rel family, which contains five mammalian Rel/NF- B proteins: Potent transactivators synthesized in their mature form • RelA (p65) • c-Rel • RelB Synthesized as precursors that are post translationally processed withnotransactivation properties • NF- B1 (p105 undergoes proteolytic maturation to p50) • NF- B2 (p100 undergoes proteolytic maturation to p52) RelB forms dimer only with p50/p52 Most Common form: p50/p65 (NF-kB1/RelA)

11. NH2 GGG C-terminal IkB-like domains Rel homology domain p105 - TAD p50 Ankyrin Repeats p100 p52 RelA(p65) +TAD cRel TransActivation domains (TAD) RelB NF- B proteins structure

12. Structural Details • Rel Homology Domain (RHD): 300aa conserved domain of Rel family which contains NLS (Nuclear localisation sequence) with several functions • DNA-binding (N-terminal half) • Dimerization (C-terminal half) • IB-interaction (C-terminal half) • IB (Inhibitor of Nf- B) blocks NLS and abolish translocation to nucleus • IkB family includes IB-,IB-, IB-and IB-, Bcl-3, etc., • They posess ankyrin-repeats which are necessary for RHD-interaction • impedes DNA-binding • Upon Signal undergoes dissociation and degradation

13. Dimerization Dimer formation is necessary for DNA-binding Each subunit interacts with one half site kB-sites are symmetric: 5´-GGGRNNYYCC-3 p50 p50 RelA RelA p50

14. Structure: NFkB (p50-p65) + DNA Side view b • --5´-GGGRNNYYCC-3´-- • - 3´-CCCYNNRRGG-5´--

15. Role as Regulator of Immune responses

16. Role in inflammation Inflammation is a process by which the body attempts to dilute, destroy, or isolate a noxious (harmful) agent and repair damage TNF-alpha Transcription TNF Il-1 Up regulation of adhesion molecules (ICAM-1, VCAM-1) Cytokines that further enhance the immune response activators of inflammatory pathways (arachidonic acid metabolites, superoxides and nitric oxide)

17. Role of IKK in Activating NF-B Pro-inflamatory cytokines, Pathogen associated molecular patterns (PAMPs), TNF Receptor (TNFR), Toll-like Receptor (TLR), Interleukin-1 receptor (IL-1R) Two types of inactive complexes in the cytoplasm Trimers = RHD-Homo-or heterodimers bound to an IkB-repressor Heterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110) Transcription NLS is exposed and translocated to Nucleus kB site

18. Two main signaling pathways Innate Adaptive Inflammatory/Immune Proteins NUCLEUS mRNA Target genes Transcription

19. Innate vs Adaptive NF-kB activation pathway • Canonical/Classical/Innate • NF-kB activation pathway • Applies to RelA-p50 and c-Rel-p50 • Retained in cytoplasm by IkB • Triggered by microbial and viral • infections and exposure to proinflammatory cytokines • Depends mainly on the IKKb subunit of the IKK complex. • Induce the phosphorylation-dependent proteolytic removal of the IkB • NonCanonical/NonClassical/Adaptive • NF-kB activation pathway • Affects NF-kB2, which preferentially • dimerizes with RELB • Triggered by members of the tumor- • necrosis factor (TNF) cytokine family • Depends selectively on activation of the IKKa subunit + another kinase NIK. • Induce the phosphorylation-dependent • proteolytic removal of the IkB-like • C-terminal domain of NF-kB2

20. Role as Regulator of Apoptosis

21. Apoptosis is Programmed Cell Death Apoptosis is needed to destroy cells that represent a threat to the integrity of the organism Inducers: • Damage-related inducers • heat shock, viral infection, bacterial toxins, oncogenes (myc, rel, E1A), tumor suppressors (p53), cytolytic T cells, oxidants • Therapy-associated agents • Chemotherapeutic drugs (e.g., cispatin, nitrogen mustard) • Antracyclines (doxorubicin), gamma radiation, UV radiation • Toxins • Ethanol, b-amyloid peptide

22. Diseases Associated with Deregulated Apoptosis Increased Apoptosis Inhibition of Apoptosis Cancer Follicular lymphomas carinomas with p53 mutations hormone dependent tumours: breast cancer, prostate cancer, ovarian cancer Autoimmune Disorders Systemic lupus erythematosus Immune-mediated glomerulonephritus Viral Infections Herpesvirus, poxvirus, adenovirus AIDS Neurodegernative disorders Alzeheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis Retinitis pigmentosa Myelodysplastic syndromes Aplastic anaemia Ischaemic Injury Myocardial infarction, Stroke, Reperfusion injury Toxin-Induced liver disease Alcohol

23. NF-kB is an anti-apoptotic factor Via NF-kB TNF blocks its own cell death potential Chemotherapy activates NF-kB within tumor cells NF-kB inhibitors augment chemotherapy

24. Cell Death Mechanism behind Anti-apoptotic activity RIP: Receptor interacting protein FADD: Fas associated Death domain IAP: Inhibitor of Apoptosis TRAF: TNFR associated protein Bid: Bcl-2 interacting domain JNK: Jun N-terminal kinase

25. Balance between life and death When NF-kB is not inhibited When NF-kB is inhibited ANTI-APOPTOTIC PROTEINS PRO-APOPTOTIC PROTEINS

26. Regulation Mechanism

27. RelA p50 Negative feedback: Attenuation of NF-kB response • Negative loop: IkB is under direct control of NF-kB IkB IkB ikb kB site • RIP and TRAF1 are cleaved Caspase 8 RIP Cleaved TRAF1

28. Therapeutic inhibition of NFkB

29. Conclusions • The NF-kB is an important pathway in regulating the stress response in the body • It plays a key role in oncogenesis • Work continues on manipulating the pathway for use in therapy • Complete elucidation of the mechanisms involved in regulation of NF-kB activation is required to generate inhibitors • Therapeutic inhibitors that selectively block NF-kB activation in cancer cells without effecting normal functions are required

30. References • Seminars in Cancer Biology 13 (2003) 107–114 • TRENDS in Immunology Vol.25 No.6 June 2004 • Clinical Chemistry 45:1 7–17 (1999) • The Journal of Clinical Investigation January 2001 Volume 107 Number 2 • Human Molecular Genetics, 2002, Vol. 11, No. 20