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The NF-  B/Rel family. MBV4230 Odd S. Gabrielsen The NF-κB/Rel family A family of signal-responsive transcription factors  rapid response som ikke requires.

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Presentation on theme: "The NF-  B/Rel family. MBV4230 Odd S. Gabrielsen The NF-κB/Rel family A family of signal-responsive transcription factors  rapid response som ikke requires."— Presentation transcript:

1 The NF-  B/Rel family

2 MBV4230 Odd S. Gabrielsen The NF-κB/Rel family A family of signal-responsive transcription factors  rapid response som ikke requires proteinsyntese 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 NFkB also a major anti-apoptopic factor  aberrant activation of NF-  B = one of the primary causes of a wide range of human diseases like in Inflammatory diseases, Rheumatoid arthritis, Asthma, Atherosclerosis, Alzheimer  Persistent activated in many cancers - help keeping cancer cells alive NFkB also promoting growth  Activated NF-  B  cyclin D expression enhanced  growth Drug against NFkB = putative anti-cancer drug

3 MBV4230 Odd S. Gabrielsen The NF-  B/Rel family Characteristic feature: homo- and heterodimeric TFs, which in non-stimulated cells are found inactive in the cytoplasm [in a complex with I  B-repressors].  Active DNA-binding form: Dimers with different members of the NF-  B/Rel family  Inactive cytoplasmic form: inhibitory factor/domain in addition Upon stimulation, active NF-  B rapidly translocates to the nucleus where it binds  B-sites and activates target genes. Rapid response - minutes  Signal  Activated NF-  B  immune defence activated

4 MBV4230 Odd S. Gabrielsen Signal transduction pathway Cytoplasm inactive Nucleus active Signals

5 NF-κB/Rel proteins

6 MBV4230 Odd S. Gabrielsen Common DBD: Rel-homology domain (RHD) RHD: 300aa conserved domain with several functions  DNA-binding (N-terminal half)  dimerization (C-terminal half)  I  B-interaction (C-terminal half)  NLS (C-terminal half)  kalles også NRD (=NF-kB, Rel, Dorsal) Spec.DNA-binding dimerization IkB-interaction NLS

7 MBV4230 Odd S. Gabrielsen Homo- and heterodimers NF-  B/Rel proteins = Homo- and hetero-dimeric TFs that in resting cells are retained in the cytoplasm in complex with I  B. Mature B-cells: constitutively nuclear activator  Bound to kappa immunoglobuline light- chain enhancer  its name

8 MBV4230 Odd S. Gabrielsen Two main classes of RHDs Rel with TAD (dimeric with ≥ 1 Rel-monomers which are potent transactivators) synthesized in their mature form  Rel or c-Rel (as well as v-Rel)  RelA (p65)  RelB  Drosophilas dorsal and Dif p50/52 without TAD (homodimers with no transactivation properties) synthesized as precursors that are processed  Precursor forms have internal I  B inhibitor function RHD linked to inhibitory domain through Gly-rich linker (protease sensitive) Blocks DNA-binding and translocation to nucleus  p105 undergoes proteolytic maturation to p50 [NF-  B1] Proteolytic degradation to p50 is signal dependent, requires ATP and occurs through a ubiquitin-dependent proteasome pathway Also transcription from an intronic promoter  expression  of IkB-   p100 undergoes proteolytic maturation to p52 [NF-  B2]  p50/52 are distinct gene products with very similar properties

9 MBV4230 Odd S. Gabrielsen Two main classes of RHDs p105 p50 p100 p52 RelA(p65) cRel RelB Rel homology domain C-terminal I  B-like domains Acitvation domains +TAD - TAD

10 MBV4230 Odd S. Gabrielsen RHD proteins Ankyrin repeats RHD

11 MBV4230 Odd S. Gabrielsen Dimer-formation Dimer-formation necessary for DNA- binding  each subunit interacts with one half site   B-sites symmetric: 5´-GGGRNNYYCC-3´ Most combinations allowed  Different heterodimers vary with respect to preference for different kB-seter Kinetics of nuclear translocation  p50/p65 rapid, p50/Rel slow abundance in different cells  Exception: RelB which forms dimer only with p50/p52 Common form: p50/p65 (NF-kB1/RelA)  most abundant, found in most cells –--5´-GGGRNNYYCC-3´-- –- 3´-CCCYNNRRGG-5´--

12 MBV4230 Odd S. Gabrielsen 3D structure - DNA interaction Crystal structures:  p50-p50-DNA and p50-p65-DNA Two distinct domains 1. N-terminal - specific DNA contact Compact core in the form of an antiparalell  -barrel from which loops protrude The loop between AB = recognition loop with base contacts in major groove Critical for specificity = R 57 -R 59 -E 63 C62 responsible for redox-sensitivity 2. C-terminal domain responsible for dimerisation + nonspecific DNA- phosphate contact Conserved interphase explains why most heterodimers are possible N-terminal domain C-terminal domain

13 MBV4230 Odd S. Gabrielsen Structure: NF  B (p50-p65) + DNA Side view  -barrel core with protrding loops The AB loop = recognition loop Specificity R57-R59-E63 C62 redox-sensitivity

14 MBV4230 Odd S. Gabrielsen 3D structure - DNA interaction Characteristic features of DNA-interaction  Each monomer contacts a separate half site  “Closing jaws” mechanism for DNA-binding The protein encloses DNA Unusual strong binding (K d = M) Dissociation requires opening of the jaws through a flexible linker

15 MBV4230 Odd S. Gabrielsen 3D structure - protein interaction Interaction with HMGI(Y)  IFN-  promoter: HMGI(Y) binds AT- rich centre of  B-sites in minor groove  The structure contains a corresponding open space Interaction with I  B  I  B binding in an opening over the dimer-interphase  I  B binding blocks DNA-binding HMG I(Y) IkB –--5´-GGGRNNYYCC-3´-- –- 3´-CCCYNNRRGG-5´--

16 The I-  B family

17 MBV4230 Odd S. Gabrielsen The I-  B proteins N-terminal Regulatory domain Ankyrin repeats

18 MBV4230 Odd S. Gabrielsen The I  B-family Inhibitory function  impedes DNA-binding  blocks NLS and abolish translocation to nucleus Several members ( at least 7 mammalian )  I  B-  and I  B-   I  B-  and I  B-   Bcl-3  p105 and p110  IkBR Common features:  ankyrin-repeats which are necessary for RHD-interaction aa motif repeated 3 - 7x  C-terminal acidic-region necessary for inhibition of DNA-binding  C-terminal PEST-sequence involved in protein-degradation Specificity Ex. IkB-  inhibits DNA-binding of p65/p50 but not of p50/p50

19 MBV4230 Odd S. Gabrielsen NF  B-I  B complex IkB HMG I(Y)

20 MBV4230 Odd S. Gabrielsen Signaling IκB - a key element in the canonical NF  B signaling pathway

21 MBV4230 Odd S. Gabrielsen Cytoplasmic retention due to interaction with I  B-family proteins Signal Two types of inactive complexes in cytoplasm  Trimers = RHD-Homo-or heterodimers bound to an I  B  Heterodimers = Rel-protein + unprocessed RHD-precursor (p105, p110) Signal→ [dissociation] → degradation  Induction signal  phosphorylation of both I  B and p105  I  B degradation or p105 processering  active dimers that are translocated to the nucleus.  One type of signal  two N-terminal serines (S32 and S36) become phosphorylated  Another type of signal  two C-terminal serines become phosphorylated in p105  phosphorylation probably more a signal for degradation than for dissociation Ubiquitin-pathway involved  Stimulation  rapid degradation of I  B complete after 10 min No traces of I  B  phosphorylation of I  B  → multiubiquitylation in K21, K22  → degradation through a ubiquitin-proteasome pathway  + proteasome-inhibitors → phospho-IkB remains associated with NFkB

22 MBV4230 Odd S. Gabrielsen Several I  B-factors with different properties I  B-  : Rapid transient response  I  B-  best characterized  all stimuli  degradation of I  B-   ex: TNF-  rapid and transient activation of NF-kB I  B-  : Sustained response  Only certain stimuli  degradation of I  B-   ex: LPS or IL-1  degradation of both I  B-  and I  B-   activation of NF- kB lasting for hours Bcl-3: repressor and activator  inhibits certain complexes like a normal I  B  But may also associate with DNA-bound p50 and p52 dimers (lacking TAD) and provide transactivation properties

23 Signaling pathways

24 MBV4230 Odd S. Gabrielsen Upstream and downstream NF- k B Signal transduction pathways Upstream Downstream

25 MBV4230 Odd S. Gabrielsen NF- k B Signal transduction pathways Multiple signalling pathways activate NF-  B Several signalling pathways converge by activation of NF-  B  NF-  B respond to a broad range of different stimuli Virus infection (HIV, hepatite B), virus proteins (tax, E1A) and dsRNA Cytokines (TNF , IL-1 and IL-2) Bacterial LPS stimulation of antigen reseptor on B- and T-cells calcium ionophores protein synthesis inhibitors UV and X-ray sphingomylenase/ceramide phorbol esters nitrogen oxide

26 MBV4230 Odd S. Gabrielsen Three signal transduction pathways Cytoplasm inactive Nucleus active Signals

27 MBV4230 Odd S. Gabrielsen Signaling hits I-  B through phosphorylation Two N-terminal serines becomes phosphorylated  TNF-signalling pathways: TNF-receptor  TRADD/TRAF  IKK  I  B  I  B-kinase complex central in the signaling pathway  A large kDa IKK (I  B-kinase) complex that is induced by cytokines  Two key subunits: IKK  and IKK 

28 MBV4230 Odd S. Gabrielsen The I  B-kinase complex central in the pathway I  B-kinase complex

29 MBV4230 Odd S. Gabrielsen The IKK  -kinase becomes activated through phosphorylation Activation loop in IKK   Two serines bocomes phosphorylated in a signal dep manner (IL1, TNF)  Ala-mutants block the signalling pathway, Glu-mutants lead to a constitutive active kinase Signal  phosphorylation  phosphorylation of loop necessary for NF  B-activation of cytokines Attenuation  phosphorylated activation loop  altered HLH-kinase domain interaction  reduced kinase-aktivitet Ser-OH Ser-P Signal Upstream kinase inactive active IKKß IBIB inactive PP P P Autophosphorylation

30 MBV4230 Odd S. Gabrielsen The first pathway - the classical pathway Receptor triggered by pro-inflammatory cytokines  such as tumour necrosis factor (TNF)- α Recruitment of various adaptors  including TRADD (TNF-receptor associated death domain protein), RIP (receptor interacting protein and TRAF2 (TNF-receptor-associated factor 2) to the cytoplasmic membrane. Recruitment and activation of the classical IκB-kinase (IKK) complex  which includes the scaffold protein NEMO (NF-kB essential modulator; also named IKK γ ), IKK α and IKK β kinases. The IKK complex phosphorylates IκBα on Ser32 and Ser36 Leading to ubiquitylation and degradation via the proteasome pathway The free p50-p65 migrates to the nucleus where it activates target genes involved in immune response

31 MBV4230 Odd S. Gabrielsen The first pathway - the classical pathway dep on IKKβ Triggered by microbial and viral infections and exposure to proinflammatory cytokines

32 MBV4230 Odd S. Gabrielsen Why two kinases? Ser-OH Ser-P Signal upstream kinase inactive active IKKß IBIB In vitro: IKK  ≈ IKK   52% identity  Similar kinase activity In vivo: IKK  ≠ IKK   Ala-mutants of IKKß  NF  B response dead  Glu-mutants of IKKß  NF  B response independent of signals  Ala-mutants of IKK  NF  B response unaffected  Glu-mutants of IKK  NF  B response unaffected Is IKK  totally unlinked to NF  B?

33 MBV4230 Odd S. Gabrielsen The next indication: KO phenotypes of IKK  ≠ IKK  Knock-out of of IKK    loss of B- and T-cell response  Normal development  Mice dead at day 13.5, liver destroyed due to massive apoptosis  Lack of IKK   lack of active NFkB  lack of protection against apoptosis  massive cell death  Lost T-cell response because Apoptosis important for T-cell development Knock-out of of IKK               , epidermis 5-10x thicker than normal, highly undifferentiated     s    l  Normal number of B- and T-cells, but B-cells not fully differentiated

34 MBV4230 Odd S. Gabrielsen A separate signaling pathway through IKK  A desparate postdoc looked at all the 50 components - all behaved normal, except one The proteolytic maturation of the p100 precursor to p52 [NF-  B2] was defective in the IKK     processing depends on NIK Hypothesis: NIK acts through IKK 

35 MBV4230 Odd S. Gabrielsen The solution Processing depends on IKK  Target of IKK 

36 MBV4230 Odd S. Gabrielsen A separate signalling pathway involving only IKKα Affects NF-κB2 (p100), which preferentially dimerizes with RelB. Triggered by by cytokines such as lymphotoxin b, B-cell activating factor (BAFF) or the CD40 ligand and by viruses such as human T-cell leukaemia virus. NEMO-independent, IKKα- dependent + another kinase NIK. Induce the phosphorylation- dependent proteolytic removal of the IkB-like C- terminal domain of NF-κB2 A role in innate immunity B-cell maturation A role in adaptive immunity

37 MBV4230 Odd S. Gabrielsen Two kinases - two main signaling pathways The canonical NF-  B activation pathway (left)  Applies to RelA-p50 and c-Rel-p50  Retained in cytoplasm by I  B  Triggered by microbial and viral infections and exposure to proinflammatory cytokines  Depends mainly on the IKK  subunit of the IKK complex. The second pathway (right)  Affects NF-  B2, which preferentially dimerizes with RELB.  Triggered by members of the tumour- necrosis factor (TNF) cytokine family  Depends selectively on activation of the IKK  subunit + another kinase NIK.  Induce the phosphorylation-dependent proteolytic removal of the I  B-like C- terminal domain of NF-  B2.

38 MBV4230 Odd S. Gabrielsen A third signalling pathway independent on both IKKs classified as atypical because it is independent of IKK proteasome still required triggered by DNA damage such as UV or doxorubicin UV radiation induces IkBa degradation via the proteasome, but the targeted serine residues are located within a C- terminal cluster, which is recognized by the p38- activated casein kinase 2 (CK2)

39 MBV4230 Odd S. Gabrielsen Connectivity map of the TNF-α/NF-κB signal transduction pathway

40 MBV4230 Odd S. Gabrielsen Seminar

41 Target genes

42 MBV4230 Odd S. Gabrielsen Upstream and downstream NF- k B Signal transduction pathways Upstream Downstream

43 MBV4230 Odd S. Gabrielsen Families of target genes Immune response  Cytokines,  Chemokines  Cytokine and immuno-receptors  Adhesion molecules  Acute-phase proteins  Stress-responsive genes NF-  B is both being activated by and inducing the expression of inflammatory cytokines NF-  B activation can spread from cell to cell

44 MBV4230 Odd S. Gabrielsen Negative feedback: Attenuation of respons Negative loop: I  B-  under direct control of NF-  B  Activated NF-  B translocated to the nucleus will activate expression of I  B-   Newly synthesized I  B-  will bind up and inactivate remaining NF-  B in the cytoplasma  Excess I  B-  will migrate to the nucleus and inactivate DNA-bound NF-  B (contains both NLS and nuclear eksport signal)  A20 protein another strongly induced negative feedback protein Immunosupressive effect of glucocorticoids  Probably a direct effect of glucocorticoids enhancing the expression of I  B-  which then binds up and inactivates NF-  B in the cytoplasm, leading to reduced immune- and inflammatory response

45 MBV4230 Odd S. Gabrielsen Target genes: Link to cancer Tumorigenesis requires 6 types of alterations  Hanahan & Weinberg 2000 Several of these can be caused by perturbation in NF-  B or linked signaling molecules  Tumour cells in which NF-  B is constitutively active are highly resistant to anticancer drugs or ionizing radiation. Angiogenesis Metastasis

46 Disease links

47 MBV4230 Odd S. Gabrielsen Viruses exploit NF-  B several patogenic viruses exploit the NF-  B system for their own profit  Incorporation of  B-sites in virus DNA cause enhanced expression of virus-genes when the immune response is activated  Virus proteins activate NF-  B

48 MBV4230 Odd S. Gabrielsen Disease links

49 MBV4230 Odd S. Gabrielsen Constitutively nuclear NF-  B Disruption of the regulatory mechanism  aberrant activation of NF  B = one of the primary causes of a wide range of human diseases  Inflammatory diseases Rheumatoid arthritis Asthma  Atherosclerosis  Alzheimer

50 MBV4230 Odd S. Gabrielsen Link: inflammation - cancer A causal connection between inflammation and cancer has been suspected for many years. NF-  B might serve as the missing link between these two processes.  NF-  B becomes activated in response to inflammatory stimuli  Constitutive activation of NF-  B has been associated with cancer,

51 MBV4230 Odd S. Gabrielsen Mechanisms of NF-  B activation promoting leukemia Mechanisms by which NF-  B activation can contribute to leukaemia and lymphogenesis 1. Input: NF-  B can be constitutively activated in myeloid and lymphoid cells in response to growth factors and cytokines or the expression of certain viral oncoproteins. 2. Gene errors: Persistent NF-  B activation can also be brought about by chromosomal rearrangements that affect genes that encode NF-  B or I-  B. 3. Autocrine loop: Once NF-  B is activated, it can lead to the production of cytokines and growth factors, such as CD40 ligand (CD40L), that further propagates its activation. 4. Growth - apoptosis: It also activates the transcription of cell-cycle regulators, such as cyclins D1 and D2, which promote G1- to S- phase transition, or inhibitors of apoptosis, such as BCL-X L, cIAPs and A1/BFL Tumour cells in which NF-  B is constitutively active are highly resistant to anticancer drugs or ionizing radiation.

52 MBV4230 Odd S. Gabrielsen Breast cancer: Signalling pathways that stimulate proliferation Signaling induction of cyclin D1.  Two signalling pathways contribute to the induction of cyclin D1 transcription in mammary epithelial cells.  One pathway, which leads to activation of transcription factor AP1, is activated by growth factors (GF), which bind to receptor tyrosine kinases (RTK). This pathway relies on activation of RAS and MAPK cascades.  The second pathway is activated by the TNF-family receptor activator of NF-  B ligand (RANKL), which binds to the receptor activator of NF-  B (RANK). This pathway, which leads to activation of NF-  B, depends on the IKK  subunit of the IKK complex. After nuclear translocation, NF-  B activates cyclin D1 expression, leading to cell-cycle progression.  The expression of GFs and RANKL is regulated by various hormonal stimuli during mammary-gland development. Aberrant and persistent activation of either pathway can lead to deregulated proliferation of mammary epithelial cells.

53 MBV4230 Odd S. Gabrielsen Blocking the response Redox-dependency  Antioxidants and alkylating agens inhibit response to many stimuli and inhibit phosphorylation and degradation of I  B  H 2 O 2 activates NF-  B  Induction of ROI (reactive oxygen intermediates) a possible common element? Proteasome inhibitors

54 MBV4230 Odd S. Gabrielsen Therapeutic inhibition of NF  B Numerous inhibitors of NF-  B under development. Difficult to develop cancer specific inhibitors. Understanding the two pathways should lead to better therapeutics.


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