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The NF-κB Pathway

Micaela Roberts Pharmacology 4AA3 1 April, 2004. The NF-κB Pathway. Introduction Mechanism Activators and target genes Inhibitors. Overview. Important signalling pathway Activated by over 150 different molecules Transcribes over 150 genes Central regulator of STRESS RESPONSE

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The NF-κB Pathway

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  1. Micaela Roberts Pharmacology 4AA3 1 April, 2004 The NF-κB Pathway

  2. Introduction Mechanism Activators and target genes Inhibitors Overview

  3. Important signalling pathway Activated by over 150 different molecules Transcribes over 150 genes Central regulator of STRESS RESPONSE Many different steps: many different targets for interruption Introduction

  4. RelA RelA RelA RelA p50 p50 p50 p50 P P IkB Mechanism: Overview IKK IkB IkB IkB ikb kB site

  5. Family of eukaryotic transcription factors Consists of several structurally related proteins RelA/p65, c-Rel, RelB, p50/p105, p52/p100 Form homo- or heterodimers Two classes of Rel/NFkB proteins related by Rel homology (RH) domain - DNA binding / dimerization domain. The transcription factor: RelA/NF-κB Family

  6. Bind to 10 base pair kB sites as dimers. NFkB commonly refers to a p50-RelA heterodimer, which is one of the most avidly forming dimers and is the major Rel complex in most cells. Rel/NF-kB complexes in inactive state in cytoplasm bound to an inhibitor Most common Rel/NF-kB dimer is RelA/p50 RelA/NF-κB cont.

  7. RelA p50 Mechanism: NF-κB ikb kB site

  8. Most commonly studied is IkBa with NFkB. IkB blocks the ability of NFkB to enter the nucleus and bind to DNA. Covers the nuclear localization sequence of NFkB and interfere with sequences important for DNA binding. The inhibitor: IκB

  9. RelA p50 Mechanism: IκB IkB ikb kB site

  10. Phosphorylation of 2 serines (S32, S36 in human IkBa) in the N-terminal regulatory domain of IkBs Polyubiquitination on lysine21 and 22. Degradation of IκB

  11. Ubiquitination occurs through a cascade of enzymatic reactions E1 ubiquitin-activating enzyme forms an E1-ubiquitin thioester E2 ubiquitin conjugating enzymes receive activated ubiquitin from E1 E3 ubiquitin-protein ligases transfer activated ubiquitin to the substrate Ubiquitination

  12. The enzymes that catalyze the ubiquitination of phospho-IkB are constitutively active so the only regulated step in the IkB degradation pathway is the phosphorylation of the two N-terminal seines in the IkB molecule Ubiquitination cont.

  13. RelA RelA RelA p50 p50 p50 P P IkB Mechanism: IκB degradation IkB ikb kB site

  14. Almost all signals that lead to activation of NFkB converge in a high molecular weight complex that contains a serine-specific IkB kinase (IKK). IKK is responsive to a number of potent NFkB activators in addition to TNFα which stimulates its activity with kinetics that match those of IkBα degradation. Extent to which IKK is activated by a given stimulus seems to dictate the kinetics of IkB degradation. The phosphorylator: IKK

  15. There are three tightly associated IKK polypeptides: IKKα and IKKβ are the catalytic subunits of the kinase IKKγ is the regulatory subunit IKK cont.

  16. IKKα and IKKβ have very similar primary structures (52% overall identity) protein kinase domains at their N-termini leucine zippers (LZ) and helix-loop-helix (HLH) motifs in their C-terminal portions IKKγ no recognizable catalytic domain three large a-helical regions, including a LZ IKK cont.

  17. The predominant form of IKK is an IKKα:IKKβ heterodimer associated with either a dimer or trimer of IKKγ IKK activation is dependent on the presence of an intact IKKγ subunit. Interesting results of IKKα and IKKβ knockout mice experiments IKK cont.

  18. RelA RelA RelA p50 p50 p50 P P IkB Mechanism: IKK IKK IkB ikb kB site

  19. NFkB dimers bind to kB sites in the DNA consensus sequence 10 bp long large number of different kB sites that display varying degrees of consensus DNA binding

  20. Each dimer subunit contains two sets of β sheet immunoglobulin folds N- terminal domain (NTD) contacts DNA C- terminal domain (CTD) mediates dimerization and some DNA contacts. NF-kB proteins use ten flexible loops extending from the secondary structure of these immunoglobulin folds to mediate DNA contacts DNA binding cont.

  21. Butterfly structure of NF-κB bound to DNA From Chen & Ghosh, 1999

  22. The long loop L1 reaches into the major groove of the DNA two arginine residues interact with conserved guanines stabilized by hydrogen bonds from a glutamate to the cytosines positively-charged arginine or lysine electrostatically interacts with the pyrimidines opposite of the guanine rich-strand. DNA binding cont.

  23. Transcriptional activation is not biphasic – but it can be finely tuned based on 2 points of regulation: Phosphorylation - phosphorylated p50 shows increased DNA binding Variations in the primary sequence - substitutions can cause decreased binding to specific kB sites DNA binding cont.

  24. RelA RelA RelA RelA p50 p50 p50 p50 P P IkB Mechanism: DNA binding IKK IkB ikb kB site

  25. Certain IkBs, such as IkBa contain both an NLS and a nuclear export sequence (NES) Newly-synthesized IkBa enters the nucleus Binds to NFkB dimers Causes their exportin-mediated transport to the cytoplasm Removal of NF-κB from nucleus

  26. RelA RelA RelA RelA p50 p50 p50 p50 P P IkB Mechanism: Removal of NF-κB IKK IkB IkB IkB ikb kB site

  27. NFkB was initially thought to be a B-cell specific transcription factor Activity could be induced in most cell types upon treatment with phorbol esters, the proinflammatory cytokines TNFα and IL-1, or bacterial LPS, dsRNA, certain viruses and ionizing radiation Activators and Target Genes

  28. Activated by wide variety of bacteria and bacterial products, viruses and their proteins Many of the genes transcribed participate in the immune response (cytokines, chemokines, and receptors required for immune recognition, and for neutrophil adhesion and transmigration across blood vessel walls NF-kB is “central to the immune system”

  29. Many viruses that induce NF-kB activity also have NFkB binding sites in their viral promoters Selective advantage because as the immune system responds to the virus being present, the activation of NFkB leads to transcription of more virus. Eg. HIV-1 has a kB site in its promoter region NF-kB induced by viruses

  30. Involved in transcription of many genes whose functions extend beyond the immune response eg. acute phase proteins (angiotensinogen) Therefore it more generally represents a regulator of stress response Beyond the immediate immune response

  31. Induced during physiological stress in ischemia/reperfusion, hemorrhagic shock Physical stress irradiation, oxidative stress induces stress response genes such as iNOS and COX-2 Environmental stress heavy metals, cigarette smoke, therapeutic drugs Beyond immune response cont

  32. Has been found that NFkB can induce both aptototic and anti-apoptotic effects in different cell types Apoptotic: several stimuli lead both to NFkB activation and to apoptosis this evidence is questionable because in RelA knockouts the apoptosis rate was higher Antiapoptotic: several growth factors and mitogens stimulate or are induced by NFkB Apoptosis

  33. Makes sense to couple a stress responder to anti-apoptotic pathways - it responds to the stress while helping the cells survive through it. Apoptosis and stress response

  34. Mostly based on which cell type is targeted Combinatorial response of promoter/enhancer regions usually need more than one transcription factor to induce transcription Selective activation different NFkB dimers have different preferences for different kB sites Specificity of the NF-kB response

  35. NFkB transcription factors regulate many important physiological processes Have become sought-after molecular targets for pharmacologial intervention Many modulators of this pathway and therefore many opportunities to disrupt it. Inhibiting the NF-kB response

  36. Inhibitors of the NFkB pathway include anti-oxidants, proteasome inhibitors, peptides, small molecules and dominant-negative or constitutively active polypeptides in the pathway Several are general inhibitors of NFkB activation, where others inhibit specific pathways Inhibiting cont.

  37. RelA RelA RelA RelA p50 p50 p50 p50 P P IkB Mechanism: Inhibitory points IKK IkB IkB IkB ikb kB site

  38. The NF-kB is an important pathway in regulating the stress response in the body Work continues on manipulating the pathway for use in therapy Summary

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