bZIP: leucine zippers

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bZIP: leucine zippers. Leucine-zipper (bZIP) -family: common DBD-structure. 60-80 aa motif found in many dimeric TFs Prototypes: GCN4, Fos, Jun, C/EBP, ATF, CREB several possible dimer-partners  numerous combinations rapid equilibrium  combinations determined by abundance
Transcript
bZIP: leucine zippersLeucine-zipper (bZIP) -family: common DBD-structure
  • 60-80 aa motif found in many dimeric TFs
  • Prototypes: GCN4, Fos, Jun, C/EBP, ATF, CREB
  • several possible dimer-partners  numerous combinations
  • rapid equilibrium  combinations determined by abundance
  • Dimer-formation through parallel coiled coils of -helices (ZIP)
  • each 7.aa = Leu
  • 3.5 aa per turn (coiled coil)  each 7.aa in equivalent positions
  • All Leu on same side  dimerization through “leucine zipper”
  • b Z in PLeucine-zipper (bZIP) -family: common DBD-structure
  • Structure - models
  • bZIP like the letter Y: paired in ZIP region, separated in b-region, grips around DNA
  • “induced helical fork” (induced structure in b)
  • Crystal structure of GCN4, Fos-Jun: -helical tweezer with a single continuous helix slightly bended
  • Almost a zipper (glidelås)
  • b Z in PgcdfLLLLaLbeVVANVThe heptad Leu-repeat
  • Example: c-Fos
  • ESQERIKAERKRMRNRIAASKCRKRKLERIAR (= basic region)
  • LEEKVKTLKAQNSELASTANMLREQVAQLKQ (=leucine zipper)
  • 1. . . . . . 7
  • Coiled-coil
  • Equivalent positions of leucines
  • Dimerization through the zipperHydrophobic interfaceContacts DNA like a tweezersbZIP-structure: Gcn4p-DNA complexTweezer-like structure wih a pair of continuous -helicesTweezers - pinsettGcn4 (Basic Region, Leucine Zipper) Complex With Ap-1 DNABasic region - DNA contact
  • Structured -helices formed upon DNA-binding
  • Extended - solvent exposed
  • Cis-element with two half sites that are contacted by each of the monomers (different half-site spacing)
  • TRE site: TGACTCA, CRE: TGACGTCA (symmetrical)
  • Sequence recognition
  • 5 contact aa: N--AA--S(C)R
  • N: H-bonds to CG
  • AA: to methyl-T
  • S: methyl-T
  • R: H-bonds to GC
  • Adaptation to TRE and CRE through DNA-distortion
  • Specific examples:The AP-1 transcription factor
  • AP-1 (activator protein 1) proteins include the protein families:
  • JUN
  • FOS
  • ATF (activating transcription factor) and
  • MAF (musculoaponeurotic fibrosarcoma)
  • These can form homodimers and heterodimers through their leucine-zipper domains.
  • The different dimer combinations recognize different sequence elements in the promoters and enhancers of target genes.
  • Specific examples:AP-1 (Jun -Fos dimer)Specific examples:CREB
  • Structure of the CREB bZIP domain bound to the somatostatin CRE.
  • residues that function in DNA recognition highlighted in yellow.
  • A magnesium ion (green) with surrounding water molecules (red) is located in the cavity between DNA and the CREB basic region.
  • Rules for specificity in dimerization: spes = f(e+g)
  • Heptad repeat: abcdefg
  • a+d = inner hydrophobic contact interface
  • d = leucines
  • a = hydrophobic (-branched preferred)
  • Shielding of the a-d-interface by e and g
  • e and g: polar, charged (AKET)
  • if charged: repulsion or salt-bridges
  • ggccddffLaaLLbbLeeLRules for dimerization- the e-g interactionLLKLIKLTLJUNHydrophobicinterphaceFOSVVANVEI-+EK-E-AE-+ERK++-+-Dimerization specificityHydrophobic interfacei+5-rule:
  • Electrostatic repulsion in e-g prevents certain dimers to form
  • ex Fos does not dimerize
  • Fos: e: QEQLE, g:EEEEI
  • Jun: e: EKARK, g: KQTQK
  • EK or KE facilitate dimerization, while KK and EE block dimerization
  • Does not cover all functional pairs
  • Doubt whether electrostatic attraction e-g facilitates dimer-formation.
  • e-g interaction: forward or backward
  • each e and g may form two saltbridges with partner (i+2 and i+5)
  • i+2 = e - g´ two positions towards the C-term,
  • i+5 = 5 positions towards the N-terminal
  • AP-1- a bZip prototypeThe AP-1 family
  • The AP-1 (activator protein 1) transcription factor is a dimeric complex that comprises members of the
  • JUN and FOS,
  • ATF (activating transcription factor) and
  • MAF (musculoaponeurotic fibrosarcoma) protein families.
  • The AP-1 complex can form many different combinations of heterodimers and homodimers,
  • The specific combination determines the genes that are regulated by AP-1
  • Jun-Jun, Fos-Jun
  • low abundance in resting cells, strongly induced upon various stimulation
  • Response element
  • Palindromic TRE (TGASTCA) - The classical DNA response element for AP-1 is the TPA-responsive element (TRE), so called because it is strongly induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).
  • DNA binding of the AP-1 complex to the TRE sequence is rapidly induced by growth factors, cytokines and oncoproteins
  • AP-1 function
  • AP-1 activity can be regulated by dimer composition, transcription, post-translational modification and interactions with other proteins.
  • Two of the components of AP-1 - c-JUN and c-FOS - were first identified as viral oncoproteins.
  • However, some JUN and FOS family proteins can suppress tumour formation.
  • The decision as to whether AP-1 is oncogenic or anti-oncogenic depends on the cell type and its differentiation state, tumour stage and the genetic background of the tumour.
  • AP-1 can exert its oncogenic or anti-oncogenic effects by regulating genes involved in cell proliferation, differentiation, apoptosis, angiogenesis and tumour invasion.
  • AP-1 might be a good target for anticancer therapy.
  • Oncogenic activation - what alterations?b ZIPb ZIPTAD
  • v-Jun
  • The protein encoded by the avian sarcoma virus 17 oncogene v-Jun shows increased transforming activity compared with c-Jun, its normal cellular counterpart.
  • v-Jun differs from c-Jun in three important ways that might explain its transforming potential: (1) deletion of the delta domain - Jnk docking?, (2) single amino-acid substitutions that change a phosphorylation sites and (3) site that is recognized by the redox factor Ref1
  • a common principle that underlies oncogenic mutations - to escape regulation by kinases or other modifying enzymes, leading to constitutive activity.RasRac1/cdc42?RafMEKK1ASK1MEKK2/3MEK1/2MKK7MKK4MKK3MKK6TYERK1/2JNK1/2p38ATF2c-JunMkn2HSF-1c-MybBCL6MAPKAPK2Elk-1Mnk1Sap1aMEF2cCHOPEnd-point of MAPK signallingMAPKKKMAPKKPPTYMAPKMAPKTranscriptional outputRegulation Jun
  • Expression / abundance determines dimer equilibrium
  • Jun: positive autoregulatory loop
  • TPA  c-Fos ass. with low abundance c-Jun  Fos/Jun dimer  binds TRE in c-Jun promoter  c-Jun more of active Fos/Jun dimer
  • Positive regulation of Jun transactivation through JNK-mediated phosphorylation of TAD
  • Kinase-docking dep on -domain (recently challenged)
  • -domain (27aa) deleted in v-Jun
  • response to various stress-stimuli
  • Negative regulation of Jun DNA-binding through CK2-phosphorylation of DBD
  • phosphorylation of T231, S243, S249  reduced DNA-binding
  • Kinase = casein kinase II (≈constitutive)
  • v-Jun har mutert S243F  hindrer phosphorylation omkr øker AP-1 aktivitet 10x
  • TPA-stimulation  rapid dephosphorylation (trolig activation of fosfatase)  økt DNA-binding
  • Transcriptional and post-translational activation of AP-1CREBThe CREB-family - bZIP-factors mediating cAMP-response in the nucleus
  • The cAMP response mediated by a classical bZIP
  • binds CRE (cAMP responsive elementer): TGACGTCA
  • Binds as dimers
  • Signalling pathway
  • Hormone or ligand  membrane receptor  G-prot stimulates adenylate cyclase  [cAMP] cAMP binds R-subunits of PKA  active catalytic C-subunit liberated  C migrates to the nucleus  RRxS-sites in target proteins becomes phosphorylated - including CREB´s TAD  CREB recruits the coactivator CBP  genes having CREs becomes activated
  • ACgabcAMPCytoplasmDissociationNuclear translocationCPKACPhosphorylationCCPCBPPPCREBNucleusTarget gene activationSignalling through cAMP and PKA to CREBSeveral genes + Alternative splicing generates several variants
  • Distinct gene products, such as:
  • CREB
  • CREBP1
  • CREM
  • ATF1-4
  • Alternative splicing in CREM
  • generates isoforms acting both as activators and repressors
  • Two main classes of CRE-binding TFs
  • Activators (CREM, ATF-1)
  • Repressors (CREM-, -, -, ICER, E4BP4, CREB-2)
  • Domain structure of cAMP-responsive factorsATGATGATGTGAATGTAATAATAATAAATGATGTGATAATAATAATAGAlternative splicing produces both activators and repressorsQ1KIDQ2bZIPCREB1CREB-aActivatorsCREB-DCREB-D14InhibitorsCREB-D35CREMCREM-tActivatorCREM-aCond.ActivatorInhibitorS-CREMICERInducibleinhibitorATF1CREB - endepunkt for flere signalveiergabbZIP (uten TAD)ICERCCompetitionInactive heterodimersRepressor TURN OFFTurning off the response- the ICER strategyACcAMPCytoplasmDissociationNuclear translocationCPKACPhosphorylationCPCBPPPCREBNucleusTarget gene activationbHLH: helix-loop-helixHelix-loop-helix-family: common DBD-structure
  • large family involved in development, differentiation etc
  • Hundreds of characterized members from yeast to humans
  • Members central in neurogenesis, myogenesis, haematopoiesis,
  • bHLH resembles bZIP, but dimerization is achieved by an interrupted coiled coil
  • two amfipathic helices separated by a loop: helix-loop-helix = dimerization interface
  • Larger dimer-interface than in bZIPs
  • basic region N-terminally like for bZIPs
  • Ferre-D'Amare et al. (1993) Recognition by Max of its Cognate DNA Through a Dimeric B/HLH/Z Domain. Nature 363 pp. 38 (1993)Helix-loop-helix-family: 3D DBD-structure
  • 3D-structure Max-Max/DNA
  • Dimer = parallel lefthanded “4-helix bundle”
  • loop binds together helix 1 and 2
  • helix 1 and 2 almost parallel
  • loop close to DNA
  • b-region = extension of helix 1
  • Ferre-D'Amare et al. (1993) Recognition by Max of its Cognate DNA Through a Dimeric B/HLH/Z Domain. Nature 363 pp. 38 (1993)HLH-structures: MyoD-DNA and Pho4p-DNAPho4pMyoDHelix-loop-helixYeast Regulatory Protein Pho4; DNA Binding Domain;Myod Basic-Helix-Loop-Helix (bHLH) domain complexed with DNASome bHLH = bHLH-ZIP
  • characteristic feature: helix 2 is extended and becomes a ZIP-helix
  • Eks Myc, Max
  • LLLLLbZIPLLLLLHLHbZIPLLLLLbZIPLLLLLbHLH binding sites = E box (CANNTG)
  • First characterized in immunoglobuline heavy chain gene enhancers (mE1-mE5)
  • Critical response element: CANNTG called E-box
  • E-boxes later found in a series of promoters/enhancers that regulate cell type specific genes (muscle-, neuronal-, pancreatic-specific genes).
  • E-boxes are recognized by E-factors, such as the dimer E12+E47 (alternative splice-variants from the E2A gene)
  • Six different classes of bHLH proteins
  • Class I: ubiquitous (E12, E47, E2-2)
  • Expressed in many tissues, form homo- and heterodimers binding E-boxes
  • Class II: tissue specific (MyoD, myogenin, Atonal...)
  • Most members inable to homodimerize, but form heterodimers with class I partners
  • Class III: growth regulators (Myc, TFE3, SREBP-1,...)
  • These are of the bHLH-ZIP type
  • Class IV: Myc-partners (Mad, Max)
  • Class V: HLH without DNA-binding properties (Id, emc,...)
  • Function as negative regulators of Class I and II
  • Class VI: bHLH with proline in basic region
  • Example.: Drosophila hairy, enhancer of split
  • Class VI: with bHLH-PAS domain
  • Eks.: Aromatic hydrocarbon receptor, hypoxia-inducible factor 1a
  • Myc- a prototype bHLHZipHLHbasicA bHLH-Zip prototype: Myc- positive regulator of cell growth
  • Structure:
  • 64 kDa b-HLH-ZIP
  • Unable to form stable homodimers
  • Found in the cell as stable heterodimers with Max
  • HLHbZIPLLLLLbZIPLLLLLBrief biology
  • Involved in an extraordinarily wide range of cancers
  • One of the earliest oncogenes identified
  • Translocated in Burkitt´s lymphoma  Myc
  • Mitogenic stimulation  Myc
  • low level (2000 molecules/cell; half life 20-30min)  after growth stimulation 5000 molecules/cell  medium level
  • +Myc  Proliferation
  • - serum, - growth factors  Myc 
  • ectopic Myc expression forces cells into S-phase
  • antisense Myc blocks S-phase entry
  • +Myc  Differentiation
  • Normally down-regulated upon differentiation
  • Myc as oncogene, enhanced expression  transforming, lymphoma
  • +Myc  Apoptosis
  • Yin-yang interaction with other TFs: Myc-Max versus Mad-Max
  • Other actors in the play :
  • Max
  • Max: abundant, stable, not regulated by growth factors
  • Max forms DNA-binding homodimers
  • Max lacks TAD and functions as a repressor
  • Mad
  • Max forms heterodimers also with Mad and Mxi1
  • Active repressor
  • Interaction with Sin3
  • Mxi1 functional analogue to Mad
  • differentiation  induction of Mad, Mxi1
  • Myc-Max proliferating  Mad-Max differentiating
  • A family of playersProliferationDifferentiationMycMaxMadMaxMaxMaxTADReprThe Myc-networkMxi-1ProliferationDifferentiationMaxMad3MaxMad4Mad1c-MycMntMad1c-MycMaxMax
  • Mad1
  • Upregulated during terminal differentiation
  • Role in cell cycle withdrawal
  • Negative regulator of proliferation-associated c-Myc target genes
  • ActivatorRepressorSin3E-boxHDACRepression ofTarget genesActivation ofTarget genesMyc-networkAn avalanche of targets
  • Patterns of target genes
  • Genes repressed = proliferation arrest genes
  • Cell cycle genes activated = cdk4, cyclin D2, Id2, cdc25A
  • Apoptosis = p19ARF induced by Myc
  • Growth - size or division rate?
  • Myc may regulate growth rate (increase in cell mass & size), not only division rate
  • Effect on increase in cell mass & size: fits with many target genes in ribosome biogenesis, energy and nucleotide metabolism, translational regulation
  • Cdk2Cdk2Cdk2PPPc-Myc controls cell cycle genesCyclin D1Cdk4pRbp107E2Fc-MycBin-1Cyclin ESeq.Pr ?Cdc25ACyclin Ep27Kip1Cyclin ECyclin ECdk2Cell cyclec-Myc controls cell cycle genesCell cycleAn extended network- role for Myc as both activator and repressorMyc repression:getting a grip on activators
  • Myc repression results, not from direct binding to DNA by Myc-Max, but rather from their interaction with positively acting factors
  • Myc = anti-Miz-1
  • Miz-1 induces arrest by induction of CDKI (p15INK4B) through binding to INR
  • Myc binding to Miz-1 block this induction
  • Down-regulation of Myc - release of Miz-1 - CDKI induction
  • Myc and Mad mediate histone acetylation and deacetylation
  • HAT/HDAC activities manifested at promoters of Myc target genes (ChIP)
  • Myc-binding correlates with increased acetylation of H4 close to E-boxes, H3 not altered, dep on box II
  • HATTRRAPTIP60INI1Swi/SnfMyc-Max network controls Histone acetylation/deacetylation
  • Mad associates with Sin3, which binds HDAC
  • Myc associates with the coactivator TRRAP
  • Myc Box II = interaction domain
  • TRRAP = subunit of several HAT-complexes
  • hGCN5/PCAF and Tip60/NuA4
  • Dominant negative TRRAP inhibits Myc transformation
  • TIP48/TIP49 also associated with Myc TAD
  • N-CoR = a corepressorRpd3= histon deacetylaseSin3= en linkMaxCloses chromatinMadMyc-Max network controls Histone acetylation/deacetylationMyc/Mad-induced local alterations in chromatinMax-Myc-TRRAP complex binds to E-boxescauses acetylation of H4leads to induction of target genesMax-Mad complex binds to E-boxescauses deacetylation of H4leads to repression of target genesWhy only H4 acetylation?
  • Interesting explanations - histone code: assuming that Myc-TRRAP specifies only a portion of the code
  • ?H4Not H3Enigma - a gap between…
  • Biological effects ≠ molecular mechanims
  • Mountain of biological effectsImplicated in wide range of cancersA relatively weak transcriptional regulator of uncertain target genes
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