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Protein networks, moonlighting and fuzziness

Protein networks, moonlighting and fuzziness. Peter Tompa. Institute of Enzymology Hungarian Academy of Sciences Budapest, Hungary. Science of networks. social networks transportation networks intern et feeding networks neuron al networks intracel lular networks

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Protein networks, moonlighting and fuzziness

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  1. Protein networks, moonlighting and fuzziness Peter Tompa Institute of Enzymology Hungarian Academy of Sciences Budapest, Hungary

  2. Science of networks • social networks • transportation networks • internet • feeding networks • neuronal networks • intracellularnetworks • metabolicnetworks • expression networks • protein-protein interactions (interactome)

  3. The TAP tag method Gene-specific casettecontaining TAP tag by PCR Insert at 3’ end of ORF by homologous recombination Extract of recombinant cells Tandem affinity purification (TAP) Analysis (SDS-PAGE, MS) Rigaut et al. (1999) Nature Biotech. 17, 1030

  4. Yeasttwo-hybrid method (Y2H) bait prey Fields and Song (1989) Nature 340, 245

  5. hub node edge The yeast interactome Uetz et al. (2000) Nature 403, 623

  6. Comparison of TAP-tag and Y2H Y2H TAP-tag • real complexes • tag interference • strength of interaction • stoichiometry • one condition • pairwise interactions • no posttransl. mod. • third partner? • bait-prey nonprod. • compartmentalisation - comparison? - partial coverage!

  7. How to relate Y2H and TAP-tag data? TAP Y2H tag B A B A C E D C A L F G H A x E K I J A H x

  8. “spoke” model better than “matrix” TAP spoke (pairwise interactions of bait): a-b, a-c, a-d, a-e tag B A C E D L F matrix (pairwise interactions of all components): a-b, a-c, a-d, a-e, b-c, b-d, b-e, c-d, c-e, d-e G H K I J Bader (2002) Nat. Biotech. 20, 991

  9. Deane et al. (2002) MCP 1, 349 Partial coverage (Y2H yeast data)

  10. Partial coverage (HTS vs. low-throughput) Gandhi (2006) Nat. Gen. 38, 285

  11. Partial coverage (all interactions) Human 25.464 Yeast 16.069 16 Worm 5.625 Fly 25.587 Gandhi (2006) Nat. Gen. 38, 285

  12. Erdősi-Rényi Barabási-Albert Desrition of real networks? Uetz et al. (2000) Nature 403, 623

  13. Random (RNN) or scale-free (SFN)network?

  14. IDP functional advantages… …may also manifest in hub function? - large binding surface - adaptability (binding promiscuity) - increased rate of interactions - regulation by post-translational mod.

  15. DIP  8

  16. HUMAN WORM YEAST FLY  10

  17. What biological processes hubs are involved in ?

  18. Further features in hubs ? HUMAN WORM YEAST FLY floating cutoff - top 10% -

  19. disorder (Yeast) hubs contain more of: repeats low-complexity length Dosztányi (2006) J.Proteome. Res. 5, 2985

  20. disorder (Yeast) hubs contain a higher frequency of: repeats low-complexity Dosztányi (2006) J.Proteome. Res. 5, 2985

  21. date party Correlation coeff. of expression „Party” and „date” hubs Han et al. (2004) Nature 430, 88

  22. Date hubs are more disordered than party hubs… Ekman (2006) Genome Biol. 7, R45

  23. Disorder in hubs Dunker (2005) FEBS J. 272, 5129

  24. HMGA (high mobility group A) PRGRP PRGRP PRGRP IRF-1 ATF/c-Jun SRF NF-Y NF-kB p50 Oct-6 HIPK-2 Reeves (2001) Gene 277, 63

  25. HMGA: architectural transcription factor citokynes IL-1b, IFN-g tumor promoters phorbol ester environm. stress hypoxia, virus diff. factors RA, Ca++ ionophores growthfactors EGF, FGF, PDGF HMGA apoptosis phosphorylation transcription enhancosome cancer overexpression Neg. gene reg. competition proliferation differentiation chromatin remodeling AT hook

  26. B C A Moonlighting: multiple functions of proteins

  27. The interplay of Ca2+ and CST in calpain regulation Ca++ Ca++ CST B C A A

  28. The interplay of Ca2+ and CST in calpain regulation Ca++ Ca++ CST C A A

  29. CST subdomains A and C activate -calpain - experiment: 2001 Sept. 11 -

  30. 0’ 1’ 2’ 4’ 1’ 2’ 4’ MAP2c Activation is a result of Ca2+-sensitisation

  31. Calpain activation in COS-7 cells 1 mM Ca2+ 1 mM Ca2+ 20 mM Ca2+ 20 mM Ca2+ 0 min PenA+PenC 30 min PenA+PenC

  32. Inhibition of Cdks in cell-cycle regulation - promoting assembly of complex - activity within the complex

  33. Excitation-contraction coupling in muscle DHPR

  34. Regulation of actin polymerisation thymosin b4 WH2 actin - promoting actin polymerisation in actobindin (WH22) and in ciboulot(WH23) - activation of other target(s)

  35. The securin story normal chromosome segregation Inhibition of separase expression: Waizenegger (2002) Curr. Biol. 12, 1368

  36. The securin story - securin knockout - Jallepalli (2001) Cell 105, 445

  37. The securin story

  38. Other multifarious effectors

  39. TiBS 1996

  40. The concept of moonlighting (multitasking)

  41. The concept of moonlighting (multitasking) PutA neuroleukin PGI neuropilin GAPDH aconitase UDG IRE-BP

  42. Structural background of „classical” moonlighting - the use of different surfaces of globular proteins - neuroleukin F6P G6P PGI Cell type 2 Cell type 1 Jeffery (2004) DDT: Targets

  43. Structural disorder throws new light on moonlighting Tompa et al. (2005) TiBS 30, 484 Binding a partner in different conformations Binding different partners DHPR C WH2 Structural reorganisation around the partner p21

  44. Fuzziness: disorder in the bound state Curr. Opin. Struct. Biol. 2002

  45. IDPs do fold upon binding

  46. IDPs do fold upon binding … CBP KIX CREB KID

  47. … but do they become fully folded? CBP KIX 2400 AAs CREB KID 365 AAs

  48. Kd: 0.7 mM … well, they don’t Kd: 3.1 mM

  49. Disorder in the bound state: fuzziness Tompa and Fuxreiter (2008) TiBS33, 2-8

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