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Cybrlecture02A

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Cybrlecture02A

  1. 1. Cybr, a Cytokine-Inducible Protein that Binds Cytohesin-1 and Regulates Its Activity Pingtao Tang MD, Ph.D
  2. 2. A simplified view of the development of T cell
  3. 3. Th2Th1 Naïve CD4+ T cell IL--4 Interleukin (IL)-12 & IL-4 promote Th1and Th2 polarization, respectively IL-12, IL-2 IFN-r
  4. 4. Cellular immunity Antihelminthic immunity, allergic disease Cytokines regulate lymphoid cells at all stages of their development, differentiation, and activation
  5. 5. TTh1 Naïve CD4+ T cell IL-12 (10ng/ml) Identification > 200 genes, Cybr  B3-1/36.3K zipper-containing protein  Cytohesin-binding protein HE  Plechstrin homology, Sec7 and Coiled/coil Domain Binding Protein (PSCBP) A cytokine-inducible protein that binds cytohesin-1 and regulates its activity Microarray slides containing 7,000 cDNA
  6. 6. Structure of Cybr • Chromosome 2 band q11.2, cDNA from human NK/T cell; This protein is expressed weekly in resting NK/T cell. • PDZ domains, 80-100 residue domains , function in the organization and localization of signalizing complexes to various membrane sites • Leucine zipper region with five heptad repeats, contains 2 leucine zipper domains • C-terminal Nuclear Targeting Singnal, and it dose not have any hydrophobic regions; No Alanine /Proline rich * General Receptor for Phosphoinositides 1-associated scaffold protein (GRASP) * Plechstrin homology, Sec7 and Coiled/coil Domain Binding Protein (PSCDBP), B3-1 =CBP=Cybr PDZ Leu 77 166 202 329 334 359 H2N H2N COOH COOH A/P PDZ Leu 15 71 96 186 196 262 392 GRASP Cybr s C-C188
  7. 7. h-Cybr MSLQRL- -LQ HSS-- ----- -NGNLAD FCA ----- ---G- PAYSSYST LT m-Grasp MTLRRLR KLQ QKEEA TAAPD PAGRAPD SEA ARAAP LPSGP PAAAAPPG AP Consens us M L RL LQ G D A G PA h-Cybr GS---LT MND ---NR RIQML ADTVATL PRG RKQLA LTRSS SLSDFSWS QR m-Grasp GEELYAA LED YHPAE LYRAL AVSGGTL PR- RKGSG F-RWK NFTQSPEQ QR Consens us G D L A TLPR RK R QR h-Cybr KLVTVEKQD N ETFGFEI QSY RPQNQNA CSS EMFTL ICKIQ EDSPAHCAGL m-Grasp KVLTLEKG DN QTFGFE IQTY GLHH REEQRV EMVTFV CRVH ESSP AQLAG L Consens us K T EK DN TFGF EIQ Y EM T C E SPA AGL h-Cybr QAGDVLA NIN GVSTE GFTYK QVVDLIRSSG NLLTI ETLNG TMILKRT ELE m-Grasp TPGDTIA SVN GLNVE GIRHR EIVDIIKASG NVLRL ETLYG TSIRK-A ELE Consens us GD A N G EG VD I SG N L ETL G T I K ELE h-Cybr AKLQVLK QTL KQKWVEYRSL QLQEHRLL HG DAANCP S-LE NMDLDELS LF m-Grasp ARLQYLK QTL YEKWGEYRSL MVQEQRLV HG LVVKDP SIYD TLESVRSC LY Consens us A LQ LKQTL KW EYRSL QE RL HG PS L h-Cybr GPLPGPG PAL VDRNR LSSES SCKSWLS SMT MDSED G-YQT CVSEDSSR GA m-Grasp GAGLLPG SLP FGP-L LAAPG SARGGAR RAK GDTDD AVYHT CFFGGAEP QA Consens us G PG L S D D Y T C A h-Cybr FSRQTST DDE CFIPK EGDD- ----FLR -RS SSRRN RSISN TS--SGSM SP m-Grasp LP-PPPP PAR ALGPS SAETP ASVLFPA PRS TLSRS ASVRC AGPGGGGG AP Consens us P F RS R S G P h-Cybr --LW-EG NLS SMFGT LPRKS RKGSVRK QLL KFIPG LHRAV EEEESRF m-Grasp GALWTEA REQ ALCGA GLRKT KYRSFRR RLL KFIPG LNRSL EEEESQL Consens us LW E G RK S R LL KFIPG L R EEE ES Amino acid sequences of human (h) and murine (m) GRASP
  8. 8. GEP GRP1/Cytohesine-3 GRASP Cytohesin-2 Cytohesin-1 ? Cybr • Enhance association with plasma membrane • May modulate signaling by GTPase GDP/GTP- exchange Protein Does it interact with GEP?
  9. 9. GEP GRP1/Cytohesine-3 GRASP Cytohesin-2 Cytohesin-1 ? Cybr • Enhance association with plasma membrane • May modulate signaling by GTPase ARF-GDP GTP GDP GAPPi GTPase-activating Protein GDP/GTP- exchange Protein (Cytosolic) (Membrane-associated) ARF-GTP
  10. 10. Objectives • Analysis of expression of Cybr mRNA in human cells and tissues. • Interaction of Cybr with cytohesin-1 and the regions of interaction between Cybr and cytohesin-1. • Determine whether Cybr influences cytohesin-1 biochemical activity.
  11. 11. Expression of Cybr mRNA Purification of Proteins • Cytohesin-1 and cytohesin-4 • Histidine / GST fusion Cybr • rARF1 and nARF • Cybr • GEP, Cytohesin family • ARFs Interaction between Cybr and Cytohesin-1 • GSH-Sepharose beads put-down: Using recombinant GST fusion Cybr bound to GSH-Sepharose beads that were then incubated with the Cytohesin-1 • Yeast Two hybrid assay (Harris, Edith,S) • Coimmunoprecipitation of overexpressed Cybr and Cytohesin-1 a) Cybr sequences required for interaction with Cytohesin-1 b) Region of Cytohesin-1 required for interaction with Cybr Introduction Effect of Cybr on Cytohesin-1 Activation of ARF • His-tagged/ GST-tagged Cybr • Concentration, Time • rARF1 and nARF1/3 • Sec7 and coiled-coil domain Summary
  12. 12. ARF-GDP GTP GDP GAP GEP Pi GTPase-activating Protein GDP/GTP- exchange Protein (Cytosolic) (Membrane-associated) Sensitive: Sec 7 domain ~200- AA (brain) Brefeldin A(BFA) Geal 1, Geal 2 ~160- kDa (yeast) ~230- AA (yeast) Insensitive: Cytohesin family ~ 50a-kDa (GRP1/Cytohesin-3) Regulation of ARF activity by GEPs and GAPs ARF-GTP ARF-GEP100 (fungal fatty acid metabolite) Big1/2, ~200/~190 kDa
  13. 13. B2-1/ cytohesin-1/Sec7A Cytohesin-2/ARNO/Sec7B Sec7 PHCC Sec7 PHCC PHCC Sec7 Sec7 PHCCGRP1/ cytohesin-3/ARNO3 Cytohesin-4/dj63g5.1 CC Sec7 PH P Coiled-coil Sec7(Arf GEF) PIP3-binding Proline-rich EFA6/PSD/cytohesin-5 Tic/cytohesin-6 HCAA67/cytohesin-7 Sec7 PH CCP P Sec7 PHP P CC P CC Sec7 PH P CC P Members of the cytohesin family
  14. 14. Class II: 180 amino acids Class III: ARF4, ARF5 ? Golgi and ER to Golgi pathways Class I: 181 amino acids, highest homologies ARF1, ARF3: Intracellular vesicle traffickingARF2 GEPs: GTP binding Membrane recycling, remodeling the cell surface, rearranging cytoskeleton? 175 amino acids, lowest homology ARF6: plasma membrane EFA6 and cytohesin-2 and ARF GEP100: GTP binding ADP-ribosylation Factors, ARFs • Size, sequence indentity, gene structure and phylogentic analysis • A specific interaction and concomitant localization with a GEP • In numerous vesicular trafficking pathways
  15. 15. Expression of Cybr mRNA • Cybr • GEP, Cytohesin family (GRP1/ Cytohesin-3) • ARFs Introduction
  16. 16. Distribution of Cybr mRNA in human cells and tissues Pripheral blood mononuclear cells, PBMCs
  17. 17. Expression of Cybr mRNA Purification of Proteins • Cytohesin-1 and cytohesin-4 • Histidine / GST fusion Cybr • rARF1 and nARF • Cybr • GEP, Cytohesin family (GRP1/ Cytohesin-3) • ARFs Introduction
  18. 18. Purification of cytohesin-4 and cytohesin-1 A. Protein Coomassie Staining B. Western blot (anti-cyto4 antibody) Cytohesin-1 Cytohesin-4 Cytohesin-1 Cytohesin-4 47 kDa 46 kDa 111.4 79.6 61.3 49.0 36.4 24.7 19.2 13.1 kDa
  19. 19. Fractionation of ARF1 on Ultrogel ACA54 31 33 35 37 38 40 42 44 46 48 50 52 54 55 56 58 60 62 64 66 68 70 72 85 86 88 90 0.5 1 1.5 GTPgSbound(pmol) 5 10 15 Protein(ug/10ul) 25 50 75 100 No. GTPrS bound Protein 79.6 9.3 13.1 19.2 36.4 61.3 111.4 24.7 kDa
  20. 20. Purification of native ARF1/3 Supernatant, treated with 25% NH4(SO4) Supernatant, dialyzed against TEDNS Bovine brain 200g, homogenization 12,000 rpm, 1h 12,000rpm, 1 h Supernatant, treated with 70% NH4(SO4) 12,000rpm, 1h Pellets, dissolved in TEDNS 37,000rpm, 1h ACA54 column SDS PAGE and Western blot Fractions contracted G25 column Fractions DEAE-Sephacel column Eluted by a gradient of 0-150 mM NaCl Assayed protein and ARF activation of CTA- catalyzed ADP- ribosylagmatine formation Fractions Contracted ACA54 column SDS PAGE, Western blot Identification of nARF1/3
  21. 21. Purification of nARF from ACA 54 Column 1 Column 2 rARF1 65 67 69 71 73 75 77 79 81 83 85 87 89 65 67 69 71 73 75 77 79 81 83 85 87 Fraction (11 ml/Fr) 19.2 13.1 24.7 36.4 49.0 61.3 79.6 172.6 111.4
  22. 22. Identification of nARF1/3 activity in fraction from DEAE chromatography nARFactivity-nmol/h() Protein-g/ml() Fraction No. 0 50 100 150 NaCl-mMol/L() 200 0 2 4 6 8 0.1 0.2 0.3 0.4 0.5 0.6 25 50 75 100 125
  23. 23. Identification of nARF1/3 112 108 104 100 112 108 104 100 96 Anti-ARF5 antibody Anti-ARF1/ 3 antibody Long AcA54 column fraction M 88 90 92 96 98 100 102 104 106 108110 112rARF1 A. Protein Staining B. Western blot
  24. 24. Cloning Cybr sequence into GST fusion vector Thrombin Stop codonsMCS PCR’ primers Sal I 5’ GAC A GT CGA C AT ATG TCT TTA CAAAGG CTC CTG C 3’ Not I 5’ AT CT G CGG CCG C AG TCA AAA GCG ACT TTC TTC CTC 3’ pGEX-4T-1PCR’ Product 5’ 3’ Sal I Not I
  25. 25. Purification of GST-Cybr 172 111.4 79.6 49 61.3 A Protein staining B. Western blot (anti-GST antibody) kDa E1 E 2 E 3 E4 E5 E6 Resin E1 E 2 E 3 E4 E5 E6 Resin
  26. 26. Purification of His-Cybr Imidazole (mM) 100 400 100 400 A. Protein staining B. Western blot (anti-5xHis antibody) C1 Mr E1 E2 E1 E2 C1 Mr E1 E2 E1 E2
  27. 27. Purification of proteins Interaction between Cybr and Cytohesin-1 GSH-Sepharose beads pull down: Using recombinant GST fusion Cybr bound to GSH-Sepharose resin that were then incubated with the Cytohesin-1 Introduction Distribution and Expression of Cybr
  28. 28. Ovalbumin (200 mg /ml) over night at 4 0C Washed with PBS, x2 GST-Cybr or GST was incubated with the resin(1h, 4 0C) BSA (100mg/ml), Ovalbumin (200 mg/ml), 1h 4 0C Washed with PBS, x 2 His-tagged C1 was incubated with the resin (1h, 40C) in PBS with BSA(10 mg/ml), Ovalbumin 20 (mg /ml) Washed with PBS, x 2 Eluted with 15 mM GSH in 50 mM Tris-HCl, pH 8.0 SDS PAGE for Silver staining, Western blotting GSH-Sepharose 4B resin Binding of the Cybr to Cytohesin-1
  29. 29. 19.2 36.4 61.3 49.0 111.4 79.6 172 61.3 79.6 111 172 49.0 36.4 24.7 19.2 kDakDa GST + - - - + - - + - - - + - - GST-Cybr - + + + - - - - + + + - - - His.TaqC1 + + + - - + + 12 48 24 + + + - - + + 12 24 48 Anti His antibody Anti GST antibody 24.7 Binding of the cytohesin-1 to immobilized GST-Cybr
  30. 30. To test the region of Cybr / Cytohesin-1 responsible for the association with cytohesin-1/ Cybr Cytohesin 1 and mutants PCR product pAS1(bait plasmid) (GAL4-binding domain) Trp-HA-Cyto1 Cybr and mutants PCR product pACT (target expression plasmid) (GAL4 trascription-activation domain II) Leu--His-CBP Y190 Leu- - Trp- -His- selective media B-galactosidase activity (blue/white) Two yeast hybrid assay
  31. 31. Interaction between cytohesin 1 and Cybr Sec7 PHCC Cytohesin-1 77 166 202 359 Leu Leu 174 357 153 208 LeuPDZ PHSec7CC (Harris, Ediths, 2000)
  32. 32. Coimmunoprecipitation of overexpressed Cybr and cytohesin-1 mutants Construct for expression of Flag-tagged Cyto-1 (and fragments), X-press-tagged Cybr
  33. 33. Coimmunoprecipitation of overexpressed cytohesin-1 and Cybr mutants Flag-tagged Cyto-1 (and fragments), X-press-tagged Cybr (fragments)
  34. 34. Purification of proteins Interaction between Cybr and Cytohesin-1 Introduction Effect of Cybr on Cytohesin-1 activation of ARF • His-tagged/ GST-tagged Cybr • Concentratin, Time • rARF1 and nARF1/3 • Sec7 • Coiled-ciol Summary
  35. 35. Effect of His-Cybr on cytohesin-1 activation of rARF1 1 2 3 GTPg[S35]bound(pmol)
  36. 36. Effect of GST-Cybr on cytohesin-1 activation of rARF1 GST - + - - - - - + - - - - - 0 0.5 1 1.5 GTPg[S35]bound(pmol) Cyto1 - - - + + - + + Sec7 + + + + ARF1 - - - - - + + + + + + + + GST-Cybr - - + + - - - - - + + + +
  37. 37. Effect of Cybr on Cytohesin-1 activation of rARF1 0.5 1 1.5 0 3 6 GST-Cybr or GST (pmol) g[S35]bound(pmol) Cybr+Cyto1+ARF1 GST+Cyto1+ARF1 Cybr+ARF1 GST+ARF1 Cybr
  38. 38. Effect of GST-Cybr on cytohesin-1 activation of nARF1/3 Cybr1+ARF+C1 Cybr2+ARF+C1 Cybr3+ARF+C1 ARF Cybr+ARF ARF+C1 GST+ARF+C1 Time (min) 0.5 1 1.5 20 40 GTPg[35S]bound(pmol)
  39. 39. Effect of His-Cybr on Cytohesin-1 activation of rARF1 2 4 6 8 0.2 0.4 0.6 0.8 1 1.2 Time (min) C1+ARF1+Cybr C1+ARF1 ARF1+Cybr ARF1
  40. 40. Interaction between cytohesin 1 and Cybr Sec7 PHCC Cytohesin-1 77 166 202 359 Leu 153 208 LeuPDZ CC
  41. 41. Effect of Cybr on Sec7 activation of rARF1 0.5 1 1.5 3 6 GST-Cybr or GST (pmol) g[S35]bound(pmol) Cybr+Cyto1+ARF1 Cybr+Sec7+ARF1 GST+Cyto1+ARF1 GST+ARF1+Sec7 Cybr+ARF1 GST+ARF1 Cybr
  42. 42. CC Sec7 PH Coiled-coil Sec7(Arf GEF) PIP3-binding Members of the cytohesin family B2-1/ cytohesin-1 Cytohesin-2 Sec7 PHCC PHCC Sec7 Sec7 PHCCCytohesin-4 Sec7 Sec7 BIG1/2 Sec7(cytohesin-1)
  43. 43. How does Cybr regulate GEP activity? • Sec7, BIG1 and BIG2 (no coiled-coil) • Cytohesin-1, Cytohesin-2 and Cytohesin-4 (coiled-coil) • others
  44. 44. Cyto1 - + - + - - + + + - - - - - Cybr - - + + - + - + ++ - ++ - + ++ nARF1/3 - - - - + + + + + - - + + + 0.5 1 1.5 2 BIG1 complex - - - - - - - - - + + + + + Effect of His-Cybr on BIG1 complex activation of nARF1/3
  45. 45. Cyto1 - + - - - + + + - - - Cyto4 - - + - - - - - + + + Cybr - - - + - - + ++ - + ++ ARF1 - - - - + + + + + + + GTPgS35bound(pmol) Effect of His-Cybr on cytohesin-4 activation of ARF1 20 min 4 min 0.5 1 1.5
  46. 46. 0.2 0.4 0.6 0.8 1 1.2 2 4 6 8 C4+ARF1+Cybr C4+ARF1 ARF1+Cybr ARF1 C 0.2 0.4 0.6 0.8 1 1.2 2 4 6 8 C2+ARF1+Cybr C2+ARF1 ARF1+Cybr ARF1 B Time (min) Time (min) Effect of His-Cybr on GEPs activation of rARF1 2 4 6 8 0.2 0.4 0.6 0.8 1 1.2 A Time (min) C1+ARF1+Cybr C1+ARF1 ARF1+Cybr ARF1
  47. 47. 0.2 0.4 0.6 0.8 1 1.2 1.4 2 4 6 8 Time (min) [S35]GTPgSbound(pmol) C2+ARF1 C2+ARF1+Cybr ARF1+Cybr ARF1 0. 2 0.4 0.6 0.8 1 2 4 6 8 C2+ARF1 ARF1+Cybr C2+ARF1+Cybr ARF1 B Time (min) A Effect of His-Cybr on Cytohesin-2 activation of ARF1
  48. 48. Summary 1. Interaction between cytohesin-1 and Cybr both In vitro and vivo, is dependent on the coiled-coil domain. 2. Effect of Cybr on cytohesin-1 activation of ARF1, is dependent on the concentration and time of Cybr incubation. 3. Effect of Cybr on cytohesin (GEPs) activation of ARF1, is dependent on the presence of coiled-coil domain of cytohesin. (PNAS March 5, 2002, vol. 99 no. 5 2625-2629)
  49. 49. ACKNOWLEDGMENT NHLBI Dr. Martha Vaughan Dr. Joel Moss Dr. Vincent C. Manganiello Dr. Gustavo Pacheco-Rodrigues Dr. Ronald Adamik NIAMSD Tammy P. Cheng, Davide Agnello, John J O’Shea and Massimo Gadina, et al. Vaccine Research Center Chang-you Wu
  50. 50. Expression of hsp70 in zebrafish embryos A B C
  51. 51. Expression of hsp70 in zebrafish embryos
  52. 52. Expression of hsp70 in trangenic zebrafish embryos

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