6. Cellular immunity Antihelminthic immunity, allergic disease
Cytokines regulate lymphoid cells at all stages
of their development, differentiation, and activation
7. 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
8. 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
9. 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
11. 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
12. 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.
13. 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
14. 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
15. 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
16. 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
17. Expression of Cybr mRNA
• Cybr
• GEP, Cytohesin family (GRP1/ Cytohesin-3)
• ARFs
Introduction
19. 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
20. 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
22. 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
25. 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
26. 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
28. 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
29. 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
30. 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
32. 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
33. 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)
36. 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
37. Effect of His-Cybr on
cytohesin-1 activation of rARF1
1
2
3
GTPg[S35]bound(pmol)
48. 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
49. 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
50. 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)
51. 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
