Research Interests
description
Transcript of Research Interests
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Fluorescent Chemosensors for Biology: Visual Snapshots of Intramolecular
Kinase Activity at the Onset of Mitosis
Zhaohua DaiDepartment of Chemistry & Physical Sciences, NY
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Fluorescent and chiropticalprobes for metal ions
Research InterestsFluorescent probes for kinaseactivity in live cells
Zn2+, Mn2+, Hg2+
Das, D.; Dai, Z.; Holmes, A. E.; Canary, J. W. Chirality, 2008, 20, 585-591. Dai, Z.; Canary, J. W. New J. Chem. 2007, 31, 1708-1718.Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc. 2005, 127, 1612-1613.Dai, Z.; Xu, X.; Canary, J.W. Chirality 2005, 17, S227-233.Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004, 126, 11760Dai, Z.; Xu, X.; Canary, J. W. Chemical Communications 2002, 1414-5.
Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem. & Biol. 2007, 14, 1254-1260.Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, 14016-14017.
Tyrosine Kinase, PKC
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Zinc in Brain• More Zn2+ in brain than in any other organ• Zn2+ and Cu2+ are implicated in Alzheimer’s, Parkinson’s, and
Amyotrophic Lateral Sclerosis (ALS)• Complicated roles• Tools needed to image Zn2+ distribution and kinetics
N
NHO2S
R1
OR2
R3
TSQ, Zinquin
High sensitivy
Poor Zn(II)/Cu(II) selectivity
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Tailoring Tripodal Ligands for Zinc Sensing
Zhaohua Dai and James W. Canary, New J. Chem., 2007, 31, 1708-1718.
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Chiral Fluorescent Probes for Zn2+
1. Higher Zn2+/Cu2+ Selectivity Stereochemical Control 2. Better contrast Fertile Optical Information:
Differential Circularly Polarized Fluorescence Excitation (CPE)
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Zn2+ 11.0 7.1 8.95
Cu2+ 16.15 7.1 7.0
10-5 1 90*
Stereochemical Approach to Improved Zn(II)/Cu(II) Selectivity
15% acetonitrile/aqueous buffer pH 7.19* Z. Dai, et al. unpublished
Zn2+/Cu2+
Selectivity:
log
NN N
NN NH
N
H
H
N
NN N
N
H
H
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Fluorescence-detected Circular Dichroism (FDCD)
J-8100 Circular Dichroism System with FDCD Attachment
Nehira; Berova; Nakanishi; et al. J. Am. Chem. Soc. 1999, 121, 8681
F =
Two channels of data
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Differential Circularly Polarized Fluorescence Excitation (CPE)
Changes in F will be very large when changes in BOTH fluorescence AND circular dichroism are large.
A
IKF10
*0
a
b
A
A
b
a
b
a
b
a
FF
1010
CPE utilized only F part of FDCD raw data for analysis.
: CD ellipticity; : Fluorescence quantum yield.
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200 220 240 260 280 300 320 340-5
-4
-3
-2
-1
0
1
2
200 220 240 260 280 300 320 340
-5
0
5
10
15
320 360 400 440 480 5200
200
400
600
800
1000
CPE Reduces Background from Free Ligand
/nm
Rel
ativ
e In
tens
ity I f
Zn2+
/nm
Ellip
ticity
/
mde
g
Zn2+
/nm
CPE
F
Zn2+
Free ligand
[Zn(L)]2+
Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004, 126, 11760
NN N
N
H H
(S,S)-17
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200 220 240 260 280 300 320 340-30
-25
-20
-15
-10
-5
0
5
300 330 360 390 420 450 480 510 5400
200
400
600
800
1000
1200
CPE SELECTS AGAINST PROTEIN-BASED BACKGROUND FLUORESCENCE
/nm
Rel
ativ
e In
tens
ity I f
Lysozyme
Zn2+
CPE
F
/nm
Zn2+
Lysozyme
Lysozyme+
[Zn(L)]2+
Dai, Z.; Proni, G.; Mancheno, D.; Karimi, S.; Berova, N.; Canary, J.W. J. Am. Chem. Soc., 2004, 126, 11760
200 220 240 260 280 300 320 340-70
-60
-50
-40
-30
-20
-10
0
10
260 280 300 320 340-4
-2
0
2
4
6
Ellip
ticity
/
mde
g
Zn2+
/nm
NN N
N
H H
(R,R)-17
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Chiral Fluorescent Sensor for Hg2+
N
SO
HO
N NO
O
HO
O
O
OH
HOOC
N
SO
HO
N N
O
OH
O
COOH
1
2O
OH
O
COOH
COOH
We intend to use these ligands to further develop CPE.
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Colorimetric Mn(II) Sensor
N
N
N N
NaO
Br
SO3Na
5-Br-PAPS-Zn(II)-EGTADisplacement system
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Summary for Metal Sensors
• Achieved solid Zn(II)/Cu(II) selectivity through a stereochemical approach
• Developed a new approach for analysis: CPE• CPE may be used to improve contrast in detecting
metal ions by fluorescent, chiral ligands with low background
• CPE may be used to diminish interference from fluorescent non-analytes
• CPE needs further development
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Caged Sensors for Kinase Activity
Dai, Z.; Dulyaninova, N. G.; Kumar, S.; Bresnick, A. R.; Lawrence, D. S. Chem.
& Biol. 2007, 14, 1254-1260. Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, 14016-14017.
Light-Regulated Sampling of Protein Tyrosine Kinase Activity
Phe Arg Arg Arg Arg Lys amide
NH
O
O
N
N
O2N
O
O2N OCH3
OCH3
Snapshots of PKC Activity at the Onset of Mitosis
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Protein Kinase C
• Cell proliferation, apoptosis, differentiation, migration• Cause cancer, etc.• Tools are needed for probing, therapeutics
Nakashima, S. J. Biochem. 2002, 132, 669-675.
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PKC in Early Mitosis (G2/M)
Review: Black, J. D. Front. Biosci. 2000, 5, 406-423P. Collas et al J. Cell Sci. 1999, 112, 977-987.
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PKC II in G2/M Transition
A. P. Fields et al. J. Biol. Chem. 1994, 269, 19074-19080.A. P. Fields et al. J. Biol. Chem. 1996, 271, 15045-15053.
Target: lamin B Ser405
85K
Km (M): 4.9 (soluble) and 3.9 (envelope). IC50: 16 M
nocodazoleChelerythrine
Chelerythrine (PKC inhibitor ????)
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NBD-based Fluorescent Sensor for PKC
Phe Arg Arg Arg Arg Lys amide
NH
O
HO
N
N
O2N
O NBD-peptide
Yeh, R.-H.; Yan, X.; Cammer, M.; Bresnick, A. R.; Lawrence, D. S. J. Biol. Chem. 2002, 277, 11527-11532
Assay PKC PKC PKC
Radioact. 9.0±1.0 9.2 ±0.4 5.0 ±1.0
Fluoresc. 29 ±3 27 ±4 30 ±5
Km(M)
VIP
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In vivo Studies in HeLa cells
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Caged PKC Sensor
Phe Arg Arg Arg Arg Lys amide
NH
O
O
N
N
O2N
O
O2N OCH3
OCH3
Veldhuyzen, W. F. et al J. Am. Chem. Soc. 2003, 125, 13358-13359
KVIP
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Why Caged Sensors
• In cuvette: investigator controls the start and stop of enzyme catalyzed rxns
• In live cell: the cell controls the timing and during
• Caged sensors can be delivered in inert forms and activated on demand
• Give precise temporal control over sensor activity
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Real-time temporal probing of PKC activity
Veldhuyzen, W. F. et al J. Am. Chem. Soc. 2003, 125, 13358-13359
Phe Arg Arg Arg Arg Lys amide
NH
O
O
N
N
O2N
O
O2N OCH3
OCH3
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Studying MitosisMicroinjection
Phe Arg Arg Arg Arg Lys amide
NH
O
O
N
N
O2N
O
O2N OCH3
OCH3
PtK2 Cells: flat
Kangroo rat didneyepithelial cells
KVIP
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PKC in PtK2
S. Kumar
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VIP PKC Activity
Other kinases: Akt-1, AurB, Cdc-2, Plk1 (do not work on VIP) Nek2 (weakly)
S. Kumar
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before 0 min injection 2 min uncaging 3 min
Green Fl NBD
Red Fl70K dextran-Texas red
Coinjection of 200 M KVIP and 5 M 70K dalton texas red-dextran
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4 min 5 min 6 min 7 min
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0 min injection 2 min uncaging 25 min
Coinjection of 200 M KVIP and 5 M 70K dalton texas red-dextran
Mmc1.mov Mmc2.mov
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Injection with 200 M KVIP before NEBD
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9
t (min) relative to NEB
Rel
ativ
e Fl
uore
scen
ce
Total cellsNEBD Large
enhancement (>40%)
Small enhancement(<40%)
No enhancement
18 Yes 15 6 9 0
No 3 3
1.PKC activityaccompaniesNEBD.Which one?
2. PKC activitylevels off afterNEBD:
PKC off? orSensor gone?
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0 min injection 2 min uncaging 11 min
Coinjection of 200 M KVIP and 5 mM 70K dalton texas red-dextran (uncaging after NEBD )
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Injection with 200 M KVIP (Uncaging after NEBD)
Total cells Large enhancement (>40%)
Small enhancement(<40%)
No enhancement(within 5%)
Very smallEnhancement(within 15%)
16 0 0 14 2
1. No PKC activityright after NEBD?
2. Both PKC and phosphatase are active?
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14
Time (after NEBD)
I f
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Incubation with 1.5 M okadaic acid
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12 14 16 18
No PKC activityright after NEBD.
Total cells NEBD Large enhancement (>40%) Small enhancement(<40%)
No enhancement Little enhancement(around 15%)
10 Yes 10 0
0 8 2
Phosphatase inhibited
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High PKC inhibitor concentration (12 M) induced or blocked cells at prophase
65% of the cells (20 out of 31) are stuck at prophase
IINek2
IC50 1.3 M 11 nM no obs. inhibition
Tanaka, M. et al. Bioorg. Med. Chem. Lett. 2004, 14, 5171-5174
S. Kumar
PKC , might be implicated in NEBD. Which one?
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Coinjection w/ 2 mM PKC inhibitor and 200 M KVIP, 5 M 70K Texas ted-dextran
PKCIC50 (M) Ki (M)
0.0019 0.00080
PKC 385-fold PKC 580-fold
PKC 2730-fol PKC 600-fol
PKC 1310-fold PKC 1210-fold
PKC 940-fold PKC 640-fold
Arg Arg Gly Ala Leu Arg Dap Ala NHCH2CH2SH
NH CO
N
ClCl
HN
O
Ala
6
Lee, Nandy, Lawrence. JACS, 2004
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0 min injection 2 min uncaging 30 min
Coinjection w/ 2 mM PKC inhibitor and 200 M KVIP, 5 M 70K rhodamine-dextran (No NEBD)
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Coinjection of 2 mM PKC inhibitor and 200 M KVIP
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25 30 35
t(injection)
If
Total cells NEBD Large enhancement (>30%)
Small enhancement(<30%)
No enhancement
10 Yes 0 0 0 0
No 10 0 0 10
When PKCs areshutdown, NEBD is blocked w/o FLenhancement.
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Co-injection of 1 M PKC inhibitor and 200 M KVIP
0 min injection
2 min
3 min
4 min
5 min
6 min
7 min
9 min
13 min
14 minTexas-redfluorescence
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Co-injection of 1 M PKC inhibitor and 200 M KVIP
11.11.21.31.41.51.61.71.81.9
2
-17
-16
-15
-14
-13
-12
-11
-10
-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5t (NEBD)
If
Total cells NEBD Large enhancement (>30%)
Small enhancement(<30%)
No ehancement(within 1%)
15 Yes 12 6 5 1
No 3 0 0 3
PKC is responsiblefor NEBD and FL
1 or 2?
PKC shutdown
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Redistribution of PKCI and PKCII
In Cell Cycle
N. G. Dulyaninova
1: associated w/ nucleusin interphase and prophase.
2: everywhere in interphase Partial relocation to nuclear boundary in prophase.Significant for NEBD?
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Conclusion for Caged PKC Sensor• Caged sensors can be used to probe PKC activity
at G2/M in live cells with temporal precision, providing a way to interrogate enzymatic activity at any point during the cell-division cycle.
• PKC is implicated in NEBD of PtK2 cells. It is active just prior to NEBD, not immediately
after.
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Acknowledgement• Mike Isaacman• Cho Tan• Amanda Mickley• Patrick Carney• Nikhil Khosla• Pace Colleagues• Prof JaimeLee I. Rizzo
• Prof. James W. Canary (NYU)• Prof. David S. Lawrence (Einstein, UNC) Dr. Williem Veldhuyzen, Dr. Sandip Nandy• Prof. Sanjai Kumar • Prof. Anne R. Bresnick (Einstein) Dr. Natalya G. Dulyaninova Dr. Zhonghua (Alice) Li
NSF (JWC) NIH (DSL, ARB, JWC)
Pace University (Startup Fund, Scholarly Research Fund, Kenan Award)
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Mechanism of Uncaging