Download - Thesis presentation gm dt480.4 p
Pharmaceutical Relevant Proteins; Studies of RBP4 and Kvβ2.
Gavin Mooney
B.Sc. Pharmaceutical Technology
Supervisor – Dr. Barry Ryan, PhD.
Overview
� Human Retinol Binding Protein -4 & its links to Diabetes
� Kvβ2; subunit of Kv1 Potassium Channel
� Research Objectives
� Laboratory Methods Employed
� Results
� Research Conclusions
� Questions
Human Retinol Binding Protein-4 (RBP4)
� Protein secreted by Hepatocytes & Adipocytes
� Links with Type 2 Diabetes Mellitus (DM2)
� Reports identify RBP4 role in insulin resistance (IR)
� ↑ RBP4 serum conc. ≈ ↓Glut-4 Transporter expression
� Glut-4 activity is Insulin mediated
3D Representation of RBP4.
Human Retinol Binding Protein-4 (RBP4)
� Population study
� Serum biomarker of IR
� Direct serum RBP4 conc.
� Indirect; serum Transthyretin conc.
� Chen, et al. (2009) reports no evidence linking RBP4 as DM2 marker
� Uric acid production and RBP4 correlation provides links between
RBP4 and eGFR
Kvβ2; Subunit of Kv1 Potassium Channel
� Cytosolic protein which is part of the Shaker family of Kv1
Potassium Channels
� Widely dispersed in CNS
� Thought to modulate K+ channel thus effecting membrane potential
� Similar homology to AKR’s and contains a bound NADPH (Cofactor)
� AKR fold aides Kvβ2 to catalyse redox reductions
Research Objectives
� Optimization of PCR conditions for RBP4 amplification
� Over expression, purification of the Kvβ2 protein
� Assess inhibitory activity of Rutin, Quercitin & Resveratrol on Kvβ2
activity through assessing the 4-N-B-alcohol output from the following
rxn:
3D Representation of Kvβ2.
Kvβ2 + NADPH + 4-N-B-aldehyde Kvβ2 + NADP+ + 4-N-B-alcohol
Methods - PCR
Step Temperature Time
Initial Denaturation 94oC 4min
PCR Cycle at 35 Cycles
Denaturation 94 oC 1min
Annealing 58 oC 45secs
Elongation 72 oC 45secs
Finish Cycle
Final Elongation 72 oC 10min
� Example of PCR condition set used -
� Annealing Temp. and gradients thereof acquired using:
TM(Av) = (TM1 – TM2)TAnneal (Average) = TM(Av) – 5oC ±3-5oC (approx)
Methods - PCR
GoTAQ® Master Mix 12.5µµµµlhRBP4 -5’ / Alu 5’ 1µµµµl to 3µµµµlhRBP4 -3’ / Alu 5’ 1µµµµl to 3µµµµlcDNA (1:50) or (1:500) 3µl
SDW to 25µµµµl
� Example of PCR Reaction mix used -
� Variations in primer and cDNA conc. used to alter binding probability
� A.G.E was carried out in 07% to 1% Agarose gel under 100-115V for 1-
1.5hr durations.
� UV Gel Images obtained at 320nm using Alpha-Imager
Methods – Expression & Purification
IPTG- induced
Kvβ2 expression in E.coli
Re-suspension in Lysis Buffer
Sonication & Centrifugation
Purified by passing Supernatant
through Ni+ column
UV analysis for Elution Profile
Elution w/ 20mM Tris-HCl
Elution Profile of Kv2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 2 4 6 8 10 12 14 16
Protin Volume (ml)
Ab
sorb
an
ce (
28
0n
m)
Methods – HPLC Assay
� Inhibition studies carried out in 0.2M Phos. Buffer containing NADPH,
4-N-Bald, Kvβ2 and inhibitor.
� Incubation: 15mins prior to post substrate addition incubation of 40min
0
20
40
60
80
100
120
0 4 8 12 16 20 24 28 32
Run Time, minutes
So
lven
t, %
Solvent A
Solvent BGradient Profile Isocratic Profile
Return to
Normal Conditions
HPLC Elution Method
Solvent A20% MeOH, 0.1% TFA
Solvent B60% MeOH, 0.1% TFA
Results - PCR
1500bp
500bp
100bp
1 2 3 4 5 6 7 8 9 10 11 12
Results - Chromatograms
1.70
2
7.58
8
9.03
6
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
NADPH
4-N-B-alc
4-N-B-ald
�Chromatogram showing a control reaction for Rutin
experiment containing 0.5mM 4-nitrobenzaldehyde, 0.2M
phosphate buffer, ~10µM Kvβ2 and 0.2mM NADPH.
�This clearly shows the enzymatic reaction taking place with
the production of 4-nitrobenzylaclohol
Concentration dependant inhibition of Kvβ2 by Inhibitors
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
0.030
Minutes7.12 7.14 7.16 7.18 7.20 7.22 7.24 7.26 7.28 7.30 7.32 7.34 7.36 7.38 7.40 7.42 7.44 7.46 7.48 7.50 7.52 7.54 7.56 7.58 7.60
700µM
500µM300µM100µM
No Rutin
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Minutes2.96 2.98 3.00 3.02 3.04 3.06 3.08 3.10 3.12 3.14 3.16 3.18 3.20
500µM
300µM
100µM
50µM
No Quercetin
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
Minutes7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.95 8.00
No Resveratrol
100µM
300µM
500µM
�Comparison of reduction in
4-N-Balc output from Kvβ2
Results – Inhibition Study
� The graph shows the
percentage-inhibition of the
Kvβ2 activity as a result of
increasing concentrations of
Rutin
� Flourometric data showing the binding of Rutin to Kvβ2 which is leading to a 64% reduction in Flouresence emission of the peak at 460nm representing the bound cofactor.
Conclusion
� PCR Optimization of RBP4 amplification needed
� Primer : cDNA conc. Ratio needs more optimization
� Three new lead compounds found for treatment in various fields such
as Cardiology via myocardium
� Further enhancement in knowledge about the physiological
processes that are on going in the Shaker potassium channel
� Successful research overall:
� Beneficial PCR guidelines &
� 1st study to report the link between Resveratrol and Kvβ2 activity
inhibition
Sincere thank you to…..
� Dr. Barry Ryan
� Ms. Alka Singh, PhD Student, DIT Cathal Brugha St
� Mr. Tony Hutchinson, Lab Technician, DIT Cathal Brugha St.
� DT480 Class
Questions??