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??