Název:
Electrochemical determination of PrP and its interractions with metals and metallothionein
Školitel:
Alžběta Cardová
Datum:
6. 2. 2014
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Prion • PrPC is glycosylphosphatidylinositol-anchored host glycoprotein normally present in brain (Dormont, 2002) • This protein with predominance of α-helix structure can be converted to an abnormal protease resistant isoform with increased ratio of β-sheet structure called prion (PrPSc) (Kong et al, 2013) • PrPSc isoform can cause a range of slow neurodegenerative disorders called transmissible spongiform encephalopathies (Tiraboschi & Tagliavini, 2013). The most famous prion caused disease is BSE Dormont D (2002) Prion diseases: pathogenesis and public health concerns. FEBS Lett 529: 17-21 Kong QZ, Mills JL, Kundu B, Li XY, Qing LT, Surewicz K, Cali I, Huang SH, Zheng MJ, Swietnicki W, Sonnichsen FD, Gambetti P, Surewicz WK (2013) Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo. Cell Rep 4: 248-254 Tiraboschi P, Tagliavini F (2013) Prion disease: a promising rating scale for prion disease clinical research. Nat Rev Neurol 9: 366-367
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Bacterial production of recombinant PrPC • pRSET B cloning kit (Invitrogen, Germany) for high-level expression of recombinant proteins in E. coli was used. For subsequent E. coli cultivation we followed the manual by Invitrogen • Expression of PrPC in E. coli was verified by gel electrophoresis and western-blot Western blot – verification of prion protein presence in harvested cells kDa Mr
0
1
Hours 2 3 4
5
Hours
6
0
1
2
3
4
5
6
75 50 37
25 20 15
160 V/40 min gradient gel 0.9 mA/cm2 membrane/50 min Blocking 1% BSA 45 min I. AB 1:847 clone 8H4 (Sigma #P0110) 2 hours II. Dako anti-mouse/HRP 1:1000 1 hour AEC
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Recombinant PrPC isolation and purification • PrPC was isolated on HisTrap excel 1 ml column (GE Healthcare, Uppsala, Sweden) using histidine protein anchors. We optimized the elutions according to the western-blot (picture below)
• Protein was purified by cut-off filtration (Amicon Ultra 3K-0,5 mL 3K Centrifugal Filters for Protein Purification and Concentration) • Finally we verified our results by MALDI-TOF Detection of PrPC in isolated fractions 250 kDa
25 kDa 20 kDa
37 kDa 25 kDa 20 kDa
15 kDa
Intens. [a.u.]
250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa
MALDI-TOF spectrum of recombinant PrPC
150 kDa 100 kDa 75 kDa 50 kDa
1200
PrPC = 20.828 kDa 1000
800
600
15 kDa 150 V, 1 hour, gradient gel 0,9 mA/cm2 membrane 50 min Blocking 1% BSA 45 min I. AB 1:847 clone 8H4 (Sigma #P0110) 2 hours II. Dako anti-mouse/HRP 1:1000 1 hour L = marker W = wash fraction E = elution fraction
400
200
0 18000
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20000
22000
24000
26000
28000 m/z
Matrix HCCA in TA30
Prions, metals and metallothionein • PrPC may play a role in cell signaling or in binding and transport of Cu(II) and Zn(II) ions (Gavier-Widen et al, 2005) (Kozlowski et al, 2012). Cu together with Zn ions are involved in the formation of amyloid plaques in case of neurodegenerative disorders (Pedersen et al, 2012). • According to some authors Cu ions can destabilise the native fold of PrPC and can facilitate the conversion to PrPSc isoform (Younan et al, 2011).
•
Metallothionein (MT) fulfils multiple functions including the involvement in zinc and copper homeostasis and protection against heavy metal toxicity and oxidative damage. Due to its physiological role, zinc and copper belong to the most investigated metal ions connected to metallothionein.
• Brain specific subtype of MT is called MT-III and this protein is able to bind copper when Cu homeostasis is disrupted.
Gavier-Widen D, Stack MJ, Baron T, Balachandran A, Simmons M (2005) Diagnosis of transmissible spongiform encephalopathies in animals: a review. J Vet Diagn Invest 17: 509-527 Kozlowski H, Luczkowski M, Remelli M, Valensin D (2012) Copper, zinc and iron in neurodegenerative diseases (Alzheimer's, Parkinson's and prion diseases). Coordin Chem Rev 256: 2129-2141 Younan ND, Klewpatinond M, Davies P, Ruban AV, Brown DR, Viles JH (2011) Copper(II)-Induced Secondary Structure Changes and Reduced Folding Stability of the Prion Protein. J Mol Biol 410: 369-382 Pedersen JT, Hureau C, Hemmingsen L, Heegaard NHH, Ostergaard J, Vasak M, Faller P (2012) Rapid Exchange of Metal between Zn-7-Metallothionein-3 and Amyloid-beta Peptide Promotes Amyloid-Related Structural Changes. Biochemistry 51: 1697-1706
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Electrochemical determination of PrPC, zinc and copper (differential pulse voltammetry)
25 nA
Peak height [nA]
PrPC Peak II Peak I PrP 250 µg/ml PrP 125 µg/ml PrP 100 µg/ml PrP 62.5 µg/ml PrP 31.25µg/ml PrP 15.125µg/ml Electrolyte
10 5
y = 0,0339x + 0,0252 R² = 0,9966
0
Peak height [nA]
0
-0.500
1 0
200
400
Potential [V]
• measured in acetate buffer pH 5 • Electrochemical signal corresponds to the concentration. • Dependence of all peaks is linear (R2 higher than 0.9)
Zinc Peak height [nA]
50 nA Cu – 100µg/ml Cu – 50µg/ml Cu – 25µg/ml Cu – 12.5µg/ml Cu – 6.25µg/ml Cu – 3.125µg/ml Electrolyte
Peak height [nA]
Copper
400
y = 0,0062x - 0,143 R² = 0,9952
0
-0.750
200
Potential [V]
Peak II
2
Potential [V]
-0.150
Peak I
500 y = 3,7125x + 51,091 R² = 0,9903
0 0
100
50 nA
10 y = 0,0495x + 3,8028 R² = 0,9974
5 0
200
0
100
200
Potential [V]
Potential [V]
-0.100
-0.050
0
-1.200
Potential [V]
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Potential [V]
-0.950
Zn 100 mM Zn 50 mM Zn 25 mM Zn 12.5 mM Zn 6.25 mM Zn 3.125 mM Electrolyte
Electrochemical determination of PrPC interactions with copper and zinc
Peak Cu
Peak Cu+PrP I Peak height [nA]
50 nA
300 200
y = 3,0749x + 22,073 R² = 0,9925
100
Peak Cu
0 0
50
100
150
Potential [V] PrP + Cu – 100µg/ml PrP + Cu – 50µg/ml PrP + Cu – 25µg/ml PrP + Cu – 12.5µg/ml PrP + Cu – 6.25µg/ml PrP + Cu – 3.125µg/ml Electrolyte -0.250
-0.200
15 10 5 0
y = 0,0698x + 5,9374 R² = 0,9947 0
-0.050
0
0.050
0.100
10
y = 0,0641x + 0,9885 R² = 0,9919
0 0
Potential [V]
100 200 Potential [V]
Peak Zn
Peak Zn+PrP I
10
Peak height [nA] -0.950
Potential [V]
• The diagram below shows ascending or descending trends of PrP and metals interactions
Peak Zn
0 0
y = 0,0681x + 2,144 0,9939 50R² =100 150 Potential [V]
50 nA -1.200
Peak height [nA]
PrPC + Zn PrP + Zn 100 mM PrP + Zn 50 mM PrP + Zn 25 mM PrP + Zn 12.5 mM PrP + Zn 6.25 mM PrP + Zn 3.125 mM Electrolyte
150
Peak Cu+PrP II
Peak Cu+PrP II
-0.100
100
Potential [V]
Peak Cu+PrP I
-0.150
50
• Constant PrPC concentration 100 µg/ml and various conc. of Cu and Zn (100, 50, 25, 12.5, 6.25, 3.125 µg/ml) measured in acetate buffer pH 5
Peak Zn+PrP I
y = -0,0043x + 0,5362 R² = 0,9917
1 0,5 0 0
100
200
Potential [V]
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16
Peak height (nA)
400
Peak height [nA]
Peak height [nA]
PrPC + Cu
14
100 µg/ml
12
50 µg/ml
10
25 µg/ml
8
12,5 µg/ml
6
6,25 µg/ml
4
3,125 µg/ml
2 0 Zn+PrP I
Cu+PrP I
Cu+PrP II
PrPC
MT
Electrolyte PrP 15 µg/ml PrP 31 µg/ml PrP 62 µg/ml PrP 125 µg/ml PrP 250 µg/ml
Peak height [nA]
Peak PrP II Peak PrP I Peak PrP II 10 8 6 4 2 0
y = 0,0379x - 0,9772 Peak PrP I R² = 0,9918 y = 0,02x + 0,0206 R² = 0,9877
0
100
200
300
Potential [V]
-0.200 -0.300 -0.400 -0.500 -0.600 -0.700 -0.800 -0.900
Peak height [nA]
10 nA
Electrolyte MT 1 µg/ml MT 2 µg/ml MT 4 µg/ml MT 8 µg/ml MT 16 µg/ml 50 y = 2,799x - 2,03 R² = 0,9964
0 0
10 Potential
[V]
-0.400
-0.500
Potential [V]
• • • •
measured in sodium phosphate buffer pH 7 AdTS – accumulation time = 120s Electrochemical signal corresponds to the concentration. Dependence of all peaks is linear (R2 higher than 0.9)
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10 nA
20 -0.600
-0.700 Potential [V]
-0.800
Electrochemical determination of PrPC interaction with MT and vice versa PrPC + MT (constant PrPC concentration = 100µg/ml) Peak PrP+MT
Peak height [nA]
10 nA Electrolyte PrP + MT 0.25 µg/ml PrP + MT 0.50 µg/ml
10
y = -0,3578x + 8,1564 R² = 0,9912
5 0
0
PrP + MT 2 µg/ml
Peak MT
PrP + MT 4 µg/ml
Peak PrP+MT
PrP + MT 8 µg/ml
-0.200 -0.300 -0.400 -0.500 -0.600 -0.700 -0.800 -0.900 -1.000
Peak height [nA]
PrP + MT 1 µg/ml
PrP + MT 16 µg/ml
10
20
Potential [V]
Peak MT
40 20
y = 1,9961x + 1,8173 R² = 0,991
0 0
10
20
• In case of PrPC and MT interaction there are two coalesced peaks • Calibration curves of peaks are linear (R2 higher than 0.9).
Potential [V]
Potential [V]
10 nA
Peak I
Peak II
Shifted Peak II
Peak I (with shifted peak I) 20 y = 0,0503x + 1,3705 R² = 0,9909
10 0
Electrolyte 0 200 400 Potential [V] MT + PrP 15,13 µg/ml Peak II (without shifted peak II) MT + PrP 31,25 µg/ml 50 MT + PrP 62.50 µg/ml y = 0,0705x + MT + PrP 125 µg/ml 14,385 R² = 1 MT + PrP 250 µg/ml 0
-0.400 -0.500 -0.600 -0.700 -0.800 -0.900 -1.000 Potential [V]
Peak height [nA]
Shifted Peak I
Peak height [nA]
MT + PrPC (constant MT concentration = 8µg/ml)
0
200 Potential [V]
400
• In case of MT and PrPC interaction there is a peak shift to the new position with increased conc. of PrPC • There is a massive change of structure
Diagram of PrPC interaction with MT and vice versa Constant conc. of PrPC + various conc. of MT
Constant conc. of MT + various conc. of PrPC
MT
PrPC
PrPC + MT
PrPC
MT + low PrPC conc.
MT + high PrPC conc.
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Conclusion • Recombinant PrPC was produced, isolated and purified • PrPC was used for electrochemical determination and for an investigation into its interactions with metals and metallothionein • Massive interaction was discovered especially in case of MT and PrPC interaction • We presume that a change of the peak position is caused by the formation of MT tetramers enclosing the PrPC molecule to the center in case of high PrPC concentration
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Acknowledgements • • • • •
Ing. Pavlina Sobrova Doc. RNDr. Vojtech Adam, Ph.D. Dr. Vladimir Pekarik, Ph.D. Mgr. Ondrej Zitka, Ph.D. Mgr. Marketa Vaculovicova, Ph.D.
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Thank you for your attention
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