Prion Alžběta Cardová. Electrochemical determination of PrP and its interractions with metals and metallothionein

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1..1 Název: Školitel: Electrochemical determination of PrP and its interractions with metals and metallothionein Alžběta Cardová Datum: 6.. 1 Reg.č.projektu: CZ.1.7/../31.

resistant isoform with increased ratio of β sheet structure called prion (PrP Sc ) (Kong et al, 13) PrP Sc isoform can cause a range of slow neurodegenerative disorders called transmissible

FEBS Lett 9: 17-1 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 (13) Thermodynamic Stabilization of the

1 1..1 Název: Školitel: Electrochemical determination of PrP and its interractions with metals and metallothionein Alžběta Cardová Datum: Reg.č.projektu: CZ.1.7/../31.3 Prion is glycosylphosphatidylinositol anchored host glycoprotein normally present in brain (Dormont, ) 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 (PrP Sc ) (Kong et al, 13) PrP Sc isoform can cause a range of slow neurodegenerative disorders called transmissible spongiform encephalopathies (Tiraboschi & Tagliavini, 13). The most famous prion caused disease is BSE Dormont D () Prion diseases: pathogenesis and public health concerns. FEBS Lett 9: 17-1 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 (13) Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo. Cell Rep : 8- Tiraboschi P, Tagliavini F (13) Prion disease: a promising rating scale for prion disease clinical research. Nat Rev Neurol 9: Reg.č.projektu: CZ.1.7/../31.3 Bacterial production of recombinant 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 in E. coli was verified by gel electrophoresis and western blot Western blot verification of prion protein presence in harvested cells Hours Hours kda Mr V/ min gradient gel.9 ma/cm membrane/ min Blocking 1% BSA min I. AB 1:87 clone 8H (Sigma #P11) hours II. Dako anti mouse/hrp 1:1 1 hour AEC Reg.č.projektu: CZ.1.7/../31.3 1

2 m/z 1..1 Recombinant isolation and purification 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, ml 3K Centrifugal Filters for Protein Purification and Concentration) Finally we verified our results by MALDI-TOF Detection of in isolated fractions MALDI-TOF spectrum of recombinant kda 1 kda 1 kda 7 kda kda 37 kda kda kda 1 kda kda 1 kda 1 kda 7 kda kda 37 kda kda kda 1 kda Intens. [a.u.] =.88 kda 1 V, 1 hour, gradient gel,9 ma/cm membrane min Blocking 1% BSA min I. AB 1:87 clone 8H (Sigma #P11) hours II. Dako anti mouse/hrp 1:1 1 hour L = marker W = wash fraction E = elution fraction Matrix HCCA in TA3 Reg.č.projektu: CZ.1.7/../31.3 Prions, metals and metallothionein may play a role in cell signaling or in binding and transport of Cu(II) and Zn(II) ions (Gavier Widen et al, ) (Kozlowski et al, 1). Cu together with Zn ions are involved in the formation of amyloid plaques in case of neurodegenerative disorders (Pedersen et al, 1). According to some authors Cu ions can destabilise the native fold of and can facilitate the conversion to PrP Sc isoform (Younan et al, 11). Metallothionein () 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 is called III and this protein is able to bind Gavier-Widen D, Stack MJ, Baron T, Balachandran A, Simmons M () Diagnosis of transmissible spongiform encephalopathies in animals: a review. J Vet Diagn Invest 17: 9-7 Kozlowski H, Luczkowski copper M, Remelli M, when Valensin D (1) Cu Copper, homeostasis zinc and iron in neurodegenerative diseases is disrupted. (Alzheimer's, Parkinson's and prion diseases). Coordin Chem Rev 6: Younan ND, Klewpatinond M, Davies P, Ruban AV, Brown DR, Viles JH (11) Copper(II)-Induced Secondary Structure Changes and Reduced Folding Stability of the Prion Protein. J Mol Biol 1: Pedersen JT, Hureau C, Hemmingsen L, Heegaard NHH, Ostergaard J, Vasak M, Faller P (1) Rapid Exchange of Metal between Zn-7-Metallothionein-3 and Amyloid-beta Peptide Promotes Amyloid-Related Structural Changes. Biochemistry 1: Reg.č.projektu: CZ.1.7/../31.3 na PrP µg/ml PrP 1 µg/ml PrP 1 µg/ml PrP 6. µg/ml PrP 31.µg/ml PrP 1.1µg/ml Copper Cu 1µg/ml Cu µg/ml Cu µg/ml Cu 1.µg/ml Cu 6.µg/ml Cu 3.1µg/ml Electrochemical determination of, zinc and copper (differential pulse voltammetry) na -. I y = 3,71x + 1,91 R² =,993 1 y =,339x +, R² =,9966 I Zinc na Reg.č.projektu: CZ.1.7/../ y =,6x -,13 R² =,99 measured in acetate buffer ph Electrochemical signal corresponds to the concentration. Dependence of all peaks is linear (R higher than.9) 1 y =,9x + 3,88 R² =,997 1 Zn 1 mm Zn mm Zn mm Zn 1. mm Zn 6. mm Zn 3.1 mm

3 Electrochemical determination of interactions with copper and zinc + Cu 3 1 Peak Cu 1 1 PrP + Cu 1µg/ml PrP + Cu µg/ml PrP + Cu µg/ml PrP + Cu 1.µg/ml PrP + Cu 6.µg/ml PrP + Cu 3.1µg/ml y = 3,79x +,73 R² =,99 na Peak Cu Peak Cu+PrP I Peak Cu+PrP II Zn PrP + Zn 1 mm PrP + Zn mm PrP + Zn mm PrP + Zn 1. mm PrP + Zn 6. mm PrP + Zn 3.1 mm na Peak Zn Peak Zn+PrP I Peak Zn Peak Cu+PrP I y =,698x +,937 R² =,997 Peak Cu+PrP II y =,61x +,988 R² =, y =,681x +,1 R² = 1, Peak Zn+PrP I 1 y = -,3x +,36, R² =, Reg.č.projektu: CZ.1.7/../31.3 Constant concentration 1 µg/ml and various conc. of Cu and Zn (1,,, 1., 6., 3.1 µg/ml) measured in acetate buffer ph The diagram below shows ascending or descending trends of PrP and metals interactions Peak height (na) Zn+PrP I Cu+PrP I Cu+PrP II 1 µg/ml µg/ml µg/ml 1, µg/ml 6, µg/ml 3,1 µg/ml PrP 1 µg/ml PrP 31 µg/ml PrP 6 µg/ml PrP 1 µg/ml PrP µg/ml na Peak PrP II Peak PrP I Peak PrP II 1 8 y =,379x -,977 6 R² =,9918 Peak PrP I y =,x +,6 R² =, µg/ml µg/ml µg/ml 8 µg/ml 16 µg/ml y =,799x -,3 R² =, na -.8 measured in sodium phosphate buffer ph 7 AdTS accumulation time = 1s Electrochemical signal corresponds to the concentration. Dependence of all peaks is linear (R higher than.9) Reg.č.projektu: CZ.1.7/../31.3 Electrochemical determination of interaction with and vice versa + (constant concentration = 1µg/ml) 1 na PrP +. µg/ml PrP +. µg/ml PrP + 1 µg/ml PrP + µg/ml PrP + µg/ml PrP + 8 µg/ml PrP + 16 µg/ml Shifted I Peak PrP Shifted I Peak (constant concentration = 8µg/ml) 1 na + PrP 1,13 µg/ml + PrP 31, µg/ml + PrP 6. µg/ml + PrP 1 µg/ml + PrP µg/ml Reg.č.projektu: CZ.1.7/../ Peak PrP+ y = -,378x + 8,16 R² =,991 1 Peak y = 1,9961x + 1,8173 R² =,991 1 (with shifted peak I) y =,3x + 1 1,37 R² =,999 I (without shifted peak II) y =,7x + 1,38 R² = 1 In case of and interaction there are two coalesced peaks Calibration curves of peaks are linear (R higher than.9). In case of and interaction there is a peak shift to the new position with increased conc. of There is a massive change of structure 3

1..1 Diagram of interaction with and vice versa Constant conc. of + various conc. of Constant conc. of + various conc. of + + low conc. + high conc. Reg.č.projektu: CZ.1.7/../31.

4 1..1 Diagram of interaction with and vice versa Constant conc. of + various conc. of Constant conc. of + various conc. of + + low conc. + high conc. Reg.č.projektu: CZ.1.7/../31.3 Conclusion Recombinant was produced, isolated and purified was used for electrochemical determination and for an investigation into its interactions with metals and metallothionein Massive interaction was discovered especially in case of and interaction We presume that a change of the peak position is caused by the formation of tetramers enclosing the molecule to the center in case of high concentration Reg.č.projektu: CZ.1.7/../31.3 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. An entire Laboratory of Metallomics and Nanotechnologies Reg.č.projektu: CZ.1.7/../31.3

5 1..1 Thank you for your attention Reg.č.projektu: CZ.1.7/../31.3

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Alžběta Cardová. Electrochemical determination of PrP and its interractions with metals and metallothionein. Název: Školitel: Datum: 6. 2. Název: Školitel: Electrochemical determination of PrP and its interractions with metals and metallothionein Alžběta Cardová Datum: 6. 2. 214 Reg.č.projektu: CZ.1.7/2.4./31.23 Prion PrP C is glycosylphosphatidylinositol-anchored