Alžběta Cardová. Electrochemical determination of PrP and its interractions with metals and metallothionein. Název: Školitel: Datum: 6. 2.

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1 Název: Školitel: Electrochemical determination of PrP and its interractions with metals and metallothionein Alžběta Cardová Datum: Reg.č.projektu: CZ.1.7/2.4./31.23

2 Prion PrP C is glycosylphosphatidylinositol-anchored host glycoprotein normally present in brain (Dormont, 22) 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, 213) PrP Sc isoform can cause a range of slow neurodegenerative disorders called transmissible spongiform encephalopathies (Tiraboschi & Tagliavini, 213). The most famous prion caused disease is BSE Dormont D (22) Prion diseases: pathogenesis and public health concerns. FEBS Lett 529: 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 (213) Thermodynamic Stabilization of the Folded Domain of Prion Protein Inhibits Prion Infection in Vivo. Cell Rep 4: Tiraboschi P, Tagliavini F (213) Prion disease: a promising rating scale for prion disease clinical research. Nat Rev Neurol 9: Reg.č.projektu: CZ.1.7/2.4./31.23

3 Bacterial production of recombinant PrP C 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 PrP C in E. coli was verified by gel electrophoresis and western-blot Western blot verification of prion protein presence in harvested cells kda Mr Hours Hours Reg.č.projektu: CZ.1.7/2.4./ V/4 min gradient gel.9 ma/cm 2 membrane/5 min Blocking 1% BSA 45 min I. AB 1:847 clone 8H4 (Sigma #P11) 2 hours II. Dako anti-mouse/hrp 1:1 1 hour AEC

4 Recombinant PrP C isolation and purification PrP C 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-,5 ml 3K Centrifugal Filters for Protein Purification and Concentration) Finally we verified our results by MALDI-TOF Detection of PrP C in isolated fractions MALDI-TOF spectrum of recombinant PrP C 25 kda 15 kda 1 kda 75 kda 5 kda 37 kda 25 kda 2 kda 15 kda 25 kda 15 kda 1 kda 75 kda 5 kda 37 kda 25 kda 2 kda 15 kda Intens. [a.u.] PrP C = kda 15 V, 1 hour, gradient gel,9 ma/cm2 membrane 5 min Blocking 1% BSA 45 min I. AB 1:847 clone 8H4 (Sigma #P11) 2 hours II. Dako anti-mouse/hrp 1:1 1 hour L = marker W = wash fraction E = elution fraction Matrix HCCA in TA3 m/z Reg.č.projektu: CZ.1.7/2.4./31.23

5 Prions, metals and metallothionein PrP C may play a role in cell signaling or in binding and transport of Cu(II) and Zn(II) ions (Gavier-Widen et al, 25) (Kozlowski et al, 212). Cu together with Zn ions are involved in the formation of amyloid plaques in case of neurodegenerative disorders (Pedersen et al, 212). According to some authors Cu ions can destabilise the native fold of PrP C and can facilitate the conversion to PrP Sc isoform (Younan et al, 211). 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 Gavier-Widen D, Stack MJ, Baron T, Balachandran A, Simmons M (25) Diagnosis of transmissible spongiform encephalopathies in animals: a review. J Vet Diagn Invest 17: Kozlowski H, Luczkowski copper M, Remelli M, when Valensin D (212) Cu Copper, homeostasis zinc and iron in neurodegenerative diseases is disrupted. (Alzheimer's, Parkinson's and prion diseases). Coordin Chem Rev 256: Younan ND, Klewpatinond M, Davies P, Ruban AV, Brown DR, Viles JH (211) Copper(II)-Induced Secondary Structure Changes and Reduced Folding Stability of the Prion Protein. J Mol Biol 41: Pedersen JT, Hureau C, Hemmingsen L, Heegaard NHH, Ostergaard J, Vasak M, Faller P (212) Rapid Exchange of Metal between Zn-7-Metallothionein-3 and Amyloid-beta Peptide Promotes Amyloid-Related Structural Changes. Biochemistry 51: Reg.č.projektu: CZ.1.7/2.4./31.23

6 Electrochemical determination of PrP C, zinc and copper (differential pulse voltammetry) PrP C 25 na PrP 25 µg/ml PrP 125 µg/ml PrP 1 µg/ml PrP 62.5 µg/ml PrP 31.25µg/ml PrP µg/ml Peak I -.5 Peak II Peak I 5 y =,339x +,252 R² =, Peak II y =,62x -,143 R² =, measured in acetate buffer ph 5 Electrochemical signal corresponds to the concentration. Dependence of all peaks is linear (R 2 higher than.9) Copper Zinc Cu 1µg/ml Cu 5µg/ml Cu 25µg/ml Cu 12.5µg/ml Cu 6.25µg/ml Cu 3.125µg/ml 5 na y = 3,7125x + 51,91 R² =, na Reg.č.projektu: CZ.1.7/2.4./ y =,495x + 3,828 R² =, Zn 1 mm Zn 5 mm Zn 25 mm Zn 12.5 mm Zn 6.25 mm Zn mm

7 Peak height (na) Electrochemical determination of PrP C interactions with copper and zinc PrP C + Cu Peak Cu y = 3,749x + 22,73 R² =, PrP + Cu 1µg/ml PrP + Cu 5µg/ml PrP + Cu 25µg/ml PrP + Cu 12.5µg/ml PrP + Cu 6.25µg/ml PrP + Cu 3.125µg/ml 5 na Peak Cu Peak Cu+PrP I PrP C + Zn Peak Cu+PrP II Peak Cu+PrP I y =,698x + 5,9374 R² =, Peak Cu+PrP II y =,641x +,9885 R² =, Constant PrP C concentration 1 µg/ml and various conc. of Cu and Zn (1, 5, 25, 12.5, 6.25, µg/ml) measured in acetate buffer ph 5 The diagram below shows ascending or descending trends of PrP and metals interactions PrP + Zn 1 mm PrP + Zn 5 mm PrP + Zn 25 mm PrP + Zn 12.5 mm PrP + Zn 6.25 mm PrP + Zn mm 5 na Peak Zn Peak Zn+PrP I 1 Peak Zn y =,681x + 2,144 5R² = 1, Peak Zn+PrP I 1 y = -,43x +,5362,5 R² =, Reg.č.projektu: CZ.1.7/2.4./ Zn+PrP I Cu+PrP I Cu+PrP II 1 µg/ml 5 µg/ml 25 µg/ml 12,5 µg/ml 6,25 µg/ml 3,125 µg/ml

8 PrP C MT PrP 15 µg/ml PrP 31 µg/ml PrP 62 µg/ml PrP 125 µg/ml PrP 25 µg/ml Peak PrP I Peak PrP II na Peak PrP II y =,379x -,9772 R² =,9918 Peak PrP I y =,2x +,26 R² =, MT 1 µg/ml MT 2 µg/ml MT 4 µg/ml MT 8 µg/ml MT 16 µg/ml 5 y = 2,799x - 2,3 R² =, na -.8 measured in sodium phosphate buffer ph 7 AdTS accumulation time = 12s Electrochemical signal corresponds to the concentration. Dependence of all peaks is linear (R 2 higher than.9) Reg.č.projektu: CZ.1.7/2.4./31.23

9 Electrochemical determination of PrP C interaction with MT and vice versa PrP C + MT (constant PrP C concentration = 1µg/ml) 1 na PrP + MT.25 µg/ml PrP + MT.5 µg/ml PrP + MT 1 µg/ml PrP + MT 2 µg/ml PrP + MT 4 µg/ml PrP + MT 8 µg/ml Peak PrP+MT Peak MT PrP + MT 16 µg/ml MT + PrP C (constant MT concentration = 8µg/ml) Peak I Shifted Peak I Peak II Shifted Peak II na MT + PrP 15,13 µg/ml MT + PrP 31,25 µg/ml MT + PrP 62.5 µg/ml MT + PrP 125 µg/ml MT + PrP 25 µg/ml Peak PrP+MT y = -,3578x + 8,1564 R² =, Peak MT 2 y = 1,9961x + 1,8173 R² =, Peak I (with shifted peak I) 2 y =,53x + 1 1,375 R² =, Peak II (without shifted peak II) 5 y =,75x + 14,385 R² = In case of PrP C and MT interaction there are two coalesced peaks Calibration curves of peaks are linear (R 2 higher than.9). In case of MT and PrP C interaction there is a peak shift to the new position with increased conc. of PrP C There is a massive change of structure

10 Reg.č.projektu: CZ.1.7/2.4./31.23 Diagram of PrP C interaction with MT and vice versa Constant conc. of PrP C + various conc. of MT PrP C Constant conc. of MT + various conc. of PrP C MT PrP C + MT MT + low PrP C conc. PrP C MT + high PrP C conc. MT

11 Conclusion Recombinant PrP C was produced, isolated and purified PrP C 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 PrP C interaction We presume that a change of the peak position is caused by the formation of MT tetramers enclosing the PrP C molecule to the center in case of high PrP C concentration Reg.č.projektu: CZ.1.7/2.4./31.23

12 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.1.7/2.4./31.23

13 Thank you for your attention Reg.č.projektu: CZ.1.7/2.4./31.23