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Krzysztof Liberek Protein Biochemistry Research Group Department of Molecular and Cellular Biology Intercollegiate Faculty of Biotechnology University.

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Presentation on theme: "Krzysztof Liberek Protein Biochemistry Research Group Department of Molecular and Cellular Biology Intercollegiate Faculty of Biotechnology University."— Presentation transcript:

1 Krzysztof Liberek Protein Biochemistry Research Group Department of Molecular and Cellular Biology Intercollegiate Faculty of Biotechnology University of Gdansk

2 Heat shock gene expression Environmental stress heat shock; amino acids analogs; heavy metals; inhibitors of energy metabolism Non-stressful conditions cell cycle; growth factors; oncogenes and proto- oncogenes Pathophysiological state fever; inflammation; ischemia; viral and bacterial infection; aging chaperones proteases

3 Molecular chaperones folding of newly synthesized proteins translocation of proteins into organella reactivation of denatured proteins degradation of proteins unfolded polipeptide Folded protein DEGRADATION AGGREGATION DEGRADATION DISAGGREGATION

4 Protein folding vs aggregation Protein folding vs aggregation

5 Molecular chaperones in protein disaggregation J Hsp70 RF + folded protein aggregated protein ATP ADP Hsp100

6 mitochondria Ssc1p DnaK Mdj1p DnaJ Mge1p GrpE Hsp78 ClpB Ssa1p Sis1p Hsp104 cytosol Saccharomyces cerevisiae Escherichia coli Purified Hsp100/Clp proteins Hsp104 MWS Hsp78 ClpB Krzewska et al.., (2001) FEBS lett.. Krzewska et al.., (2001) J. Mol. Biol. Hsp78

7 wtK149TK547TK149T/K547T monomer oligomer dimer ATP Effects of the mutations in the putative ATP binding domains on Hsp78 oligomerization and ATPase activity Krzewska et al.., (2001) J. Mol. Biol NBD1 NBD GXXXXGKT TT

8 Chaperone mediated refolding of firefly luciferase Krzewska et al.., (2001) FEBS lett..

9 Order of chaperone action Hsp70 Hsp100 folded protein aggregated protein ? ? ? J J J Preincubation Reactivation

10 Green fluorescent protein as a substrate for chaperone dependent disaggregation measurements B DnaK, DnaJ,GrpE, ClpB Time (min) Fluorescence AU DnaK, DnaJ, GrpE ClpB Reactivation

11 Kinetics of GFP refolding following preincubation with different chaperones Time (min) Fluorescence AU Preincubation/ Reactivation K,J / E,B K,J,E / B J / K,E,B K / J,E,B - / K,J,E,B Preincubation Reactivation Reactivation

12 J Hsp70 RF Hsp100 folded protein aggregated protein ? ? ? Small heat shock proteins

13 ,1 14,4 MWS IbpA IbpB luciferase activity (%) KJE-B System KJE System denaturation step: - IbpA - IbpA IbpB IbpB reactivation step: KJEB KJEB KJE KJE Presence of small heat shock proteins during the denaturation step increases the luciferase refolding by chaperone systems

14 HEAT SHOCK EXPERIMENT: Culture samples fixed with formaldehyde Fluorescent / confocal microscopy analysis of microscope preparates –statistics counting –photographs taking Yeast cell pVT100U-mtGFP GFP-labeled mitochondria samples to fix Time (h) Recovery 25 o C 37 o C Temperature of cells growth 46 o C /- CHX

15 % of cells with altered mitochondrial morphology time after heat shock [min] HSP78 gene supplied on a plasmid restores wild type behaviour in Δhsp78 mutant Mutant strain was co-trasformed with plasmid carrying mtGFP and pYES-HSP78 plasmid with wt copy of HSP78 gene or with empty pYES2.0 vector. 46 o C25 o C before heat-shock 90 min reactivation directly after heat-shock

16 Igor Konieczny Jarosław Marszałek Grażyna Konopa Ewa Laskowska Joanna Krzewska Agnieszka Lewandowska Marlena Matuszewska Szymon Ziętkiewicz Krzysztof Liberek Protein Biochemistry Research Group Department of Molecular and Cellular Biology Liu, Q, Krzewska, J., Liberek, K., Craig, E. A. (2001) Mitochondrial Hsp70 Ssc1: Role in protein folding. J. Biol. Chem 276, Krzewska, J., Langer, T., Liberek, K. (2001) Mitochondrial Hsp78, a member of the Clp/Hsp100 family in Saccharomyces cerevisiae, cooperates with Hsp70 in protein refolding. FEBS Lett. 489, Krzewska, J., Konopa, G., Liberek, K. (2001) Importance of two ATP binding sites for oligomerization, ATPase activity and chaperone function of mitochondrial Hsp78 protein. J. Mol. Biol. 314, Konieczny, I., Liberek, K. (2002) Cooperative action of Escherichia coli ClpB protein and DnaK chaperone in the activation of a replication initiation protein. J. Biol. Chem. 277, Germaniuk, A., Liberek, K., Marszalek, J. (2002) A bi-chaperone (Hsp70-Hsp78) system restores mitochondrial DNA synthesis following thermal inactivation of Mip1p polymerase. J. Biol.Chem. 277, Selected publications Financing: Polish State Committee for Scientific Research collaboration


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