Krzysztof Liberek Protein Biochemistry Research Group Department of Molecular and Cellular Biology

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

2. 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 Heat shock gene expression chaperones proteases

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

4. Protein folding vs aggregation Protein folding vs aggregation

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

6. Hsp104 Hsp78 MWS ClpB Saccharomyces cerevisiae Escherichia coli mitochondria 93 cytosol 67 43 Ssa1p Sis1p Hsp104 Ssc1p DnaK Mdj1p DnaJ Mge1p GrpE Hsp78 ClpB 30 1 2 3 4 Purified Hsp100/Clp proteins Hsp78 Krzewska et al.., (2001) FEBS lett.. Krzewska et al.., (2001) J. Mol. Biol.

7. wt K149T K547T K149T/K547T oligomer dimer NBD1 NBD2 1 758 monomer - + - + - + - + ATP 143 150 541 548 GXXXXGKT GXXXXGKT T T Effects of the mutations in the putative ATP binding domains on Hsp78 oligomerization and ATPase activity Krzewska et al.., (2001) J. Mol. Biol.

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

9. ? Preincubation Reactivation 100 100 100 Hsp70 Hsp100 + + ? ? 70 70 70 J J J + + aggregated protein + + folded protein Order of chaperone action

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

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

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

13. MWS IbpA IbpB 94 67 43 30 20,1 14,4 KJE-B System KJE System luciferase activity (%) 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. GFP-labeled mitochondria 46oC +/- CHX Temperature of cells growth Recovery 37oC Time (h) 25oC -0.5 0 0.5 1 2 samples to fix Yeast cell pre GFP URA pVT100U-mtGFP • HEAT SHOCK EXPERIMENT: • Culture samplesfixed with formaldehyde • Fluorescent / confocal microscopy analysis of microscope preparates • statistics counting • photographs taking

15. 90 min reactivation before heat-shock 46oC 25oC directly after heat-shock HSP78 gene supplied on a plasmid restores wild type behaviour in Δhsp78 mutant Mutant strain was co-trasformedwith plasmid carrying mtGFPand pYES-HSP78 plasmidwith wt copy of HSP78 geneor with empty pYES2.0 vector. % of cells with altered mitochondrial morphology time after heat shock [min]

16. Protein Biochemistry Research Group Department of Molecular and Cellular Biology Joanna Krzewska Agnieszka Lewandowska Marlena Matuszewska Szymon Ziętkiewicz Krzysztof Liberek collaboration Igor Konieczny Jarosław Marszałek Grażyna Konopa Ewa Laskowska Selected publications Liu, Q, Krzewska, J., Liberek, K., Craig, E. A. (2001) Mitochondrial Hsp70 Ssc1: Role in protein folding. J. Biol. Chem 276, 6112-6118. 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, 92-96. 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, 901-910. 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, 18483-18488. 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, 27801-2780 Financing: Polish State Committee for Scientific Research