Krzysztof LiberekProtein Biochemistry Research GroupDepartment of Molecular and Cellular BiologyIntercollegiate Faculty of BiotechnologyUniversity of Gdansk

Heat shock gene expression

Environmental stressheat 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

Molecular chaperones• folding of newly synthesized proteins• translocation of proteins into organella• reactivation of denatured proteins• degradation of proteins

unfolded polipeptide

Folded protein

DEGRADATIONAGGREGATION

DEGRADATIONDISAGGREGATION

Protein folding vs

aggregation

Protein foldingvs

aggregation

Molecular chaperones in protein disaggregation

JHsp70RF

+

folded proteinaggregated protein

ATP

ADP

Hsp100

mitochondria

Ssc1p DnaKMdj1p DnaJMge1p GrpE

Hsp78 ClpB

Ssa1pSis1p

Hsp104

cytosol

Saccharomyces cerevisiae Escherichia coli

Purified Hsp100/Clp proteins

Hsp104

MWS

Hsp78

ClpB

93

67

43

30

1 2 3 4

Krzewska et al.., (2001) FEBS lett..

Krzewska et al.., (2001) J. Mol. Biol.

Hsp78

wt K149T K547T K149T/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.

Time (min)

0 5 10 15 20

AT

P h

ydro

lysi

s (%

)0

5

10

15

20

25wt Hsp78K149T K547TK149T/K547T

143 150

758 1

NBD1 NBD2

541 548

GXXXXGKT GXXXXGKT

T T

Time (min)

0 20 40 60 80 100 120 140

Luci

fera

se a

ctiv

ity (

%)

0

10

20

30

40

50

78/S/M/G78S/M/G

Mitochondrial system

Time (min)

0 20 40 60 80 100 120 140

Luci

fera

se a

ctiv

ity (

%)

0

10

20

30

40

50

B/K/J/EBK/J/E

Bacterial system

Chaperone mediated refolding of firefly luciferase

Krzewska et al.., (2001) FEBS lett..

Order of chaperone action

Hsp70 Hsp100

folded protein

aggregated protein

? ?

?

J70

+

+

+

J70

J70

Preincubation

+

+

+

100

100

100

Reactivation

450 500 550 600

0

200

400

600

800

1000

1200

[nm]

A.U.

native GFP denatured GFP

Green fluorescent protein as a substrate for chaperone dependent disaggregation measurements

B

0

50

100

150

200

250

0 10 20 30 40 50

DnaK, DnaJ,GrpE, ClpB

Time (min)

Flu

ores

cen

ce A

U

DnaK, DnaJ, GrpEClpB

Reactivation

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70

Kinetics of GFP refolding following preincubation with different chaperones

7

9

11

13

15

17

19

15 16 17 18 19

Time (min) Time (min)

Flu

ores

cen

ce A

U

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

JHsp70RF

Hsp100

folded protein

aggregated protein

? ?

?

Small heat shock proteins

94 67

43

30

20,1

14,4

MWS IbpA IbpB

0

25

50

75

100

luci

fera

se a

ctiv

ity (

%)

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

HEAT SHOCK EXPERIMENT:• Culture samples fixed with formaldehyde • Fluorescent / confocal microscopy analysis

of microscope preparates – statistics counting

– photographs taking

Yeastcell

pVT100U-mtGFP

GFP-labeled mitochondria

samples to fix

Time (h) Recovery

25oC

37oC Temperature of cells growth

46oC

-0.5 0 0.5 1 2

+/- CHX

0

10

20

30

40

50

60

70

80

90

100

0 30 60 90

WT

Δhsp78 [pYES2.0]

Δhsp78 [pYES-HSP78]% o

f c

ell

s w

ith

alt

ere

d m

ito

ch

on

dri

al

mo

rph

olo

gy

time after heat shock [min]

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.

46oC 25oC

before heat-shock 90 min reactivation

directly after heat-shock

Igor KoniecznyJarosław MarszałekGrażyna KonopaEwa Laskowska

Joanna KrzewskaAgnieszka LewandowskaMarlena MatuszewskaSzymon ZiętkiewiczKrzysztof Liberek

Protein Biochemistry Research GroupDepartment 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, 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

Selected publications

Financing:Polish State Committee for Scientific Research

collaboration