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Hsp70 and Hsp40 Chaperone Activities in the Cytoplasm and the Nucleus of Mammalian Cells

(Received for publication, April 18, 1997, and in revised form, September 23, 1997) Annemieke A. Michels, Bart Kanon, Antonius W. T. Konings, Kenzo Ohtsuka, Olivier Bensaude, and Harm H. Kampinga From the Department of Radiobiology, Faculty of Medicine, University of Groningen 9713 B2 Groningen, The Netherlands, Ge ne tique Mole culaire, Centre de la Recherche Scientifique Unite 1302, Ecole Normale Supe rieure, Paris Cedex 05, France, and the Laboratory of Experimental Radiology, Aichi Cancer Center Research Institute, Chikusaku, Nagoya 464, Japan

Chaperones vs. Heat shock proteins (Hsps)


Chaperones Different types of proteins that help with folding/unfolding of proteins Two classes:
Monomeric chaperones: Hsp70/Hsp40 Multisubunit complexes: chaperonins

Hsp70/Hsp40

A class of monomeric chaperones Upregulated in response to temperature stress

Hsp70

Chaperonin

Hsp40

Important Players
O23 hamster fibroblasts hu-Hsp70/hu-Hsp40 Firefly cyt-luciferase and nuc-luciferase Plasmids:
nuc- and cyt-luciferases: pRSVnlsLL/V, pRSVLL/V heat shock proteins: pCMV5, pCMV70, pCMV40

Purpose
To examine the role of human Hsp70 and Hsp40 in mammalian cells - Protection against denaturation - Recovery from heat-induced protein denaturation

Knowledge prior to this experiment


HSPs work to protect the cells against heat Transient thermotolerant state results in increase of Hsps Cells subjected to heat shock is less susceptible to a following heat shock

Experimental Procedures
Plasmids and Cloning Techniques Cell Culture, Transfection, Heat Shock, and Cell Lysis Western Blotting Insolubilization Experiments Immunohistochemistry

Plasmid Cloning
Luciferases:
Cytoplasmic: pRSVLL/V Nuclear: pRSVnlsLL/V

Vector: pCMV5 HSPs 40 & 70:


pCMV40 pCMV70

Recognition Sites:
HindIII-BamHI BglII-HindIII

Cell Culture, Transfection, Heat Shock, and Cell Lysis


1) O23 hamster fibroblasts grown in Dulbeccos modified Eagles medium 2) Transfected with luciferases plasmids with/without pCMV40&70 3) Inactivation experiments (heat shock) 4) Recovery experiments 5) Cell Lysis 6) Measured luciferases activities

Western Blotting
Hsp70 Detection
1 Ab: monoclonal antibody 2 Ab: peroxidase-conjugated sheep-anti-mouse

Hsp40 Detection
1 Ab: anti-Hsp40 antiserum 2 Ab: peroxidase-conjugated donkey-anti-rabbit

Insolubilization Experiments
Heated and unheated cells lyzed in buffer A Fractionated by centrifugation at 12,000 x g into supernatants and pellets Both dissolved in SDS & 2-mercaptoethanol Run a western blot to analyze presence of luciferase

Immunohistochemistry
Process of detecting antigens in cells using Ab Cells transfected with pCMV40 or pCMV70 Components used:
PBS, paraformaldehyde, BSA

Hsp70 Detection
1 Ab: anti-Hsp70 2 Ab: Cy3-conjugated antimouse antibody

Hsp40 Detection
1 Ab: anti-Hsp40 2 Ab: fluorescein-conjugated (FITC) anti-rabbit antibody

Controls & Variables

Figure 2
Controls: Positive control: unstressed, nontransfected 023 fibroblasts Negative control: thermotolerant cells (heat-shocked for 20min at 44C) Variables: Cells transfected with: 1) pCMV5 2)pCMV40

3)pCMV70

*All cells were transfected with either pRSVLL/V or pRSVnlsLL/V

Protection Factor (PF)


Ratio that allows comparing luciferase inactivation in the absence or presence of HSPs When PF < 1, inactivation of luciferase is faster than the control When PF > 1, inactivation of luciferase is slower than the control

Thermal Denaturation of Luciferases in Vivo


Cells transfection:
Cyt-luciferase: Fibroblasts + pRSVLL/V Nuc-luciferase: Fibroblasts + pRSVnlsLL/V

Heated at various temperatures


2-10min heating at 40C 2-6min heating at 42C 2-4 min heating at 44C

Inactivation rate constants determined

Inactivation kinetics of nuc- and cytluciferase at different temps.


o Open symbols: nuc-luciferase Closed symbols: cyt-luciferase

Figure 1

Expression of the Human Hsp40 Sensitizes Luciferase Denaturation


Cells transfected with pCMV40 Underwent heat shock followed by recovery at 37C Expression of hu-Hsp40 was evaluated by Western blotting
Lysates from cells were used

Nontransfected Cells:

Transfected Cells:

Co-transfected Cells:
- Cyt-/Nuc-luciferase w/ pCMV40 - no protective effect on luciferase - higher inactivation rates

-Substantial increase in Hsp40 expression levels Unstressed -No induction of ham-Weak Hsp40 band Hsp70 - No Hsp70 detected -Expression of huHsp40 did not Thermotolerant induce a stress - High amounts of response Hsp40 and Hsp70

Expression of the hu-Hsp70 Attenuates Luciferase Denaturation


Cells transfected with pCMV70 Underwent heat shock followed by recovery at 37C Expression of hu-Hsp70 was evaluated by Western blotting Lysates from cells were used

Nontransfected Cells:
Unstressed -Weak Hsp40 band - No Hsp70 detected Thermotolerant - High amounts of Hsp70 and Hsp40

Transfected Cells:
-Substantial increase in Hsp70 expression levels -No induction of hamHsp40 - did not induce a stress response

Co-transfected Cells:
- Cyt-/Nuc-luciferase w/ pCMV70 - both luciferases were protected against heat inactivation at all temps - level of protection: nuc-lucif < cyt-lucif

Expression of human Hsp40 and Hsp70 in O23 cells after transient transfection
Non-transfected cells Control: unstressed
Weak Hsp40 band; no Hsp70 band

TT: stressed
High amounts of Hsp40 and Hsp70

Transfected cells pCMV70: stressed


High amounts of Hsp70

Figure 2 hu-Hsp40

pCMV40: stressed
High amounts of Hsp40
hu-Hsp70

Effect of hu-Hsp40 on the Protective Action of hu-Hsp70


Combined hu-Hsp40 and hu-Hsp70 expression Heat-shocked at 40C, 42C and 44C

Effects of hu-Hsp40, hu-Hsp70 and huHsp40+hu-Hsp70 on heat inactivation of luciferase

Figure 3

PF for hu-HSP40, hu-Hsp70 and hu-Hsp40+huHsp70

Table I
Thermal inactivation rates of nuclear and cytoplasmic luciferase and effects of hu-Hsp40 hu-Hsp40 hu-Hsp70 hu-Hsp70 + hu-Hsp40

Solubility of Recombinant Proteins


Indicator of protein denaturation Solubility of recombinant luciferases, Hsp40 and Hsp70 was examined Unstressed and heat-shocked cells were fractionated
Unstressed cells Heat-shocked cells (0, 5, 10, and 20 mins at 43C)

Cyt-/Nuc-luciferases, Hsp40 and Hsp70 were detected in the soluble fraction

Both luciferases and Hsp40 detected in insoluble fraction Hsp70 remained in soluble fraction

Supernatant (S) The bands become lighter as time passes indicating that the proteins starts to disappear from the supernatant and into the pellet Pellet (P) The bands become darker as time passes indicating the presence of proteins in the pellet

hu-Hsp40 and hu-Hsp70 Localization


Overexpression and localization of hu-Hsp40 or hu-Hsp70 affect the inactivation kinetics of cyt- and nuc-luciferase Performed indirect immunofluorescence on transfected cells and examined under confocal microscopy

Immunolocalization of transfected Hsp40 and Hsp70 in O23 cells

pCMV40 Figures 5

pCMV70

Recovery of Luciferases upon hu-Hsp70 and hu-Hsp40 Coexpression


I Recovery of Luciferases w/out the hu-Hsps O23 cells transiently transfected with pRSVnlsLL/V or pRSVLL/V were heated for 30min at 44C and allowed to recover at 37C for 060mins Control cells were kept at 37C Cell lysates were loaded on 10% polyacrylamide gels and transferred to nitrocellulose These were probed with an anti-luciferase antibody

Figure 6

Figure 7

Circles nuc-lucif. Squares cyt-lucif. Open symbols heat-shocked for 15mins at 42C Closed symbols heatshocked for 15mins at 43C

Recovery was less for nuc-luciferase compared with cyt-luciferase

The total amount of both luciferase proteins did not change during heating or up to 60min thereafter

II Recovery of Luciferases w/ hu-Hsps O23 cells transiently transfected with


pRSVnlsLL/V or pRSVLL/V alone or with pCMV40, pCMV70 or both pCMV40 and pCMV70

Heated for 30min at 44C and recovered at 37C for 0-60min with cycloheximide

Effects of hu-Hsp40, hu-Hsp70, and huHsp40 + hu-Hsp70 transfection on recovering luciferase

Figure 8A

More recovery time for hu-Hsp40 transfected cells


Black column: O23 cell transfected with pRSVnlsLL/V or pRSVLL/V alone

White column: above cells transfected with pCMV40


ALL cells heat shocked for 30min at 44C and allowed 4hr recovery at 37C

Figure 8B

Coexpression of hu-Hsp40 and huHsp70


In vitro studies: level of Hsp70-mediated recovery of luciferase from its denatured state is highly dependent on the stoichiometry of Hsp70 and luciferase Tested whether the coexpression of hu-Hsp40 might have changed the Hsp70/luciferase ratio in the cell Transfected cells with o either luciferase and pCMV70 alone or oLuciferase and both pCMV70 and pCMV40 Lysates containing equal luciferase activities were probed for Hsp70 levels

Figure 9 The level of Hsp70 per active luciferase protein is independent of the absence or presence of co-transfected hu-Hsp40

Enhancement of hu-Hsp70 functions in the presence of hu-Hsp40 during recovery


The importance of differences in initial inactivation on the recovery in the presence of hu-Hsp70 and hu-Hsp40 was investigated Observed in nuclear luciferase only based on Fig 8A O23 cells transiently transfected with pRSVnlsLL/V or pRSVLL/V together with pCMV70 or both pCMV40 and pCMV70 were heated for 30 min at 42, 43, 44, 45 and 46C in the presence of cycloheximide

At 42C, the nuc-luciferase was inactivated to a level comparable to cyt-luciferase in cells heated at 44C

Table 2

In the presence of hu-Hsp70, hu-Hsp40 enhanced the recovery of both luciferases, but this enhancement was more pronounced when the inactivating heat-shock, and thus the initial inactivation, was more severe.

Hsp70-mediated Protection Against Denaturation


hu-Hsp70 yielded protective effect on both luciferases Protection decreased with increasing temp. The degree of protection by Hsp70 decreases with increasing protein denaturation force Expression of hu-Hsp70 enhances luciferase recovery

Expression of the Human Hsp40 Sensitizes Luciferase Denaturation and Decreases Recovery
hu-Hsp40 sensitizes luciferase for heat inactivation
hu-Hsp40 overexpression prevents the endogenous Hsp70 from protecting the luciferase

No recovery in cells expressing hu-Hsp40 Hsp40 overexpression may cause imbalance and disrupts other protein-protein interactions and their ratios

Hsp40 Cooperate with Hsp70 for an Accelerated Recovery


Overexpression of hu-Hsp40 accelerated recovery of luciferase when hu-Hsp70 was also expressed
Hu-Hsp40 may act as a co-chaperone of Hsp70 in assisting the refolding of luciferase

More prominent for severe heat treatments

QUESTIONS???