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Hsp40 couples with the CSPalpha chaperone complex upon induction of the heat shock response.

Gibbs SJ, Barren B, Beck KE, Proft J, Zhao X, Noskova T, Braun AP, Artemyev NO, Braun JE - PLoS ONE (2009)

Bottom Line: Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s).This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins.In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics & Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

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Related in: MedlinePlus

Effect of heat shock on the CSPα dimer.CAD cells were transfected with CSPα, treated as indicated with 200 µM quercetin and then heat shocked for 40 min at 42°C and allowed to recover for 5 hours. CSPα was detected by western analysis. Quantification of CSPα dimer. N values are indicated in brackets.
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pone-0004595-g004: Effect of heat shock on the CSPα dimer.CAD cells were transfected with CSPα, treated as indicated with 200 µM quercetin and then heat shocked for 40 min at 42°C and allowed to recover for 5 hours. CSPα was detected by western analysis. Quantification of CSPα dimer. N values are indicated in brackets.

Mentions: Although CSPα is present in neural cell lines, its expression is ∼10 fold lower (10±1.7, n = 3, data not shown) than that found in rat brain homogenates. To conduct a more detailed analysis of the CSPα chaperone complex, CSPα was examined in CAD mouse neuroblastoma cells transiently transfected with CSPα in order to bring CSPα levels up to those found in adult rat brain. After transfection a 70 kDa CSPα immunoreactive band was observed (Figure 4). 70 kDa CSPα dimers have previously been reported in rat brain [13], [20], rat hippocampus [12], rat pancreas [13], PC12 cells transiently expressing CSPα [14], [21], and HEK293 cells transiently expressing CSPα [22]. The cellular role of the CSPα dimer is not known. Figure 4 shows that in CAD cells transfected with CSPα the 70 kDa CSPα dimer is stable, SDS-resistant and maintained after incubation in sample buffer at 80°C for 10 min. Figure 4 clearly demonstrates that following 40 minutes of conditioning heat shock at 42°C, there is a decline in the CSPα dimer detected. In contrast, quercetin increases the CSPα-CSPα complex in control and heat shocked cells (Figure 4). The upper panel in Figure 4 is an overexposure of the 70 kDa CSPα dimer demonstrating its presence in control but not heat shocked CAD cells. In transfected (Figure 4) but not untransfected (Figure 1) CAD cells, quercetin was observed to increase expression levels of the CSPα monomer. Taken together, these data show that the extremely stable CSPα dimer is regulated by quercetin as well as by a conditioning heat shock.


Hsp40 couples with the CSPalpha chaperone complex upon induction of the heat shock response.

Gibbs SJ, Barren B, Beck KE, Proft J, Zhao X, Noskova T, Braun AP, Artemyev NO, Braun JE - PLoS ONE (2009)

Effect of heat shock on the CSPα dimer.CAD cells were transfected with CSPα, treated as indicated with 200 µM quercetin and then heat shocked for 40 min at 42°C and allowed to recover for 5 hours. CSPα was detected by western analysis. Quantification of CSPα dimer. N values are indicated in brackets.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2643527&req=5

pone-0004595-g004: Effect of heat shock on the CSPα dimer.CAD cells were transfected with CSPα, treated as indicated with 200 µM quercetin and then heat shocked for 40 min at 42°C and allowed to recover for 5 hours. CSPα was detected by western analysis. Quantification of CSPα dimer. N values are indicated in brackets.
Mentions: Although CSPα is present in neural cell lines, its expression is ∼10 fold lower (10±1.7, n = 3, data not shown) than that found in rat brain homogenates. To conduct a more detailed analysis of the CSPα chaperone complex, CSPα was examined in CAD mouse neuroblastoma cells transiently transfected with CSPα in order to bring CSPα levels up to those found in adult rat brain. After transfection a 70 kDa CSPα immunoreactive band was observed (Figure 4). 70 kDa CSPα dimers have previously been reported in rat brain [13], [20], rat hippocampus [12], rat pancreas [13], PC12 cells transiently expressing CSPα [14], [21], and HEK293 cells transiently expressing CSPα [22]. The cellular role of the CSPα dimer is not known. Figure 4 shows that in CAD cells transfected with CSPα the 70 kDa CSPα dimer is stable, SDS-resistant and maintained after incubation in sample buffer at 80°C for 10 min. Figure 4 clearly demonstrates that following 40 minutes of conditioning heat shock at 42°C, there is a decline in the CSPα dimer detected. In contrast, quercetin increases the CSPα-CSPα complex in control and heat shocked cells (Figure 4). The upper panel in Figure 4 is an overexposure of the 70 kDa CSPα dimer demonstrating its presence in control but not heat shocked CAD cells. In transfected (Figure 4) but not untransfected (Figure 1) CAD cells, quercetin was observed to increase expression levels of the CSPα monomer. Taken together, these data show that the extremely stable CSPα dimer is regulated by quercetin as well as by a conditioning heat shock.

Bottom Line: Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s).This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins.In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics & Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

ABSTRACT
In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

Show MeSH
Related in: MedlinePlus