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Human Molecular Genetics Advance Access originally published online on May 6, 2005
Human Molecular Genetics 2005 14(12):1659-1669; doi:10.1093/hmg/ddi174
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells

Serena Carra1, Mitchel Sivilotti1, Aura T. Chávez Zobel2, Herman Lambert1 and Jacques Landry1,*

1Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, 9 rue McMahon, Québec, Canada G1R 2J6 and 2Universidad Centroccidental Lisandro Alvarado, Decanato de Medicina, Departamento de Morfología, Sección de Anatomía Microscópica, Avenida Libertador con Avenida Andrés Bello, Barquisimeto, Estado Lara, Venezuela

* To whom correspondence should be addressed: Tel: +418 6915281; Fax: +418 6915439; Email: jacques.landry{at}med.ulaval.ca

Received March 1, 2005; Revised April 19, 2005; Accepted April 26, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The family of small heat shock proteins (sHsp) is composed of 10 members in mammals, four of which are found mutated in diseases associated with the accumulation of protein aggregates. Though many sHsp have demonstrated molecular chaperone activity in vitro in cell-free conditions, their activity in vivo in the normal cellular context remains unclear. In the present study, we investigated the capacity of the sHsp, HspB8/Hsp22, to prevent protein aggregation in the cells using the polyglutamine protein Htt43Q as a model. In control conditions, Htt43Q accumulated in perinuclear inclusions composed of SDS-insoluble aggregates. Co-transfected with Htt43Q, HspB8 became occasionally trapped within the inclusions; however, in most cells, HspB8 blocked inclusion formation. Biochemical analyses indicated that HspB8 inhibited the accumulation of SDS-insoluble Htt43Q as efficiently as Hsp40 which was taken as a positive control. Htt43Q then accumulated in the SDS-soluble fraction, provided that protein degradation was blocked by proteasome and autophagy inhibitors. In contrast, the other sHsp Hsp27/HspB1 and {alpha}B-crystallin/HspB5 had no effect. This suggested that HspB8 functions as a molecular chaperone, maintaining Htt43Q in a soluble state competent for rapid degradation. Analyses of Hsp27–HspB8 chimeric proteins indicated that the C-terminal domain of HspB8 contains the specific sequence necessary for chaperone activity. Missense mutations in this domain at lysine 141, which are found in human motor neuropathies, significantly reduced the chaperone activity of the protein. A decrease in the HspB8 chaperone activity may therefore contribute to the development of these diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The accumulation and aggregation of misfolded proteins can be highly cytotoxic and is at the origin of several human degenerative diseases characterized by neuronal inclusions such as Alzheimer's, Parkinson's, prion-like and polyglutamine diseases (1Go–4Go). Under normal circumstances, chaperone proteins involved in protein quality control can prevent protein aggregation by binding to misfolded proteins as soon as they are produced during translation or later during their organization into supramolecular structures, thereby assisting in the protein refolding or else in targeting for degradation (3Go,5Go). Under proteotoxic stresses produced either by extrinsic factors such as heat shock or intrinsic causes such as gene alterations yielding modifications to protein structure, the overall chaperone activity of the cell may be exceeded. As a consequence, denatured proteins aggregate and accumulate as toxic insoluble plaques, trapping chaperones, ubiquitin and other elements of the proteasome system, with dramatic consequences for the cells (4Go,6Go–9Go).

Mammalian cells possess a number of proteins, which are induced in response to proteotoxic stresses and display protective chaperone activity. Hsp70 proteins and associated co-chaperones CHIP and the Hsp40 proteins Hdj-1 and Hdj-2 are the best characterized among these proteins (5Go,10Go). Functionally, Hsp70 are ATPases that can bind newly exposed hydrophobic sequences on denatured proteins. In this reaction, ATP hydrolysis provides the energy for cycles of association and disassociation, a process allowing the denatured protein with repeated opportunities to achieve native conformation. The co-chaperones of the Hsp40 family play essential roles in this process by stimulating the ATPase activity of Hsp70. Hsp40 may also directly bind denatured substrates and prevent their aggregation independently of Hsp70 (11Go). When correct refolding is not possible, Hsp70/Hsp40 target bound substrates to proteasomal degradation in collaboration with other Hsp70 interacting proteins such as CHIP, an ubiquitin E3-ligase (12Go). Another group of heat shock proteins (Hsp), called the small Hsp (sHsp) consists 10 members in human and mouse (HspB1–10), of which, Hsp27 (HspB1), {alpha}A-crystallin (HspB4) and {alpha}B-crystallin (HspB5) are the best-known representatives (13Go,14Go). Common to the C-terminal of all sHsp is the {alpha}-crystallin domain, a conserved stretch of 100 amino acids known to play an essential role in sHsp structure (15Go,16Go). Like Hsp70, sHsp also display chaperone activity in vitro where they can prevent the aggregation of denatured proteins (17Go–19Go). In contrast to Hsp70, however, sHsp have no ATPase activity and binding to substrates appears to be modulated by the de-oligomerization of the sHsp, a process itself modulated by mechanisms such as phosphorylation or temperature change (20Go–22Go). The essential role of these chaperones therefore is to maintain target proteins in a non-aggregated folding-competent state (17Go–19Go). Renaturation of the target proteins eventually requires the action of ATP-dependent chaperones such as Hsp70 to complete the refolding process (18Go).

As expected from their in vitro chaperone activity, a role for Hsp70 and co-chaperones has been demonstrated in several experimental models of protein conformational diseases. Hsp70 and Hsp40 localize to the aggregates formed by mutated Ataxin-1, Huntingtin (htt) and androgen receptor, proteins responsible for Spinocerebellar Ataxia type-1, Huntington's disease and Kennedy's disease, respectively (23Go–25Go). Moreover, Hsp40 overexpression suppresses the aggregation of various mutant polyQ proteins, such as androgen receptor, Ataxin-1, Ataxin-3 and htt, resulting in protein resolubilization or enhanced degradation, both processes critical to cellular protection. (23Go–32Go). In another example, overexpression of CHIP enhances the proteasomal degradation of mutated cystic fibrosis transmembrane conductance regulator, a protein responsible for cystic fibrosis (33Go). Finally, Hsp70 and CHIP or Hsp40 (Hdj-1) prevent the formation of inclusions composed of {alpha}BR120G, the {alpha}B-crystallin mutant responsible for desmin related myopathy (34Go).

In contrast to Hsp70 and co-chaperones for which a chaperone activity is well demonstrated both in vitro and in vivo (35Go), direct evidence of in vivo sHsp chaperone activity is lacking. Hsp27 has been shown to protect against Htt43Q-induced cell death, however, this observation did not coincide with the prevention of Htt43Q aggregation and, instead, supported a role for Hsp27 in protection against the oxidative stress generated downstream of aggregate formation (36Go). Hsp27 was also found ineffective in preventing aggregation of a mutant of Ataxin-3 (26Go). Hsp27 and {alpha}B-crystallin efficiently protect against heat shock induced-cell death, however, it is not known whether this protection occurs through the prevention of protein aggregation or through other specialized functions also attributed to these proteins (37Go,38Go). Nevertheless, in many protein conformation diseases, sHsp including {alpha}B-crystallin and Hsp27, similar to Hsp70 are upregulated and localize to cellular inclusion bodies. This is observed for example in the Rosenthal fibers of Alexander disease, the Mallory bodies of alcoholic liver disease, the cortical Lewy bodies, and into plaques and neurofibrillary tangles of Alzheimer disease. (3Go,39Go–43Go). Perhaps the strongest evidence for a role of the sHsp in protein conformational diseases is the existence of several hereditary neurodegenerative diseases associated with mutations in sHsp namely Hsp27, {alpha}B-crystallin, {alpha}A-crystallin and HspB8/Hsp22 (44Go–49Go). All these mutations compromise the structural stability of the affected proteins, resulting in their aggregation. It remains unclear whether the disease phenotype associated with sHsp aggregation is a product of a gain of function (cellular toxicity of the aggregate body) or a loss of chaperone activity.

HspB8 is a new member of the sHsp family highly expressed in the heart, brain and striated and smooth muscles (50Go). In a recent paper, we showed that HspB8 overexpression inhibits aggregate formation and rescues wild-type oligomeric organization of the {alpha}B-crystallin R120G mutant (34Go). These data provided evidence that HspB8 may act as a chaperone in vivo. However, as {alpha}B-crystallin and HspB8 are both sHsp, the general significance of the results were unclear. Here, we tested the ability of HspB8 and other sHsp family members to block the formation of aggregates by Htt43Q, a mutant htt fragment with a stretch of 43 glutamines. We show that HspB8, but not Hsp27 or {alpha}B-crystallin, prevented the accumulation of Htt43Q in the SDS-insoluble fraction of CCL39 and HEK293 cells. Moreover, when protein degradation was inhibited, Htt43Q accumulated only in the soluble fraction of the cells overexpressing HspB8. The results suggest that HspB8 can accomplish a chaperone function in vivo, keeping Htt43Q in a non-aggregated state competent for degradation. We also show that HspB8 containing C-terminal mutations equivalent to those found in hereditary motor and sensory neuropathies had significantly reduced chaperone activity, signaling the potential contribution of this activity in the progression of these diseases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HspB8 blocks the formation of Htt43Q aggregates
The effect of recombinant HspB8 on the in vitro thermal aggregation of citrate synthase (CS) was evaluated by measuring at intervals the turbidity (light scattering at 320 nm) of a solution of containing CS alone or CS in the presence of increasing concentrations of HspB8. Hsp27 was also evaluated as a control (Fig. 1A). A 50% protection level was attained at a HspB8/CS molecular ratio of 4:1, whereas a similar protection was attained in the case of Hsp27 at a molecular ratio of 1:1 (all molecules considered as monomer). Thus, although less efficient that Hsp27 for protecting CS, HspB8 demonstrated clear in vitro chaperone activity. To determine whether HspB8 could also show chaperone activity in the normal cellular context, we used a fragment of the htt protein containing a polyglutamine extension of 43 residues (Htt43Q) as a model of unstable protein. Htt containing more than 37 consecutive glutamines is known to form insoluble intracellular aggregates, a phenotype associated with Huntington's disease (51Go). Using immunofluorescence microscopy in CCL39 cells, we analyzed the effect of HspB8 overexpression on the intracellular aggregation of Htt43Q. Transfection of pHDQ43-HA, a plasmid encoding a HA-tagged version of Htt43Q (36Go), induced the formation of perinuclear aggregates in 90% of the cells, some of which also contained soluble materials as indicated by a diffuse signal (Fig. 1B and 1E). Co-expression of HspB8 with Htt43Q dramatically reduced Htt43Q aggregation as >90% of CCL39 cells expressing both HspB8 and Htt43Q showed a diffuse staining for Htt43Q with no aggregate (Fig. 1C and 1E). In cells where inclusion bodies were observed in spite of the presence of HspB8, HspB8 was found co-localized with the Htt43Q aggregates (Fig. 1D). Hence, HspB8 activity resembles that of the chaperones Hsp70 and Hsp40, which can prevent inclusion formation by polyglutamine proteins but are often found trapped in refractory aggregates (26Go).



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  		Figure 1. Effect of HspB8 expression on the aggregation of citrate synthase (CS) and Htt43Q. (A) CS was heated at 43°C for up to 30 min in the presence of various concentrations of recombinant Hsp27 (white circle) or HspB8 (black circle). The relative light scattering was calculated as the ratio of light scattering at 320 nm of the CS solution heated for 30 min in the presence of the test protein over the light scattering of the CS solution heated for the same period of time in the absence of other proteins. Heated alone, >90% of CS was aggregated after 30 min at 43°C. CCL39 cells were transfected with either pHDQ43-HA (Htt43Q) and an empty vector (B and E) or pHDQ43-HA and pMycHspB8 (Htt43Q+HspB8-myc) (C–E). The cells were fixed and processed for immunofluorescence staining 44 h after transfection using anti-HA and anti-HspB8 antibodies. The percent numbers of cells expressing Htt43Q either as aggregates (illustrated in B), in a diffuse pattern (illustrated in C) or both, were determined under the microscopes (E). In cells where inclusion bodies were observed in spite of the presence of HspB8, HspB8 was found co-localized with the Htt43Q aggregates (D). Data are mean+SD of five independent experiments. P<0.001 (One-way ANOVA) comparing aggregates (*) or diffuse signal (**) between the Htt43Q and the Htt43Q+HspB8-myc groups.

 
HspB8 prevents the accumulation of SDS-insoluble Htt43Q
Aggregates of proteins with extended polyglutamine stretches are insoluble in SDS-lysis buffer (31Go). Employing this principle, we investigated whether the reduction of Htt43Q aggregation observed by immunofluorescence in CCL39 cells overexpressing HspB8 would correlate with a decrease of SDS-insoluble Htt43Q. CCL39 cells were transiently transfected with pHDQ43-HA alone or with pMycHspB8. The activity of HspB8 was compared to that of two other sHsp Hsp27 and {alpha}B-crystallin. Hsp40, a known chaperone of Htt43Q (24Go,32Go) which likely work through distinct mechanisms was used as a positive control. CHIP, a E3-ligase and co-chaperone of Hsp70 with unknown activity in this system was also evaluated and presented here as negative control. Following transfection, the cells were lysed in an SDS-containing buffer and the extracts were filtered through a cellulose acetate membrane (31Go). The SDS-insoluble proteins retained on the membrane were probed with an anti-HA antibody. As expected, expression of Hsp40 had a major inhibitory effect on the accumulation of co-transfected Htt43Q, totally eliminating Htt43Q from the SDS-insoluble fraction at 44 h post-transfection. CHIP demonstrated little or no activity, while HspB8, like Hsp40, almost completely eliminated Htt43Q in the SDS-insoluble fraction. The effect of HspB8 contrasted with that of the other sHsp, Hsp27 and {alpha}B-crystallin, which had no or very little activity (Fig. 2A). A similar effect of HspB8 on the accumulation of SDS-insoluble Htt43Q was observed in HEK-293 cells, indicating that the activity of HspB8 was not cell-type specific (described later in Fig. 5).



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  		Figure 2. Effect of HspB8 expression on Htt43Q solubility. CCL39 cells were transfected with pHDQ43-HA and an empty vector (Htt43Q), with pHDQ43-HA and either pMycHspB8 (+HspB8-myc), pCMV40 (+Hsp40), pMycCHIPWT (+CHIP-myc), pMycHsp27 (+Hsp27-myc) or pMycCRYS (+HspB5-myc) or left untransfected (control). (A) Extracts were prepared for filter trap analyses at 44 h post-transfection. The samples were dot-blotted at four different concentrations (relative concentrations of 1, 2/3, 1/3 and 1/6,) on a cellulose acetate membrane and probed with anti-HA. (B and C) SDS-soluble and formic acid-disaggregated SDS-insoluble extracts were prepared at 24 and 44 h post-transfection, separated by SDS–PAGE and analyzed by western blot using the anti-HA antibody to detect levels of Htt43Q (B). The SDS-soluble extracts were also analyzed with the anti-myc antibody to determine the level expression of the co-expressed chaperones (C).

 


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  		Figure 5. Effect of chimeric Hsp27–HspB8 on Htt43Q solubility. CCL39 or HEK 293 cells were transfected with pHDQ43-HA and an empty vector (Htt43Q) or with pHDQ43-HA and either pHspB8 (+HspB8), pN27CB8a, pNB8C27a, pN27CB8b or pNB8C27b. pN27CB8a expressed residues 1–72 of Hsp27 fused to residues 74–196 of HspB8, pN27CB8b expressed residues 1–91 of Hsp27 fused to residues 93–196 of HspB8, pNB8C27a expressed residues 1–73 of HspB8 fused to residues 73–205 of Hsp27 and pNB8C27b expressed residues 1–92 of HspB8 fused to residues 92–205 of Hsp27. Extracts were prepared for filter trap analyses at 44 h post-transfection. The samples were dot-blotted at four different concentrations (relative concentrations of 1, 2/3, 1/3 and 1/6) on a cellulose acetate membrane and probed with anti-HA.

 
Accumulation of SDS-soluble Htt43Q in cells overexpressing HspB8
To determine the fate of the polyglutamine proteins in cells overexpressing HspB8, cellular extracts prepared in SDS-buffer were centrifuged to separate SDS-soluble (supernatant) from SDS-insoluble (pellet) proteins. The SDS-insoluble fractions were dissolved in formic acid as described in Materials and Methods, allowing subsequent SDS-solubilization (52Go,53Go). Both fractions were then analyzed by electrophoresis and western blotting. Cells transfected with Htt43Q alone showed at 24 h an equal distribution of Htt43Q between the soluble and insoluble fractions. With time Htt43Q progressively shifted to the insoluble fraction, where the majority of the proteins could be found by 44 h in most experiments (Fig. 2B). HspB8, like Hsp40, prevented the accumulation of the protein in the insoluble fraction and also caused a marked decrease in the level of the soluble protein at 44h. CHIP, Hsp27 and {alpha}B-crystallin (HspB5), expressed at concentrations equivalent to, or even higher than that of HspB8, produced little effect on either fraction (Fig. 2B and 2C).

These results suggested that HspB8 might facilitate the degradation of misfolded proteins. As both autophagy and degradation by the proteasome have been implicated in the elimination of mutated htt (54Go,55Go), we tested the effects of 3-methyladenine (3-MA), an inhibitor of autophagy, and MG132, a proteasome inhibitor, on the expression of Htt43Q in the presence or absence of HspB8. As expected, a treatment with the inhibitors in the absence of HspB8 caused an accumulation of Htt43Q in the insoluble fraction (Fig. 3). In the presence of HspB8, the inhibitors prevented the disappearance of Htt43Q. Htt43Q, which was then not degraded, persisted in the soluble fraction. These results suggested that HspB8 acts as a chaperone, keeping Htt43Q in a soluble form that is more efficiently eliminated by degradation.



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  		Figure 3. Htt43Q accumulates in the soluble fraction in the presence of HspB8 and inhibitors of the proteasomal and autophagic degradation pathways. CCL39 cells were transfected with pHDQ43-HA and either an empty vector (Htt43Q) or pMycHspB8 (+HspB8-myc). SDS-soluble and SDS-insoluble extracts were prepared at 24 or 44 h post-transfection. When indicated (3-MA+MG), 10 mM 3-MA and 10 µM MG132 were added 20 h before protein extraction. Htt43Q was revealed by western blot using the anti-HA antibody.

 
HspB8 also prevents AR65Q aggregation
To test whether the chaperone-like activity of HspB8 could be generalized to other polyglutamine proteins, CCL39 cells were transfected with AR65Q, the androgen receptor protein containing a 65 glutamine extension, alone or together with HspB8. The expansion of the polyglutamine repeat sequence in the androgen receptor has been linked to Spinal and bulbar muscular atrophy (56Go,57Go). As expected (Fig. 4), AR65Q accumulated in the SDS-insoluble fraction when transfected alone, however, SDS-insoluble AR65Q was absent in cells co-transfected with HspB8 or the positive control Hsp40 (29Go,30Go). Similar to the observations made with Htt43Q, co-expression with Hsp27 triggered no reduction in the accumulation of SDS-insoluble AR65Q (Fig. 4).



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  		Figure 4. Effect of HspB8 expression on the mutated androgen receptor (AR65Q) solubility. CCL39 cells were transfected with pHDQ43-HA or pAR65Q-HA and either an empty vector (PolyQ), pMycHspB8 (+HspB8-myc), pCMV40 (+Hsp40), pMycHsp27 (+Hsp27-myc) or pHspB8 (+HspB8). Formic acid-disaggregated SDS-insoluble extracts were prepared 44 h post-transfection and analyzed by western blot using the anti-HA antibody to detect the Htt43Q and AR65Q proteins.

 
The {alpha}-crystallin domain of HspB8 possesses chaperone activities
It is intriguing that Hsp27 showed no in vivo chaperone activity in spite of a high degree similarity in structure to HspB8. The structure of sHsp can be divided into two principle regions. In HspB8, the N-terminal domain extends from residues 1–85. The C-terminal or {alpha}-crystallin domain of HspB8 comprises residues 86–176. To determine which domain of HspB8 possesses the chaperone activity, we constructed chimeric molecules containing either the N-terminal domain of Hsp27 fused to the {alpha}-crystallin domain of HspB8 (N27CB8a and b) or, conversely, the N-terminal domain of HspB8 fused to the {alpha}-crystallin domain of Hsp27 (NB8C27a and b). The results of co-transfection experiments of Htt43Q with the chimeric proteins indicated that only the proteins containing the {alpha}-crystallin domain of HspB8 were active (Fig. 5).

Effect of K141N and K141E HspB8 mutants on Htt43Q aggregation
Recently, two missense mutations (K141N and K141E) were found in the {alpha}-crystallin domain of HspB8 associated with motor neuropathies (47Go,49Go). We used Htt43Q as an in vivo substrate to investigate whether these mutations alter HspB8's chaperone activity. CCL39 or HEK293 cells were transfected with Htt43Q alone or with either K141N- or K141E-HspB8. A significant reduction in the apparent chaperone activity of the disease-causing mutants compared with the wild-type protein was observed by immunofluorescence analyses of CCL39 cells transfected with Htt43Q and the HspB8 proteins (Fig. 6). Htt43Q was expressed as aggregates in only 10% of the cells also expressing HspB8, whereas ~25% of the cells expressing the K141N- or the K141E-mutant contained Htt43Q inclusions. The increase was significant in both cases (P<0.01). This reduced activity of the HspB8 mutants, however, could not be demonstrated consistently using either the filter trap assay of SDS-insoluble Htt43Q or the western blot analysis of soluble versus formic acid-solubilized SDS-insoluble materials (Fig. 7A and 7B). We therefore investigated whether or not a difference could be measured in the amount of Htt43Q that accumulated in the cell debris collected from cell growth medium at 44 h post-transfection. This analysis provides a relative measurement of the fraction of cells expressing Htt43Q that detach from the petri dishes and was used as a measure of toxicity. In the case of Hsp40, the chaperone activity results in protection at the cellular level (58Go). As expected, cultures transfected with Htt43Q were characterized by a large accumulation of Htt43Q in cellular debris, a result that was inhibited by expressing wild-type HspB8 as well as Hsp40. In three independent experiments where the activity of the HspB8 mutants was compared with that of the wild-type protein, cells overexpressing the HspB8 mutants accumulated more Htt43Q in the cellular debris collected from the growth medium when compared with cells overexpressing wild-type HspB8. The results suggested that the reduced chaperone activity of the HspB8 mutants had a significant impact upon Htt43Q toxicity (Fig. 7C).



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  		Figure 6. Effect of the HspB8 mutants K141N and K141E on Htt43Q aggregation. CCL39 cells were transfected with pHDQ43-HA and an empty vector (Htt43Q) or with pHDQ43-HA and either pMycHspB8 (+HspB8-myc), pHspB8 (+HspB8), pHspB8-K141N (+HspB8-K141N) or pHspB8-K141E (+HspB8-K141E). The cells were fixed at 44 h post-transfection and processed for immunofluorescence microcopy to determine the percentage of cells containing aggregates of Htt43Q (either aggregates alone or aggregates plus diffuse) or only a diffuse staining for Htt43Q. Data are mean+SD of three independent experiments. P<0.01 (One-way ANOVA) comparing aggregates (*) or diffuse signal (**) between the +HspB8-K141N or +HspB8-K141E group and the +HspB8 group.

 


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  		Figure 7. Effect of the HspB8 mutants K141N and K141E on Htt43Q solubility and accumulation in cellular debris. CCL39 cells were transfected with pHDQ43-HA and an empty vector (Htt43Q), with pHDQ43-HA and either pMycHspB8 (+HspB8-myc), pHspB8 (+HspB8), pHspB8-K141N (+HspB8-K141N), pHspB8-K141E (+HspB8-K141E) or pCMV40 (+Hsp40) or left untransfected (control). (A) SDS-soluble and formic acid-disaggregated SDS-insoluble extracts were prepared at 24 and 44 h post-transfection, separated by SDS–PAGE and analyzed by western blot using the anti-HA and anti-HspB8 antibodies to detect levels of Htt43Q and HspB8 expression, respectively. Arrowheads indicate the position of HspB8 (filled) and myc-tagged HspB8 (empty). (B) Extracts were prepared at 44 h post-transfection and processed for filter trap analyses. The samples were dot-blotted at four different concentrations (relative concentrations of 1, 2/3, 1/3 and 1/6) on a cellulose acetate membrane and probed with anti-HA. (C) The growth medium was collected 44 h after transfection and centrifuged to obtain the cellular debris. Samples combining both SDS-soluble and SDS-insoluble extracts were analyzed by western blot using the anti-HA antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Chaperones play an essential role in protein quality control as demonstrated by their ability to detect unfolded proteins and subsequently prevent their aggregation through the promotion of refolding or degradation. The importance of chaperones in protein conformational diseases has been well illustrated for Hsp70 and its associated co-chaperones (23Go–34Go). The sHsp also possess chaperone activity in vitro and strong evidence exists supporting their role in several conformational diseases. For example, Hsp27 and {alpha}B-crystallin are upregulated in several disorders characterized by protein aggregation and accumulate in inclusions containing the misfolded proteins (3Go,39Go–43Go). In addition, mutations affecting key members of the sHsp family (HspB8, Hsp27, {alpha}A- and {alpha}B-crystallin) are known to cause hereditary degenerative diseases in human (44Go–49Go). Nonetheless, evidence that any individual sHsp displays chaperone activity over a broad protein clientele in vivo remains illusive. For example, desmin related myopathy caused by a well-characterized mutation in {alpha}B-crystallin was suggested to result from a gain of toxic function by the destabilized {alpha}B-crystallin protein. A loss of function mutation was also considered, however, a specialized chaperone function towards desmin filaments rather than a generalized chaperone activity was proposed (34Go,59Go). In another example, cataracts caused by mutations in {alpha}A-crystallin appear to result from the aggregation of the {alpha}A-crystallin and its lens partner {alpha}B-crystallin (48Go). Previously, we demonstrated that HspB8 could promote the in vivo folding of the {alpha}BR120G mutant of {alpha}B-crystallin (34Go). HspB8 was not found in the final structure formed by {alpha}BR120G suggesting true chaperone activity, though this required clarification because HspB8 also had the ability to interact with the wild-type {alpha}B-crystallin protein. We showed here that HspB8, but not the highly related sHsp Hsp27 and {alpha}B-crystallin, could block the aggregation of polyglutamine proteins in vivo and promote their degradation. These findings provide strong evidence that a member of the sHsp family may have a physiological chaperone activity in vivo. Whether polyglutamine proteins represent real physiological substrates of HspB8 is not known and was not considered in this paper.

Little is known concerning the biochemical mechanisms of HspB8's observed chaperone activity. HspB8 appears to promote the solubilization of unfolded proteins as suggested by immunofluorescence data that showed diffuse staining of Htt43Q in the majority of HspB8 expressing cells in contrast with the formation of aggregates in the absence of HspB8. Biochemical analyses also revealed that Htt43Q accumulates in SDS-soluble fractions in the presence of HspB8 (when treated with inhibitors of proteasomal and autophagic degradation), whereas accumulation was observed in the SDS-insoluble fraction under conditions without HspB8. Similar results were obtained with the {alpha}BR120G mutant of {alpha}B-crystallin, where HspB8 also prevented mutant protein aggregation as examined by immunofluorescence microscopy and produced a shift of {alpha}BR120G from the insoluble to the soluble fraction (34Go). The fate of the HspB8 substrates differs in the two cases reviewed. In the case of the {alpha}B-crystallin mutant, HspB8 promoted the folding into a stable supramolecular structure and the protein achieved a native conformation (34Go). In the case described here, the polyglutamine mutants were found in majority in the degradation pathways. Such divergent fates for chaperone substrates have been previously described, specifically with Hsp70 and associated chaperones. Hsp70, for example, was found to mediate the refolding of luciferase following a heat shock treatment (60Go), whereas it enhanced the solubility and caused the proteasome-mediated degradation of a mutated androgen receptor (29Go). It has been shown that such decision between refolding or degrading likely depends on multiple factors including co-factor availability (such as CHIP) and the ability of the substrate to rapidly refold (61Go,62Go). sHsp possess no ATPase activity. From studies in vitro, it is proposed that their primary role is to keep substrates in a refolding-competent state until chaperones such as Hsp70 become available (18Go,19Go). HspB8 may therefore also require Hsp70. Hsp70-mediated mechanisms would then also dictate whether refolding or degradation prevails.

HspB8 and Hsp27 are highly similar in sequence and based of the 3D-structure established for related non-mammalian sHsp, their 3D-structure should be very similar (15Go,16Go,38Go). It is intriguing that Hsp27, {alpha}B-crystallin and HspB8 possess chaperone activity in vitro (17Go,63Go–65Go), whereas only HspB8 exhibits chaperone activity in vivo. The in vitro chaperone activity of sHsp depends on the presence of highly conserved hydrophobic sequences that serve as binding sites for denatured substrates. In {alpha}-crystallins, these sites have been localized to a short sequence in the C-terminal domain corresponding to residues 94–125 in HspB8 (reviewed in 15,66). The importance of these sequences is supported by the finding that the deletion of the entire N-terminus of {alpha}B-crystallin leaves a protein that is still active in vitro (67Go). Our finding that the C-terminal domain also provides the specificity of action of HspB8 relative to Hsp27 implies that these binding sites although highly conserved among Hsp27, {alpha}B-crystallin and HspB8, still contain some important differences. Another possibility is that the C-terminus of HspB8 contains specific regulatory rather than active sequences. The oligomeric structure of the sHsp is highly dynamic, showing rapid subunit exchanges and variable extents of oligomerization (20Go,68Go). This structural dynamics appears essential to the activity of sHsp as mutations that induce a higher oligomeric stability blocked the activity (22Go). Subunit exchange or de-oligomerization may augment the exposure of the binding sites, which are thought to be hidden in the normal oligomeric proteins, or change the binding properties of the chaperones (reviewed in 66Go). The activator of the chaperone activity of HspB8 in vivo is unknown. The possibility of constructing chimeric molecules between inactive Hsp27 and active HspB8 offers a powerful tool to identify sequences in HspB8 that contribute to the functional difference between Hsp27 and HspB8 and later identify the mechanisms involved.

Recently, mutations at Lys141 of HspB8 have been associated to hereditary motor neuropathies. Little is known about the physiological functions of HspB8 and the pathogenic role of these mutations. It has been suggested that the HspB8 mutants might gain a toxic function (47Go). Here, we reported that mutations at Lys141 produced a small but significant effect on the chaperone activity of HspB8. The results showed a 2–3-fold increase in the frequency of aggregate formation by Htt43Q in cells overexpressing HspB8-K141N or HspB8-K141E mutants when compared with cells expressing wild-type HspB8. The mutations also resulted in a reduction in the protective activity of HspB8 as determined from the increase in the Htt43Q-containing cell debris collected from cells expressing the mutants when compared with wild-type HspB8. Although modest, such a reduction in activity may be important in the etiology and/or progression of hereditary motor neuropathies particularly during aging when the total levels of Hsp and the general heat shock response to stress may be reduced (69Go,70Go). Hence, neurons from aged human expressing mutated HspB8 may not successfully control mutated or damaged protein accumulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and reagents
pHDQ43-HA encoding a HA-tagged htt exon 1 fragment with 43 CAG repeats and pAR65Q-HA encoding a HA-tagged androgen receptor with 65 CAG repeats were from Dr Rubinsztein (36Go) and Dr Fischbeck (56Go), respectively. pCINHspB8, pCINMycHspB8, pCRYSWT, pCRYSBR120G, pCINMic{alpha}B, pCINHU27 and pCINMycHU27 were previously described (20Go,34Go,71Go). They code for human wild-type and myc-tagged HspB8, wild-type, Arg120 to Gly120 mutant and myc-tagged wild-type {alpha}B-crystallin, and control and myc-tagged human Hsp27, respectively. pCIHspB8 contained the sequence of HspB8 cloned in pCI (Promega). pMycCHIPWT expressing human myc-tagged Chip-1 was a gift from Dr Cam Patterson (72Go). pCMV40-encoding Hdj1 (Hsp40) was a gift from Dr Kampinga (73Go). Plasmids expressing Hsp27–HspB8 chimera were constructed by polymerase chain reaction using appropriate primers and sequences contained in pCIHspB8 and pCINHU27. pN27CB8a expressed residues 1–72 of Hsp27 fused to residues 74–196 of HspB8, pN27CB8b expressed residues 1–91 of Hsp27 fused to residues 93–196 of HspB8, pNB8C27a expressed residues 1–73 of HspB8 fused to residues 73–205 of Hsp27 and pNB8C27b expressed residues 1–92 of HspB8 fused to residues 92–205 of Hsp27. pCIHspB8-K141N and pCIHspB8-K141E were constructed by polymerase chain reaction using appropriate primers and sequences contained in pCIHspB8. They express HspB8 with Lysine 141 changed to Asparagine or Glutamate, respectively. MG132 and 3-MA were from Sigma Chemicals.

Antibodies
The rabbit polyclonal antibody anti-HspB8 was raised against the C-terminal peptide KNELPQDSQEVTCT of human HspB8 coupled to the keyhole limpet hemocyanin. The rabbit polyclonal antibody anti-{alpha}B (anti-{alpha}B-crystallin) and the rabbit polyclonal antibody anti-Hu27 (anti-Hsp27) were described previously (34Go,74Go). The mouse monoclonal antibodies anti-HA (HA.11) and anti-myc (9E10) were from Sigma-Aldrich and American Type Culture Collection, respectively.

Cell culture and transfection
CCL39 (Chinese hamster lung fibroblast) and HEK 293 (human embryonic kidney cells) cells were grown in Dulbecco's modified Eagle medium (Gibco BRL) supplemented with 5 or 10% fetal bovine serum, respectively. Twenty-four hours after plating, the cells were transfected by calcium phosphate precipitation as previously described using 1.0–12 µg of plasmid DNA (37Go). The cells were incubated for 5 h with the precipitate and 50–100 µM chloroquine.

Preparation of recombinant Hsp27 and HspB8 proteins and in vitro thermal aggregation assay
Chinese hamster Hsp27 recombinant protein was prepared as previously described (38Go). Vector for the expression of the recombinant human HspB8 was obtained by the insertion of the HspB8 wild-type sequence at the BamHI restriction site of the pGEX6P vector (Amersham Biosciences). The resulting plasmid was subcloned in Escherichia coli strain BL21 Codon+RIL (Stratagene). Recombinant HspB8 was prepared as described earlier (38Go), with the exception that affinity purified GST–HspB8 was digested with PreScission Protease (Amersham Biosciences), instead of thrombin. The resulting protein contained five additional residues (Gly–Pro–Leu–Gly–Ser) at the N-terminus. HspB8 protein concentration was determined using an extinction coefficient of 1.2 for a 1 mg/ml solution at 280 nm (64Go). The thermal denaturation kinetics of CS were measured from the light scattering at 320 nm during incubation at 43°C using a Varian spectrophotometer (model Cary 1 Bio) equipped with a temperature-controlled cell holder. CS was diluted at a concentration of 150 µM in 40 mM HEPES, pH 7.5 buffer, in the absence or presence of various concentration of Hsp27 or HspB8. Concentrations are calculated for monomeric proteins (CS, 44 kDa; Hsp27, 24 kDa; HspB8, 22 kDa).

Immunofluorescence microscopy
At indicated times following transfection, cells were washed twice with cold phosphate-buffered saline (PBS) (10 mM NaH2PO4, 130 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, pH 7.5), fixed in 3.7% formaldehyde for 15 min at room temperature, washed twice in cold PBS and incubated at 37°C with 5% bovine serum albumin in PBS for 45 min to block non-specific sites. After incubation for 1 h at 37°C with primary antibodies anti-HA (1:150) or anti-HspB8 (1:100), the cells were washed three times with 0.05% Tween-20 PBS and subsequently incubated for 1 h with the appropriate secondary antibodies labeled with either Alexa 488 or Alexa 594 (Molecular Probes). Observations were made with a Nikon Eclipse E600 upright microscope (Tokyo, Japan) equipped with a 60x 0.85 NA objective lens. Images were captured as 16 bit TIFF files with a Micromax 130 YHS cooled (–30°C) CCD camera (Princeton Instruments, Trenton, NJ, USA) driven by Metamorph software version 4.5 (Universal Imaging Corp., Downinton, PA, USA).

Cell extracts and western blot analysis
Extracts prepared by scraping cells in SDS–polyacrylamide gel electrophoresis loading buffer supplemented with 1 mM dithiothreitol were heated for 10 min at 100°C and then centrifuged at 13 000 rpm for 20 min at room temperature. The supernatant was used as the SDS-soluble fraction. To prepare the SDS-insoluble fraction, the pellet was incubated with 100% formic acid for 30 min at 37°C and lyophilized overnight. The lyophilized proteins were finally resuspended in the same volume of SDS–polyacrylamide gel electrophoresis loading buffer as the SDS-soluble fraction. For debris analysis, the growth medium was collected and centrifuged at 13 000 rpm for 20 min at 4°C, the pellet was washed with cold PBS and processed as above to obtain the SDS-soluble and -insoluble fractions. In all cases, SDS-soluble and -insoluble fractions were separated on SDS–polyacrylamide gel electrophoresis and transferred onto Biotrace nitrocellulose membranes (Pall Life Sciences). Proteins were revealed using horseradish-peroxidase conjugated secondary antibodies and visualized with the Supersignal Chemiluminescent substrate kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Chemiluminescence was visualized and digitalized using the Fluor-S Multimager (Biorad Lab, Mississauga).

Filter trap assay
SDS-insoluble proteins were analyzed by a filter trap assay essentially as described previously (29Go,31Go). Briefly, the cells from 9 cm2Go dishes were scraped in 300 µl FTA buffer (10 mM Tris–Cl pH 8.0, 150 mM NaCl and 50 mM dithiothreitol) supplemented with 2% SDS and homogenized by three passages through a 25-gauge needle. Samples were diluted as indicated in the figures (1 volume corresponding to 30 µl of cellular extract), heated at 98 C for 3 min and immediately applied with mild suction onto 0.22 µm AcetatePlus cellulose acetate membrane (Osmonics Inc.) pre-washed with FTA buffer containing 0.1% SDS. The membrane was then washed three times with the same buffer and processed for western blot analyses as described earlier.


   ACKNOWLEDGEMENTS
 
We thank K.H. Fischbeck, R.R. Kopito, H.H. Kampinga, C. Patterson and D.C. Rubinsztein for providing plasmids. This work was supported by the Canadian Institutes of Health Research grant MT-7088 (J.L.), the Canada Research Chair in Stress Signal Transduction (J.L.) and the Universidad Centroccidental Lisandro Alvarado, Venezuela (A.T.C.Z.).

Conflict of Interest statement. None declared.


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