Exposure of cells to environmental or chemical stressors will initiate the heat shock response, which is mediated by heat shock proteins. Heat shock proteins are molecular chaperones which are classified by size into six main families: HSP100, HSP90, HSP70, HSP60, HSP40 and the small heat shock proteins (sHsps). The sHsp family members bind to denatured proteins and maintain them in a folding competent state such that they may be refolded by other molecular chaperones. <br /><br /> The present study examined the expression and function of two amphibian sHsps, namely, <em>Rana catesbeiana</em> HSP30 and <em>Xenopus laevis</em> HSP27. Initially, an antisense riboprobe was produced to study the mRNA accumulation of <em>Rana hsp30</em> in cultured tongue fibroblast (FT) cells. Results showed that <em>Rana hsp30</em> mRNA was optimally induced when maintained at 35&deg;C for 2 h. An antibody to the recombinant <em>Rana</em> HSP30 protein was also produced in order to study HSP30 protein accumulation in <em>Rana</em> FT cells. Analysis showed that <em>Rana</em> HSP30 was heat-inducible and accumulated maximally at 4 h when maintained at 35&deg;C and then allowed to recover at 22&deg;C for 2 h. Immunocytochemical analysis indicated that <em>Rana</em> HSP30 protein was present primarily in the nucleus, with diffuse localization in the cytoplasm. Additional immunocytochemical analysis showed that <em>Rana</em> HSP30 remained in the nucleus following heat stress and extended periods of recovery. <br /><br /> The molecular chaperone function of <em>Rana</em> HSP30 was also studied. Recombinant <em>Rana</em> HSP30 was found to inhibit the heat induced aggregation of various target proteins including citrate synthase, luciferase and malate dehydrogenase. Also, no major difference was detected between <em>Rana</em> HSP30 and <em>Xenopus</em> HSP30C in the inhibition of heat-induced aggregation of target proteins. <br /><br /> This study also examined the expression and function of <em>Xenopus laevis</em> HSP27. Analysis of the putative amino acid sequence of the <em>Xenopus hsp27</em> cDNA revealed that it had an identity of 71% with chicken, 65% with zebrafish, 63% with human and 53% with topminnow. Most of the identity was located within the &alpha;-crystallin domain of the protein. Interestingly, <em>Xenopus</em> HSP27 shared only a 19% identity with 2 other <em>Xenopus</em> sHsps, HSP30C and HSP30D. <br /><br /> Western blot analysis using an anti-<em>Xenopus</em> HSP27 antibody revealed that HSP27 was not detectable in cultured kidney epithelial cells. However, examination of early <em>Xenopus</em> embryos revealed that HSP27 was first detected in tadpole embryos (stage 44). Heat-inducible HSP27 was also first detected at this stage. The accumulation pattern of <em>Xenopus</em> HSP27 protein was distinct from <em>Xenopus</em> HSP30, which was heat-inducible at midtailbud stage 26, approximately two and a half days earlier in development. <br /><br /> Analysis of recombinant HSP27 by native pore exclusion limit electrophoresis showed that it formed high molecular weight, multimeric complexes. The molecular chaperone function of HSP27 was assessed by means of thermal aggregation assays employing citrate synthase, luciferase and malate dehydrogenase. <em>Xenopus</em> HSP27 inhibited the heat-induced aggregation of all of these target proteins. This study has revealed that <em>Xenopus</em> HSP27 is a member of the HSP27 subfamily of small heat shock proteins in <em>Xenopus</em> and distinct from the HSP30 family. The accumulation of HSP27 under constitutive and stress-inducible conditions is developmentally regulated. Finally, this sHsp appears to function as a molecular chaperone.

Exposure of cells to environmental or chemical stressors will initiate the heat shock response, which is mediated by heat shock proteins. Heat shock proteins are molecular chaperones which are classified by size into six main families: HSP100, HSP90, HSP70, HSP60, HSP40 and the small heat shock proteins (sHsps). The sHsp family members bind to denatured proteins and maintain them in a folding competent state such that they may be refolded by other molecular chaperones. <br /><br /> The present study examined the expression and function of two amphibian sHsps, namely, <em>Rana catesbeiana</em> HSP30 and <em>Xenopus laevis</em> HSP27. Initially, an antisense riboprobe was produced to study the mRNA accumulation of <em>Rana hsp30</em> in cultured tongue fibroblast (FT) cells. Results showed that <em>Rana hsp30</em> mRNA was optimally induced when maintained at 35&deg;C for 2 h. An antibody to the recombinant <em>Rana</em> HSP30 protein was also produced in order to study HSP30 protein accumulation in <em>Rana</em> FT cells. Analysis showed that <em>Rana</em> HSP30 was heat-inducible and accumulated maximally at 4 h when maintained at 35&deg;C and then allowed to recover at 22&deg;C for 2 h. Immunocytochemical analysis indicated that <em>Rana</em> HSP30 protein was present primarily in the nucleus, with diffuse localization in the cytoplasm. Additional immunocytochemical analysis showed that <em>Rana</em> HSP30 remained in the nucleus following heat stress and extended periods of recovery. <br /><br /> The molecular chaperone function of <em>Rana</em> HSP30 was also studied. Recombinant <em>Rana</em> HSP30 was found to inhibit the heat induced aggregation of various target proteins including citrate synthase, luciferase and malate dehydrogenase. Also, no major difference was detected between <em>Rana</em> HSP30 and <em>Xenopus</em> HSP30C in the inhibition of heat-induced aggregation of target proteins. <br /><br /> This study also examined the expression and function of <em>Xenopus laevis</em> HSP27. Analysis of the putative amino acid sequence of the <em>Xenopus hsp27</em> cDNA revealed that it had an identity of 71% with chicken, 65% with zebrafish, 63% with human and 53% with topminnow. Most of the identity was located within the &alpha;-crystallin domain of the protein. Interestingly, <em>Xenopus</em> HSP27 shared only a 19% identity with 2 other <em>Xenopus</em> sHsps, HSP30C and HSP30D. <br /><br /> Western blot analysis using an anti-<em>Xenopus</em> HSP27 antibody revealed that HSP27 was not detectable in cultured kidney epithelial cells. However, examination of early <em>Xenopus</em> embryos revealed that HSP27 was first detected in tadpole embryos (stage 44). Heat-inducible HSP27 was also first detected at this stage. The accumulation pattern of <em>Xenopus</em> HSP27 protein was distinct from <em>Xenopus</em> HSP30, which was heat-inducible at midtailbud stage 26, approximately two and a half days earlier in development. <br /><br /> Analysis of recombinant HSP27 by native pore exclusion limit electrophoresis showed that it formed high molecular weight, multimeric complexes. The molecular chaperone function of HSP27 was assessed by means of thermal aggregation assays employing citrate synthase, luciferase and malate dehydrogenase. <em>Xenopus</em> HSP27 inhibited the heat-induced aggregation of all of these target proteins. This study has revealed that <em>Xenopus</em> HSP27 is a member of the HSP27 subfamily of small heat shock proteins in <em>Xenopus</em> and distinct from the HSP30 family. The accumulation of HSP27 under constitutive and stress-inducible conditions is developmentally regulated. Finally, this sHsp appears to function as a molecular chaperone.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. The present study examined the pattern of accumulation of HSP30 and HSP70 in cells recovering from heat shock as well as the effect of proteasome inhibition on cytoplasmic/nuclear and endoplasmic reticulum (ER) molecular chaperone accumulation, large multimeric HSP30 complexes, stress granule and aggresome formation in Xenopus laevis A6 kidney epithelial cells. Initial immunoblot analysis revealed the presence of elevated levels of HSP30 after 72 h of recovery. However, the relative levels of HSP70 declined to near control levels after 24 h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24 h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. To verify if proteasome inhibition in A6 cells induced the formation of similar HSP30 granules, immunoblot and immunocytochemical analyses were performed. MG132, celastrol and withaferin A enhanced ubiquitinated proteins, inhibited chymotrypsin-like activity of the proteasome and induced the accumulation of cytoplasmic/nuclear HSPs, HSP30 and HSP70 as well as ER chaperones, BiP and GRP94 and heme oxygenase-1. Northern blot experiments determined that proteasome inhibitors induced an accumulation in hsp30, hsp70 and bip mRNA but not eIF1α. The final part of this study demonstrated that treatment of A6 cells with proteasome inhibitors or sodium arsenite or cadmium chloride induced HSP30 multimeric complex formation primarily in the cytoplasm. Moreover, these stressors also induced the formation of RNA stress granules, pre-stalled translational complexes, which were detected via TIA1 and polyA binding protein (PABP), which are known stress granule markers. These stress granules, however, did not co-localize with large HSP30 multimeric complexes. In comparison, proteasome inhibition or treatment with sodium arsenite or cadmium chloride also induced the formation of aggresome-like structures, which are proteinaceous inclusion bodies formed as a result of an abundance of aggregated protein. Aggresome formation was identified by monitoring the presence of vimentin and γ-tubulin, both of which are cytoskeletal proteins and serve as markers of aggresome detection. Aggresome formation, which was also verified using the ProteoStat assay, co-localized with large HSP30 multimeric complexes. Co-immunoprecipitation experiments revealed that HSP30 associated with γ-tubulin and β-actin in cells treated with proteasome inhibitors or sodium arsenite or cadmium chloride suggesting a possible role in aggresome formation. In conclusion, this study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation. Finally, proteasome inhibition, sodium arsenite and cadmium chloride treatment of A6 cells induced cytoplasmic/nuclear and ER chaperones as well as resulting in the formation stress granules and aggresome-like structures which associated with large HSP30 multimeric complexes.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.