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The hsp30 small heat shock protein family is a stress-inducible group of molecular chaperones in the frog, Xenopus laevis. Hsp30 genes are intronless and present in clusters. Expression of these genes are developmentally regulated likely at the level of chromatin structure. Also heat-induced hsp30 transcripts and protein are enriched in selected embryonic tissues. In vitro studies revealed that multimeric hsp30 binds to heat denatured target protein, inhibits their aggregation and maintains them in a folding-competent state until reactivated by other cellular chaperones. Finally optimal chaperone activity and secondary structure of hsp30 can be inhibited by phosphorylation or mutagenesis of the C-terminal end.
Fig. 1. Pattern of hsp30 mRNA accumulation during early Xenopus development. Whole mount in situ hybridization with DIG-labeled hsp30 antisense
riboprobe was carried out with control (A) and heat shocked (B) (1 h at 33 â—¦C) Xenopus midtailbud stage embryos. Preferential heat-induced accumulation
of hsp30 mRNA was observed in lens placode (LP), somites (S), proctodeum (P) and cement gland (CG). Constitutive hsp30 mRNA accumulation was
also observed in cement gland. (C) Treatment of Xenopus embryos with HDIs resulted in premature heat-induced accumulation of hsp30 mRNA at the
gastrula stage rather than at the late neurula–early tailbud stages as found in non-HDI-treated embryos. HDI treatment enhanced heat-induced hsp30
mRNA accumulation in neurula and tailbud embryos. These experiments suggest that hsp30 gene expression during development is regulated at the level
of chromatin structure. Xenopus embryos were incubated with (+) or without (−) 30 nM trichostatin A (TSA), 5mM valproic acid (VPA), or 5mM
sodium butyrate (NaB). Total RNA was isolated from control (C; 22 â—¦C) and heat shocked (H; 1 h at 33 â—¦C) embryos and subjected to Northern blot
analysis using an hsp30 antisense riboprobe. Arrows indicate the positions of hsp30 mRNA.
Fig. 2. Molecular chaperone activity of Xenopus hsp30. (A) The presence of hsp30D inhibited heat-induced aggregation of citrate synthase (CS). CS at
a 150nM monomer concentration was mixed with various molar amounts of recombinant hsp30D protein or incubated alone in a 50mM HEPES–KOH
buffer, pH 7.5 and heated at 42 â—¦C. Light scattering was determined at 10 min intervals in a Beckman DU7 spectrophotometer at 320 nm. An increase in
absorbance was indicative of protein aggregation. Data were calculated as percentage of the maximum aggregation of CS after 60 min and were expressed
as the mean ± S.E. CS was heat-treated alone ( , 0.1 M) or in the presence of either hsp30D ( , 0.1 M; , 0.5 M) or IgG (×, 0.5 M). (B)
Luciferase (LUC) heat denatured in the presence of hsp30C was refolded in vivo after microinjection into Xenopus oocytes. However, LUC heat-treated
alone or with BSA did not regain enzyme activity after injection. In these experiments LUC (0.2 M) was incubated at 22 â—¦C ( ) or heat denatured
alone at 42 ◦C (×) or in the presence of either 6 M BSA ( ) or hsp30C at hsp30C:LUC molar ratios of 1:1 ( ), 10:1 ( ) or 30:1 ( ) for 15 min.
Mixtures (containing 1.38 fmol of LUC in 26.7 nl) were microinjected into oocytes and LUC activity in the oocytes was monitored over time. Data are
representative of three to five trials and shown as the mean ± S.E.
hsp30e (heat shock protein 30E) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
