The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates.

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity.

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more ß-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.

Saccharomyces cerevisiae Hsp30 is necessary for homeostasis of a set of thermal stress response functions.

Saccharomyces cerevisiae Hsp30 is a plasma membrane heat shock protein which is induced by various environmental stress conditions. However functional role of Hsp30 during diverse environmental stressors is not presently known. To gain insight into its function during thermal stress, we have constructed and characterized a hsp30 strain during heat stress. BY4741Deltahsp30 cells were found to be more sensitive compared to BY4741 cells when exposed to a lethal heat stress at 50 degrees Celsius. When budding yeast is exposed to either heat shock or weak organic acid, it inhibits Pma1p activity. In this study we measured the levels of Pma1p in mutant and Wt cells both during optimal temperature and heat shock temperature. We observed that BY4741Deltahsp30 cells showed constitutive reduction of Pma1p. To gain further insights into the role of Hsp30 during heat stress, we compared total protein profile by 2D gel electrophoresis followed by identification of differentially expressed spots by LC-MS. We observed that contrary to that expected from thermal stress induced changes in gene expression, the Deltahsp30mutant maintained elevated levels of Pdc1p, Trx1p and Nbp35p and reduced levels of Atp2p and Sod1p during heat shock. In conclusion, Hsp30 is necessary during lethal heat stress, for the maintenance of Pma1p and a set of thermal stress response functions.

Identification of 30-kDa heat shock protein gene in Trichophyton rubrum.

Small heat shock proteins (sHSPs) are chaperones that are crucial in the heat shock response but also have important non-stress roles within the cell. HSP70 in Trichophyton rubrum is already detected and carefully characterised; however, no study was carried out for HSP30 in this pathogenic fungus. In the present study, T. rubrum was obtained from patients with dermatophytosis and cultured in appropriate conditions. High-molecular-weight DNA was extracted using standard extraction methods. Pairs of 21 nt primers were designed from highly conserved regions of the similar genes in other eukaryotic cells. Mentioned primers were utilised in PCR using isolated genomic DNA and extracted RNA templates of T. rubrum. The PCR fragments were then sequenced and 415 nucleotides of HSP30 in this pathogenic fungus were detected; the open reading frame had 156 nucleotides and was coding 51 amino acids. This gene (called TrHSP30) is registered in GenBank at National Center for Biotechnology Information (NIH, USA) database. Detection of TrHSP30 gene may open the way to determination of its possible role in the pathogenesis of dermatophyte infections due to T. rubrum.

Differential subcellular localization of members of the Toxoplasma gondii small heat shock protein family.

The results of this study describe the identification and characterization of the Toxoplasma gondii alpha-crystallin/small heat shock protein (sHsp) family. By database (www.toxodb.org) search, five parasite sHsps (Hsp20, Hsp21, Hsp28, Hsp29, and the previously characterized Hsp30/Bag1) were identified. As expected, they share the homologous alpha-crystallin domain, which is the key characteristic of sHsps. However, the N-terminal segment of each protein contains unique characteristics in size and sequence. Most T. gondii sHsps are constitutively expressed in tachyzoites and fully differentiated bradyzoites, with the exception of Hsp30/Bag1. Interestingly, by subcellular localization we observed that T. gondii sHsps are located in different compartments. Hsp20 is located at the apical end of the cell, Hsp28 is located inside the mitochondrion, Hsp29 showed a membrane-associated labeling, and Hsp21 appeared throughout the cytosol of the parasites. These particular differences in the immunostaining patterns suggest that their targets and functions might be different.