A proteolytic activity has been shown to be associated with the heat shock protein 70 (hsp70). In order to study this, I have constructed RNA transcribing vectors with the coding sequences of the D. melanogaster (pBUG7) and the human (pMAN70) genes coding hsp70, and with an internal deletion (pBUG301) in D. melanogaster. Proteins from 37 kDa to 70 kDa were translated in a rabbit reticulocyte lysate in the presence of 35S-methionine from RNA synthesized in vitro off the full length templates (pBUG7, and pMAN70), or altered templates. Restriction digestion of pBUG7 with BamH I and Nar I yields templates that produce carboxy-terminal truncated proteins of 37 kDa and 61 kDa respectively. The full length and the truncated proteins contain a proteolytic activity when assayed by SDS/PAGE in two dimensions. The internally deleted protein does not maintain the proteolytic activity. The proteolytic activity was shown not to be the result of non-enzymatic cleavage. A general serine proteinase inhibitor eliminates the proteolytic activity of the full length human and D. melanogaster hsp70. This evidence shows that the proteolytic activity is directly connected to hsp70.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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Ho, Sin Chu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 112-135). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgement --- p.iii / Table of Contents --- p.iv / List of Abbreviations --- p.vii / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Test Organisms: Daphnia magna and Moina macrocopa --- p.1 / Chapter 1.2 --- Biomarkers --- p.2 / Chapter 1.3 --- Heat shock protein (HSP) --- p.4 / Chapter 1.3.1 --- Regulation of hsp70 gene expression --- p.5 / Chapter 1.3.2 --- Review of studies on hsp70 of various species --- p.6 / Chapter 1.3.3 --- Review of studies using molecular methods in Daphnia and Moina --- p.7 / Chapter 1.4 --- Environmental stress studied --- p.9 / Chapter 1.5 --- Objectives --- p.11 / Chapter Chapter 2 --- Materials and Methods --- p.12 / Chapter 2.1 --- Experimental organisms --- p.12 / Chapter 2.2 --- "The effects of temperature, metals and nutritional stress on cladocerans" --- p.13 / Chapter 2.3 --- Demographic measurement --- p.15 / Chapter 2.4 --- Cloning and sequencing of D. magna and M. macrocopa hsp70 --- p.17 / Chapter 2.4.1 --- Acute heat shock --- p.17 / Chapter 2.4.2 --- Total RNA extraction --- p.17 / Chapter 2.4.3 --- Reverse transcription and polymerase chain reaction (PCR) --- p.18 / Chapter 2.4.4 --- Degenerative PCR --- p.21 / Chapter 2.4.5 --- Cloning and sequencing of hsp70 partial sequence --- p.25 / Chapter 2.4.6 --- Rapid amplification of cDNA ends (RACE) --- p.28 / Chapter 2.4.7 --- Confirmation of D. magna and M. macrocopa hsp70 sequences --- p.30 / Chapter 2.5 --- Molecular measurements - heat shock protein 70 gene expression --- p.31 / Chapter 2.5.1 --- Sample collection --- p.31 / Chapter 2.5.2 --- Quantification of cDNA level by real-time PCR --- p.32 / Chapter 2.5.2.1 --- Primer design --- p.32 / Chapter 2.5.2.2 --- Validation of real-time PCR conditions --- p.32 / Chapter 2.5.2.3 --- Determination of hsp70 gene expression levels --- p.35 / Chapter 2.6 --- Statistical analysis --- p.36 / Chapter Chapter 3 --- Results --- p.37 / Chapter 3.1 --- Demographic measurements --- p.37 / Chapter 3.1.1 --- Effects of temperature on life history parameters of M macrocopa --- p.37 / Chapter 3.1.2 --- Effects of temperature on life history parameters of D. magna --- p.40 / Chapter 3.1.3 --- Effects of Metals on life history parameters of M. macrocopa --- p.42 / Chapter 3.1.4 --- Effects of Anabaena on life history parameters of M. macrocopa --- p.47 / Chapter 3.2 --- Cloning and sequencing of hsp70 --- p.50 / Chapter 3.2.1 --- Partial sequences of D. magna and M. macrocopa hsp70 --- p.50 / Chapter 3.2.2 --- Sequences of 3´ة and 5´ة RACE amplified products --- p.53 / Chapter 3.2.3 --- Full sequences of D. magna and M. macrocopa hsp70 --- p.53 / Chapter 3.2.4 --- Phylogenetic relationships of the hsp70 gene --- p.60 / Chapter 3.3 --- Molecular measurements - heat shock protein 70 gene expression --- p.71 / Chapter 3.3.1 --- Validation of real-time PCR conditions --- p.71 / Chapter 3.3.2 --- Effect of temperature stress on hsp70 gene expression levels --- p.76 / Chapter 3.3.3 --- Effect of metals on hsp70 gene expression levels --- p.78 / Chapter 3.3.4 --- Effect of Anabaena on Hsp70 Gene Expression Levels --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Demographic responses of M. macrocopa and D. magna by different stress --- p.85 / Chapter 4.1.1 --- Effects of temperature on life history parameters --- p.85 / Chapter 4.1.2 --- Effects of metal stress on life history parameters --- p.87 / Chapter 4.1.3 --- Effects of Anabaena on life history parameters --- p.89 / Chapter 4.2 --- Characterization of hsp70 of M. macrocopa and D. magna --- p.92 / Chapter 4.3 --- Hsp70 gene expression in response to different stress --- p.94 / Chapter 4.3.1 --- Effect of temperature stress on hsp70 gene expression levels --- p.94 / Chapter 4.3.2 --- Effect of metals on Hsp70 gene expression levels --- p.97 / Chapter 4.3.3 --- Effect of Anabaena on hsp70 gene expression levels --- p.101 / Chapter 4.4 --- Comparison of demographic and molecular measurements --- p.102 / Chapter 4.5 --- Performance of hsp70 gene expression as a biomarker of environmental stress --- p.106 / Chapter 4.6 --- Conclusion --- p.111 / References --- p.112 / Appendix --- p.136

Prokaryotic and eukaryotic organisms respond to various stressors with the production of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is constitutively expressed in most adult mammalian tissues. In the present study, we have examined for the first time the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. The Xenopus hsp110 cDNA encodes an 854 amino acid protein, which shares 74% identity with mice and humans. In Xenopus A6 kidney epithelial cells hsp110 mRNA was detected constitutively and was heat inducible. Enhanced hsp110 mRNA levels were detected within 1 h, and remained elevated for at least 6 h. A similar accumulation of hsp70 mRNA was observed, but only in response to stress. Treatment of A6 cells with sodium arsenite and cadmium chloride also induced hsp110 and hsp70 mRNA accumulation. However, while ethanol treatment resulted in the accumulation of hsp70 mRNA no effect was seen for hsp110. Similarly, HSP110 and HSP70 protein increased after a 2 h heat shock and 12 h sodium arsenite treatment. The elevation in HSP110 and HSP70 protein in response to heat was detectable for up to 6 h. Recent studies with mice suggest an important role for HSP110 during development. Analysis of Xenopus embryos revealed that hsp110 mRNA was present in unfertilized eggs, indicating that it is a maternal mRNA, unlike the hsp70 message which was only detectable in response to heat shock. Heat shock-induced hsp110 mRNA accumulation was developmentally regulated, similar to hsp70, since it was not detectable until after the midblastula stage of development. Enhanced hsp110 mRNA accumulation was evident with heat shock at the blastula stage, and levels continued to increase reaching a maximum at the late tailbud stage. Message for the small heat shock protein, hsp27, was not detectable until the early tailbud stage, indicating that this hsp was not present maternally and was developmentally regulated. In situ hybridization analysis revealed that hsp110 mRNA was present in control embryos in the lens placode, spinal cord and somites, but increased upon heat shock in the anterior and posterior region, the lens placode, as well as in the somites and spinal cord. A similar distribution was observed for the hsp27 message, although it was not detectable until the early tailbud stage in control or heat-shocked embryos. The intracellular localization of HSP110 protein in response to stress was also investigated. HSP110 was detected predominantly in the cytoplasm in either a diffuse pattern or in long spindle-shaped fibres. Additionally, HSP110 was present in the nucleus. In heat shocked Xenopus A6 cells, HSP110 localized in distinct patterns surrounding the nucleus and was enhanced in the nucleus after prolonged heat stress. Sodium arsenite-treated cells displayed a similar pattern in which HSP110 localized on opposite ends of the nucleus. In contrast, in response to stress HSP30 was homogeneously distributed in the cytoplasm, moving into the nucleus only upon intense stress. This study presents, for the first time, a characterization of HSP110 in Xenopus laevis, adding to the growing knowledge of HSPs in this important model organism.

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.

Heat shock proteins (HSPs) are stress-inducible and evolutionarily conserved molecular chaperones that are involved in protein binding and translocation. As molecular chaperones, HSPs bind to denatured proteins, inhibit their aggregation, maintain their solubility, and assist in refolding. This process inhibits the formation of protein aggregates which can be lethal to the cell. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most non-native proteins. Furthermore, proteasome inhibition has been shown to induce hsp gene expression. Celastrol, a quinone methide triterpene, was shown to have an inhibitory effect on proteasome function in mammalian cells. The present study determined that celastrol induced the accumulation of ubiquitinated proteins and reduced proteasomal chymotrypsin-like activity in Xenopus laevis A6 kidney epithelial cells. In addition, incubation of A6 cells with celastrol induced the accumulation of HSP30 and HSP70 in a dose- and time-dependent manner with maximal levels of HSP accumulation occurring after 18 h of exposure. In A6 cells recovering from celastrol, the relative levels of HSP30 and HSP70 accumulation remained elevated for 18-24 h after removal of celastrol. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in celastrol-induced hsp gene expression in A6 cells, since the HSF1 inhibitor, KNK437, repressed the accumulation of HSP30 and HSP70. Exposure of A6 cells to simultaneous celastrol and mild heat shock treatment enhanced the accumulation of HSP30 and HSP70 to a greater extent than the sum of both stressors individually. Additionally, concurrent treatment of A6 cells with low concentrations of both celastrol and MG132 produced different patterns of HSP30 and HSP70 accumulation. While combined treatment with celastrol and MG132 acted synergistically on HSP30 accumulation, relative levels of HSP70 were similar to those observed with MG132 alone. Immunocytochemical analysis of celastrol- or MG132-treated A6 cells revealed HSP30 accumulation in a punctate pattern primarily in the cytoplasm with some staining in the nucleus. Also, in some cells treated with celastrol or MG132 large HSP30 staining structures were observed in the cytoplasm. Lastly, exposure of A6 cells to celastrol induced rounder cell morphology, reduced adherence and disorganization of the actin cytoskeleton. In conclusion, this study has shown that celastrol inhibited proteasome activity in amphibian cultured cells and induced HSF1-mediated expression of hsp genes.