Use of fish liver PLHC-1 cells and zebrafish embryos in cytotoxicity assays.

Heat shock proteins (HSPs) indicate exposure to cellular stress and adverse cellular effects, thus serving as biomarkers of these effects. The highly conserved Hsp70 proteins are expressed under proteotoxic conditions, whereas small HSPs are expressed in response to stressors acting on the cytoskeleton and cell signaling pathways. Poeciliopsis lucida hepatocellular carcinoma line 1 (PLHC-1) cells have been used extensively for studying effects of cytotoxicity. A number of assays have been developed to examine DNA levels, protein levels, growth rate, morphological changes, and viability. The boundary between sub-lethal and lethal effects of particular stressors has been determined. The methodology and analytical framework for these techniques along with sample assays using cadmium stressed PLHC-1 cells are described. A range of methodologies have been developed in the past decade that allow the analysis and interpretation of gene expression and function in vivo in zebrafish embryos, and many of these are now being applied to the development of embryotoxicity assays. Here we provide the theoretical background and methodology for utilizing Hsp70 expression as an indicator of toxicity in the zebrafish embryo. Hsp70 expression is activated in a tissue-specific manner in zebrafish larvae following exposure to a number of different toxicants, including cadmium. This has allowed the development of an hsp70/eGFP reporter gene system in stable transgenic zebrafish that serves as a reliable yet extremely quick indicator of cell-specific toxicity in the context of the multicellular, living embryo.

Effects of heat shock, stannous chloride, and gallium nitrate on the rat inflammatory response.

Heat and a variety of other stressors cause mammalian cells and tissues to acquire cytoprotection. This transient state of altered cellular physiology is nonproliferative and antiapoptotic. In this study, male Wistar rats were stress conditioned with either stannous chloride or gallium nitrate, which have immunosuppressive effects in vivo and in vitro, or heat shock, the most intensively studied inducer of cytoprotection. The early stages of inflammation in response to topical suffusion of mesentery tissue with formyl-methionyl-leucyl-phenylalanine (FMLP) were monitored using intravital microscopy. Microvascular hemodynamics (venular diameter, red blood cell velocity [Vrbc], white blood cell [WBC] flux, and leukocyte-endothelial adhesion [LEA]) were used as indicators of inflammation, and tissue levels of inducible Hsp70, determined using immunoblot assays, provided a marker of cytoprotection. None of the experimental treatments blocked decreases in WBC flux during FMLP suffusion, an indicator of increased low-affinity interactions between leukocytes and vascular endothelium known as rolling adhesion. During FMLP suffusion LEA, an indicator of firm attachment between leukocytes and vascular endothelial cells increased in placebo and gallium nitrate-treated animals but not in heat- and stannous chloride-treated animals, an anti-inflammatory effect. Hsp70 was not detected in aortic tissue from placebo and gallium nitrate-treated animals, indicating that Hsp70-dependent cytoprotection was not present. In contrast, Hsp70 was detected in aortic tissues from heat- and stannous chloride-treated animals, indicating that these tissues were in a cytoprotected state that was also an anti-inflammatory state.

Tissue-level cytoprotection.

In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.

A mutational study of the peptide-binding domain of Hsc70 guided by secondary structure prediction.

The abundant, cytoplasmic molecular chaperones of eukaryotic cells, of which mammalian Hsc70 is a member, have central roles in protein folding pathways in cells. Although substantial information is now available on substrate interactions and ATPase activity, neither the crystal structure of the intact Hsc70 molecule nor its isolated peptide-binding domain is known. Recently, the crystal structure of the isolated peptide-binding domain of an evolutionary relative of mammalian Hsc70, the DnaK protein of Escherichia coli, was solved. We have generated several rat Hsc70 mutants using site-directed and cassette mutagenesis guided by secondary structure predictions to test the hypothesis that the peptide-binding domains of mammalian Hsc70 and DnaK have similar molecular structures. Biochemical properties along with the ATPase and peptide binding activities of the resulting recombinant proteins were determined. Biochemical analyses included one- and two-dimensional gel electrophoresis, electrospray mass spectrometry, and N-terminal amino acid sequencing. The results of our study suggest that the DnaK molecular structure is a useful working model for the mammalian Hsc70 peptide-binding domain. Evidence is provided that (i) small additions to the N terminus of Hsc70 alter the function of the peptide-binding domain, (ii) alterations in the C-terminal tetrapeptide EEVD result in dramatic increases in basal ATPase activity, (iii) polyalanine substitution of a helical connector segment compensates for changes at the C terminus to restore near-normal function, (iv) specific side chain interactions involving this connector segment are not required for peptide-stimulated ATPase activity, and (v) disruption of the cap homologue region inhibits peptide binding by Hsc70.

Low-molecular-weight heat shock proteins in a desert fish (Poeciliopsis lucida): homologs of human Hsp27 and Xenopus Hsp30.

The heat shock response of a fish which inhabits a highly stressful environment (Poeciliopsis lucida, a minnow from river systems of the Sonoran desert in northwestern Mexico) was investigated. Cells derived from this fish exhibited a typical heat shock response when exposed to elevated temperature, synthesizing high levels of 90 kDa, 70 kDa, and 30 kDa heat shock proteins (Hsp90, Hsp70, and Hsp30), as well as lower amounts of other heat shock proteins. Additional small heat shock proteins (sHSPs), including Hsp27, were induced after a prolonged heat shock at a time when synthesis of Hsp70 and Hsp30 was decreasing. Characterization of cDNA clones for hsp27 and hsp30 revealed that both are members of the alpha-crystallin/sHSP superfamily but belong to separate lineages within this gene family. The multiple isoforms of P. lucida Hsp30 appear to be members of a multigene family and are most closely related to salmon and Xenopus Hsp30s. In contrast, Hsp27 is highly similar to mammalian and avian sHSPs; it was synthesized as three isoforms which represented differentially phosphorylated forms of a single polypeptide. In Poeciliopsis, the various sHSPs may each perform a subset of the roles attributed to mammalian sHSPs. The conservation of phosphorylation sites in Hsp27 may indicate an involvement in signal transduction to the actin cytoskeleton. The hsp30 genes appear to have diverged more rapidly than the corresponding hsp27 genes; the various members of the Hsp30 family may function as molecular chaperones and, in this role, may be less evolutionarily constrained. Finally, the presence of these two classes of sHSP in a single taxon indicates that these two lineages arose by gene duplication early in the evolution of vertebrates and raises questions about the fate of homologs of Hsp30 in mammals and of Hsp27 in Xenopus.

A 16-kDa protein functions as a new regulatory protein for Hsc70 molecular chaperone and is identified as a member of the Nm23/nucleoside diphosphate kinase family.

Cytoplasmic Hsc70 is a multifunctional molecular chaperone. It is hypothesized that accessory proteins are used to specify the diverse chaperone activities of Hsc70. A 16-kDa cytosolic protein (p16) co-purified with Hsc70 obtained from a fish hepatocyte cell line, PLHC-1. Hsc70 also co-immunoprecipitated with p16 from PLHC-1 cells and fish liver. p16 was identified as a member of the Nm23/nucleoside diphosphate (NDP) kinase family based on its amino acid sequence similarity, NDP kinase activity, and recognition by anti-human NDP kinase-A antibody. This antibody also co-immunoprecipitated Hsc70 and NDP kinase from human HepG2 cells. p16 monomerized Hsc70 and released Hsc70 from pigeon cytochrome c peptide (Pc) but not from FYQLALT, a peptide specifically designed for high affinity binding. Therefore, p16 may modulate Hsc70 function by maintaining Hsc70 in a monomeric state and by dissociating unfolded proteins from Hsc70 either through protein-protein interactions or by supplying ATP indirectly through phosphate transfer. p16 did not affect basal or unfolded protein-stimulated ATPase activity of bovine brain Hsc70 using in vitro assays. Interestingly, bovine liver NDP kinase did not dissociate the Hsc70.Pc complex. In addition, two nonconservative amino acid subsitutions were found near the amino terminus of p16. Therefore, p16 may be a unique Nm23/NDP kinase that functions as an accessory protein for cytosolic Hsc70 in eukaryotes.

Quantitative evidence that both Hsc70 and Hsp70 contribute to thermal adaptation in hybrids of the livebearing fishes Poeciliopsis.

The 70-kilodalton heat shock protein family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. While the role of the constitutively expressed stress proteins in thermotolerance is largely unknown, de novo expression of stress proteins in response to elevated temperatures has been associated with increased thermotolerance in many cell lines, developing embryos and adult organisms. Distinct, hemiclonal hybrids between the livebearing fish species Poeciliopsis monacha and P. lucida varied in their abilities to survive temperature stress, with survival being greatest when rates of temperature increase to 40 degrees C were slowest and when P. monacha genomes were combined with a sympatric P. lucida genome. Quantification of Hsp70 under heat shock conditions and Hsc70 under normal physiological conditions indicated that variation in survival among hemiclones was best explained by the combined effects of these two proteins. Similar complex interactions between maternal and paternal genomes and rate of temperature increase were found to underlie patterns of survival, Hsp70 accumulation and Hsc70 abundance. These data suggest that the relationship between Hsps and thermotolerance is more intricate than previously thought and that Hsps contribute to thermal adaptation in these fishes through genetic interactions specific to particular environments.

Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer.

Differential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70). Hsc70 had endothermic transitions with midpoints (Tm) at 59 degrees C and 63 degrees C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan. Combined with increased exposure at 60 degrees C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70. An exothermic transition (Tm = 66 degrees C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes. An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c. A novel finding was an exothermic transition of Hsc70 beginning at about 30 degrees C (Tm = 41 degrees C). No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range. Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation. However, Hsc70 was protease-resistant at 20 degrees C, sensitive at 40 degrees C and resistant when returned to 20 degrees C, indicating that this exotherm is associated with a reversible conformational change. As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c, a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK. For all of these substrates, the amount of complex formed increased with increasing temperature over the same range as the 41 degrees C exotherm. It is proposed that a conformational change exposes polar and charged residues in Hsc70 which subsequently become hydrated, resulting in an active chaperone. Hsc70 may be a thermal sensor that matches the supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells. Thermal activation of Hsc70 may also have a role in acquired thermotolerance.

Stress proteins as molecular biomarkers for environmental toxicology.

Biomarkers are increasingly being used in environmental monitoring to provide evidence that organisms have been exposed to, or affected by, xenobiotic chemicals. Usually, these biomarkers rely on biochemical, histological, morphological, and physiological changes in whole organisms; however, changes at the cellular and molecular levels of organization, especially in nucleic acids and proteins, are increasingly being used to supplement these more traditional biomarkers. This chapter starts by giving a brief overview of biomarkers and some of the basic requirements for their effective use. Then stress-inducible proteins that are potentially useful as environmental biomarkers are explored, and some examples of their application as biomarkers and methods of detecting them are presented.

Variation in heat shock proteins within tropical and desert species of poeciliid fishes.

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30-kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.

Effects of dexamethasone, heat shock, and serum responses on the inhibition of Hsc70 synthesis by antisense RNA in NIH 3T3 cells.

A dexamethasone (Dex)-inducible antisense RNA expression vector was constructed that contains the 5'-untranslated region and one third of the coding sequence for the bovine hsc70 protein. This vector was used to transfect NIH 3T3 cells from which clonal cell lines expressing hsc70 antisense RNA were developed. Quantitative Northern blot analysis with strand-specific probes was used to demonstrate the Dex-inducible accumulation of hsc70 antisense RNA in proliferating cell cultures and the inhibition of hsc70 RNA levels. Surprisingly, antisense RNA was either much less effective in reducing the amounts of hsc70 RNA in Dex-treated cultures than in untreated controls or cells compensated by producing more hsc70 RNA in response to increasing amounts of antisense RNA. Hsc70 protein synthesis did not decrease in either Dex-treated or untreated cultures: it actually increased, again suggesting the activation of a compensatory response. In Dex-treated cultures subjected to heat shock, hsc70 antisense RNA blocked the induction of hsp70, indicating that newly synthesized RNA was targeted effectively before it became translationally active. To test this hypothesis further, Dex-treated cultures were made quiescent by serum deprivation and then restimulated with serum, which causes a burst of RNA and protein synthesis. Consistent with this hypothesis, increased synthesis of hsc70 was blocked in serum-stimulated cultures expressing antisense RNA.

Hsc70-binding peptides selected from a phage display peptide library that resemble organellar targeting sequences.

A 15-mer phage display random peptide library was screened with purified bovine Hsc70, and nucleotide sequence analysis of the selected clones showed a large enrichment for peptides containing basic sequences with at least KK, KR, or RR. Binding affinity for Hsc70 of representative peptides increased dramatically for heptamers compared with hexamers. The peptide NIVRKKK had the highest affinity for Hsc70, and substitution analyses showed that hydrophobic residues followed by basic residues play important roles in maintaining this affinity. In contrast, NIVRKKK was a weaker stimulator of the Hsc70 ATPase activity compared with pigeon cytochrome c peptide and FYQLALT, a peptide optimized for binding to Hsc70. FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, possibly by causing a conformational change that masked Hsc70's binding site for the basic peptide. Two hypotheses are offered to explain the two different peptide motifs. First, it is proposed that Hsc70 recognizes two different amino acid sequence motifs in its dual roles of chaperoning proteins to organelles (NIVRKKK-like sequences) and facilitating protein folding (FYQLALT-like sequences). Second, the NIVRKKK motif may be used to bind certain folded proteins with which Hsc70 interacts, such as itself, p53, and Dnaj2.