Wound-induced PAL activity is suppressed by heat-shock treatments that induce the synthesis of heat-shock proteins

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45°C reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R² > 0.90) in tissue developing the normal wound response and in tissue subjected to 0–180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45°C for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.     

Characterization of a new high-temperature-induced 66-kDa heat-shock protein,antigenically related to heat-shock protein 72

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major “classic” heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45°C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)⁺ RNA extracted from cells treated at 45°C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)⁺ RNA extracted from cells heated at 42°C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro, hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer. © 1996 Wiley-Liss, Inc.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Natural thermal stress and heat-shock protein expression in Drosophila larvae and pupae

1. Whether Drosophila larvae and pupae naturally experience temperatures that can cause heat damage or death is poorly understood, but bears directly on numerous investigations of the thermal biology and heat-shock response in Drosophila . Accordingly, the temperatures of necrotic fruit, which Drosophila larvae and pupae inhabit, the temperatures of larvae and pupae outside the laboratory, and the levels of the heat-shock protein hsp 70 expressed by larvae in nature were examined.
2. When necrotic fruit was sunlit, internal temperatures rose to levels that can harm indwelling insects. Fruit size and evaporative water loss affected these temperatures. Temperatures of larvae and pupae in the field commonly exceeded 35 °C, with living larvae recorded at >44°C and pupae at >41°C. Natural mortality was evident, presumably because of heat.
3. In the laboratory, these temperatures kill larvae rapidly, with LT₅₀s (time taken for half the sample to be killed) of 30 min at 39 °C, 15 min at 40 °C and 8·5 min at 41 °C. Gradual transfer from 25°C to these temperatures resulted in no lesser mortality than did direct transfer.
4. Hsp 70 levels in lysates of whole larvae were measured by ELISA (enzyme-link immunosorbent assay) with an hsp 70-specific antibody. For larvae within necrotic apples experimentally transferred from shade to sun and within necrotic fruit in situ , hsp 70 levels equalled or exceeded levels detected in parallel laboratory studies of whole larvae or cells in culture.
5. These data provide an ecological context for studies of thermal stress and the heat-shock response in Drosophila that has heretofore been lacking.     

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-beta-galactosidase hybrid gene in cultured Drosophila cells

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli beta-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific beta-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.     

The arrest of embryogenesis at gastrula stage in the diapausing silkwormBombyx mori is related to the synthesis of protein P61

Summary Two-dimensional protein gels are used to assess systematically changes in protein synthesis in diapausing and non-diapausing early embryos ofBombyx mori throughout natural breakage of diapause by chilling and after artificial prevention of diapause by HCl+ heat-shock treatment. A set of proteins, the heat-shock protein (hsp) 70 family previously described, was synthesized in diapausing and non-diapausing development at the early germ-anlage stage; by contrast, protein 61 (P61; 61 kDa) was synthesized only in the diapausing gastrula stage. The synthesis of P61 decreased during days at 5°C. Thereafter, we observed the synthesis of the hsp 70 family at 5°C. After the artificial prevention of diapause, P61 synthesis was not induced at the gastrula stage, whereas the hsp 70 family was enhanced in the 6 h following the preventive treatment. P61 was only synthesized after treatments were ineffective in preventing diapause and in inducing hsp 70. Thus, P61 can be described as a diapause arrest-associated protein.     

Cellular localization of HSP23 during Drosophila development and following subsequent heat shock

The low-molecular-weight heat-shock protein HSP23 is synthesized in the absence of heat shock during Drosophila development. Here, I present a quantitative analysis of this phenomenon and describe the cellular localization of this protein during normal development and after a subsequent heat shock. HSP23 is first detected in the late third instar larvae and continues to accumulate reaching a maximum level in late pupae. In a 1-week-old adult, HSP23 can no longer be detected. Following lysis of whole pupae, HSP23 is found in the soluble lysate fraction in a form which sediments between 10 and 20 S. Exposure of larvae, pupae, and the adult fly to heat stress (37 degrees C) results in an increased amount of HSP23 which, however, is recovered in an insoluble particulate form following insect lysis. During recovery from heat shock, HSP23 is again found in the soluble 10- to 20-S lysate fraction. In pupae which are exposed to a severe heat stress (41 degrees C) HSP23 remains in the pellet fraction after the heat stress and no pupae are able to emerge as adult flies. However, when pupae are first exposed to a mild heat-shock treatment prior to the 41 degrees C stress, the thermotolerance process is induced and HSP23 is again rapidly found in the soluble lysate fraction during the recovery from heat shock. These observations suggest a possible correlation between the survival of pupae after heat shock and the recovery of HSP23 in the soluble lysate fraction as 10- to 20-S structures after the heat shock.     

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

Regulation of the synthesis of heat-shock proteins in heat-resistant variants of Chinese hamster fibroblasts

The synthesis of the major heat-shock proteins (hsp) was compared in normal and heat-resistant Chinese hamster fibroblasts which express higher levels of the 70 kDa heat-shock protein (hsp70). Following exposure to a variety of experimental conditions that induce the elevated synthesis of the hsp, higher relative levels of hsp70 and lower relative levels of hsp89 and hsp110 were found in the heat-resistant variants. This effect was observed with all inducers tested. The relatively greater synthesis of hsp70 and relatively lower synthesis of hsp89 occurred at all temperatures tested and was found to be independent of cell culture conditions. The relatively greater increase in the levels of hsp70 in the heat-resistant variants after a mild heat shock was found to be a reflection of elevated levels of messenger RNA coding for this polypeptide. These results indicate that the heat-shock response in mammalian cells displays coordinate regulatory features and that the alteration of the expression of one of the hsp may affect the expression of the others.     

Examining the function and regulation of hsp 70 in cells subjected to metabolic stress

Members of the heat-shock protein (hsp) 70 family, distributed within various cellular compartments, have been implicated in facilitating protein maturation events. In particular, related hsp 70 family members appear to bind nascent polypeptides which are in the course of synthesis and/or translocation into organelles. We previously reported that in normal, unstressed cells, cytosolic hsp 70 (hsp 72/73) interacted transiently with nascent polypeptides. We suspect that such interactions function to prevent or slow down the folding of the nascent polypeptide chain. Once synthesis is complete, and now with all of the information for folding present, the newly synthesized protein appears to commence along its folding pathway, accompanied by the ATP-dependent release of hsp 72/73. Herein, we examined how these events occur in cells subjected to different types of metabolic stress. In cells exposed to either an amino acid analog or sodium arsenite, two potent inducers of the stress response, newly synthesized proteins bind to but are not released from hsp 70. Under these conditions of metabolic stress, we suspect that the newly synthesized proteins are unable to commence proper folding and consequently remain bound to their hsp 70 chaperone. In cells subjected to heat shock, a large number of both newly synthesized as well as mature proteins are rendered insoluble. Within this insoluble material are appreciable amounts of hsp 72/73. Finally, we show that in cells depleted of ATP, the release of hsp 70 from maturing proteins is inhibited. Thus, in cells experiencing metabolic stress, newly synthesized proteins unable to properly fold, as will as mature proteins which begin to unfold become stably bound to hsp 72/73. As a consequence and over time, the free or available levels of pre-existing hsp 72/73 are reduced. We propose that this reduction in the available levels of hsp 72/73 is the trigger by which the stress response is initiated.     

Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.