Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

The role of molecular chaperones in human misfolding diseases

Human misfolding diseases arise when proteins adopt non-native conformations that endow them with a tendency to aggregate and form intra- and/or extra-cellular deposits. Molecular chaperones, such as Hsp70 and TCP-1 Ring Complex (TRiC)/chaperonin containing TCP-1 (CCT), have been implicated as potent modulators of misfolding disease. These chaperones suppress toxicity of disease proteins and modify early events in the aggregation process in a cooperative and sequential manner reminiscent of their functions in de novo protein folding. Further understanding of the role of Hsp70, TRiC, and other chaperones in misfolding disease is likely to provide important insight into basic pathomechanistic principles that could potentially be exploited for therapeutic purposes.     

Distinct chemical contaminants induce the synthesis of Hsp70 proteins in green microalgae Desmodesmus subspicatus: Heat pretreatment increases cadmium resistance

Using western-blotting techniques, we examined the effect of differently acting contaminants, such as anthracene (PAH), cadmium (heavy metal) and chloridazone (herbicide), as well as heat shock on the production of two Hsp70 proteins (cytoplasmic and stromal) in planktonic algae Desmodesmus subspicatus. All contaminants applied stimulated production of both Hsp70s in a concentration-dependent manner, but heat shock treatment turned out to be the most effective. Heat shock pretreatment (for 1 h at 40 °C) induced tolerance to cadmium in algal cells (measured by changes in growth rate), but not to anthracene or chloridazone. Two Hsp70s from D. subspicatus cells representing cytoplasmic and stromal proteins were purified by ATP-affinity chromatography.     

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.     

Expression analysis of heat shock protein genes during Aeromonas hydrophila infection in rohu,Labeo rohita,with special reference to molecular characterization of Grp78

Heat shock protein (Hsp) genes are stress-related genes that activate the host immune system during infection. Hsp genes expression in fish, studied during bacterial infections, is mostly confined to Hsp70 and Hsp90. The present study is an expression analysis of seven Hsp genes: Apg2, Hsp90, Hsp70, glucose-regulated protein 78 (Grp78), heat shock cognate 70 (Hsc70), Grp75, and Hsp30 during Aeromonas hydrophila infection in rohu, Labeo rohita. Forty-eight rohu juveniles were challenged with 3 × 10⁷ cfu bacteria per 20 g of body weight intraperitoneally. The expression of these genes was studied in infected liver, anterior kidney, and spleen tissues of rohu at different time periods: 0, 1, 3, 6, 12, 24, 48, 72 h, 7, and 15 days post-infection by qPCR. The Hsp gene modulation was greater in liver as compared to spleen and kidney tissues. Induced expression of Apg2, Hsp90, Grp78, Grp75, and Hsc70 was noticed during peak periods (3 to 24 h post-challenge) of bacterial infectivity. Hsp70 was found to be down-regulated during the process of infection. In contrast to the other six genes, Hsp30 showed a variable expression pattern in all three tissues. Grp78 was found to be the most highly (1,587-fold) expressed gene in liver at 12 h post-challenge. Further, molecular characterization of Grp78 revealed it to be a highly conserved Hsp gene, essential not only during infection but also during early developmental stages of rohu, and its expression was noticed in all organs studied except in gill tissues, which indicated its potential immune regulatory role during infection process.     

The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s

Both the Grp170 and Hsp110 families represent relatively conserved and distinct sets of stress proteins, within a more diverse category that also includes the Hsp70s. All of these families are found in a wide variety of organisms from yeasts to humans. Although Hsp110s or Grp170s are not Hsp70s any more than Hsp70s are Hsp110s or Grp170s, it is still reasonable to refer to this combination of related families as the Hsp70 superfamily based on arguments discussed above and since no obvious prokaryotic Hsp110 or Grp170 has yet been identified. These proteins are related to their counterparts in the Hsp70/Grp78 family of eukaryotic stress proteins but are characterized by significantly larger molecular weights. The members of the Grp170 family are characterized by C-terminal ER retention sequences and are ER localized in yeasts and mammals. As a Grp, Grp170 is recognized to be coregulated with other major Grps by a well-known set of stress conditions, sometimes referred to as the unfolded protein response (Kozutsumi et al 1988; Nakaki et al 1989). The Hsp110 family members are localized in the nucleus and cytoplasm and, with other major Hsps, are also coregulated by a specific set of stress conditions, most notably including hyperthermic exposures. Hsp110 is sometimes called Hsp105, although it would be preferable to have a uniform term. The large Hsp70-like proteins are structurally similar to the Hsp70s but differ from them in important ways. In both the Grp170 and Hspl10 families, there is a long loop structure that is interposed between the peptide-binding ,-domain and the alpha-helical lid. In the Hsp110 family and Grp170, there are differing degrees of expansion in the alpha-helical domain and the addition of a C-terminal loop. This gives the appearance of much larger lid domains for Hsp110 and Grp170 compared with Hsp70. Both Hsp110 and Grp170 families have relatively conserved short sequences in the alpha-helical domain in the lid, which are conserved motifs in numerous proteins (we termed these motifs Magic and TedWylee as discussed earlier). The structural differences detailed in this review result in functional differences between the large (Grp170 and Hspl10) members of the Hsp70 superfamily, the most distinctive being an increased ability of these proteins to bind (hold) denatured polypeptides compared with Hsc70, perhaps related to the enlarged C-terminal helical domain. However, there is also a major difference between these large stress proteins; Hsp110 does not bind ATP in vitro, whereas Grp170 binds ATP avidly. The role of the Grp170 and Hsp110 stress proteins in cellular physiology is not well understood. Overexpression of Hsp110 in cultured mammalian cells increases thermal tolerance. Grp170 binds to secreted proteins in the ER and may be cooperatively involved in folding these proteins appropriately. These roles are similar to those of the Hsp70 family members, and, therefore, the question arises as to the differential roles played by the larger members of the superfamily. We have discussed evidence that the large members of the superfamily cooperate with members of the Hsp70 family, and these chaperones probably interact with a large number of chaperones and cochaperones in their functional activities. The fundamental point is that Hsp110 is found in conjunction with Hsp70 in the cytoplasm (and nucleus) and Grp170 is found in conjunction with78 in tha ER in every eucaryotic cell examined from yeast to humans. This would strongly argue that Hsp110 Grp170 exhibit functions in eucaryotes not effectively performed by Hsp70s or Grp78, respectively. Of interest in this respect is the observation that all Hsp110s loss of function or deletion mutants listed in the Drosophila deletion project database are lethal. The important task for the future is to determine the roles these conserved molecular chaperones play in normal and physiologically stressed cells.     

TAT-Hsp40 inhibits oxidative stress-mediated cytotoxicity via the inhibition of Hsp70 ubiquitination

Heat shock protein 40 (Hsp40) functions as a co-chaperone of mammalian Heat shock protein 70 (Hsp70) and facilitates the ATPase activity of Hsp70, and also promotes the cellular protein folding and renaturation of misfolded proteins. In an effort to assess the effects of Hsp40, we generated TAT-fused Hsp40 (TAT-Hsp40). The cells were transduced with TAT-Hsp40 and exposed to H(2)O(2). We demonstrated that the TAT-Hsp40-transduced cells were more resistant to cellular cytotoxicity and cell death. In particular, the degradation of Hsp70 was significantly reduced in TAT-Hsp40-containing cells as a consequence of reduced ubiquitin-proteasome activity after oxidative injury. These data support the notion that Hsp40 may confer resistance to oxidative stress via the prevention of proteasome activity.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Dobutamine mediates cytoprotection by induction of heat shock protein 70 in vitro

Aims

Dobutamine is cytoprotective when applied before a subsequent stress. However, the underlying molecular mechanism is unknown. Dobutamine also inhibits nuclear factor (NF)-κB in human T lymphocytes. Other inhibitors of NF-κB induce a so-called heat shock response. We hypothesized that dobutamine mediates protection from apoptotic cell death by the induction of a heat shock response.

Main methods

Jurkat T lymphoma cells were preincubated with dobutamine (0.1, 0.5 mM) before the induction of apoptosis (staurosporine, 2 μM). DNA-binding of heat shock factor (HSF)-1 was analyzed by electrophoretic mobility shift assay, mRNA-expression of heat shock protein (hsp)70 and hsp90 by Northern Blot, activity of caspase-3 by fluorogenic caspase activity assay and cleavage of pro-caspase-3 by Western Blot. Apoptosis was assessed by flow cytometry after annexin V-fluorescein isothiocyanate staining. Hsp70 and hsp90 were inhibited using N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolaetam and 17-allylamino-17-demethoxygeldana-mycin, respectively. All data are given as median and 25/75% percentile.

Key findings

Pre-incubation with dobutamine inhibited staurosporine-induced annexin V-fluorescence (28 [20–32] % vs. 12 [9–15] % for dobutamine 0.1 mM and 7 [5–12] % for dobutamine 0.5 mM, p < 0.001), cleavage of pro-caspase-3 as well as caspase-3-like activity (0.46 [0.40–0.48] vs. 0.32 [0.27–0.39] for Dobutamine 0.1 mM and 0.20 [0.19–0.23] for Dobutamine 0.5 mM, p < 0.01). Dobutamine induced DNA-binding of HSF-1 and mRNA-expression of hsp70 and hsp90. While inhibition of Hsp90 had no effect, inhibition of Hsp70 increased the number of annexin V-positive cells (33 [32–36] % vs. 18 [16–24] %) and caspase-3-like activity (0.21 [0.19–0.23] vs. 0.16 [0.13–0.17], p < 0.05).

Significance

Dobutamine protects from apoptotic cell death via the induction of Hsp70.     

Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.     

Hsp60 protein pattern in coral is altered by environmental changes in light and temperature

The heat shock protein Hsp60 exhibited marked oscillation during a 12-hour day period when the coral Turbinaria reniformis was maintained in the laboratory under constant conditions of light (200 μE) and temperature (27 °C). A biphasic pattern of Hsp60 was apparent, punctuated by a low protein level at the midpoint of the 12-hour day period. Oscillation of Hsp60 was also apparent when coral was kept in darkness in lieu of a scheduled light period. The pattern of Hsp60 was altered when coral was exposed to increased light intensity (400 μE) or temperature elevation (32 °C). These observations suggest that Hsp60 in coral exhibits oscillation that is altered by increased light and temperature elevation.     

Interdomain interactions dictate the function of the Candida albicans Hsp110 protein Msi3

Heat shock proteins of 110 kDa (Hsp110s), a unique class of molecular chaperones, are essential for maintaining protein homeostasis. Hsp110s exhibit a strong chaperone activity preventing protein aggregation (the “holdase” activity) and also function as the major nucleotide-exchange factor (NEF) for Hsp70 chaperones. Hsp110s contain two functional domains: a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). ATP binding is essential for Hsp110 function and results in close contacts between the NBD and SBD. However, the molecular mechanism of this ATP-induced allosteric coupling remains poorly defined. In this study, we carried out biochemical analysis on Msi3, the sole Hsp110 in Candida albicans, to dissect the unique allosteric coupling of Hsp110s using three mutations affecting the domain–domain interface. All the mutations abolished both the in vivo and in vitro functions of Msi3. While the ATP-bound state was disrupted in all mutants, only mutation of the NBD-SBDβ interfaces showed significant ATPase activity, suggesting that the full-length Hsp110s have an ATPase that is mainly suppressed by NBD-SBDβ contacts. Moreover, the high-affinity ATP-binding unexpectedly appears to require these NBD-SBD contacts. Remarkably, the “holdase” activity was largely intact for all mutants tested while NEF activity was mostly compromised, although both activities strictly depended on the ATP-bound state, indicating different requirements for these two activities. Stable peptide substrate binding to Msi3 led to dissociation of the NBD-SBD contacts and compromised interactions with Hsp70. Taken together, our data demonstrate that the exceptionally strong NBD-SBD contacts in Hsp110s dictate the unique allosteric coupling and biochemical activities.     

Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes puniceus, via high throughput proteomics

Cucujus clavipes puniceus is a freeze avoiding beetle capable of surviving the long, extremely cold winters of the Interior of Alaska. Previous studies showed that some individuals typically supercool to mean values of approximately − 40 °C, with some individuals supercooling to as low as − 58 °C, but these non-deep supercooling (NDSC) individuals eventually freeze if temperatures drop below this. However, other larvae, especially if exposed to very cold temperatures, supercool even further. These deep supercooling (DSC) individuals do not freeze even if cooled to − 100 °C. In addition, the body water of the DSC larvae vitrifies (turns to a glass) at glass transition temperatures of − 58 to − 70 °C. This study examines the proteomes of DSC and NDSC larvae to assess proteins that may contribute to or inhibit the DSC trait. Using high throughput proteomics, we identified 138 proteins and 513 Gene Ontology categories in the DSC group and 104 proteins and 573 GO categories in the NDSC group. GO categories enriched in DSC include alcohol metabolic process, cellular component morphogenesis, monosaccharide metabolic process, regulation of biological quality, extracellular region, structural molecule activity, and antioxidant activity. Proteins unique to DSC include alpha casein precursor, alpha-actinin, vimentin, tropomyosin, beta-lactoglobulin, immunoglobulins, tubulin, cuticle proteins and endothelins.     

Electrostatic interactions play a critical role in Mycobacterium tuberculosis Hsp16.3 binding of substrate proteins

Mycobacterium tuberculosis Hsp16.3, a member of a small heat shock protein family, has chaperone-like activity in vitro and suppresses thermally or chemically induced aggregation of proteins. The nature of the interactions between Hsp16.3 and the denatured substrate proteins was investigated. A dramatic enhancement of chaperone-like activity of Hsp16.3 upon increasing temperature was accompanied by decreased ANS-detectable surface hydrophobicity. Hsp16.3 exhibited significantly enhanced chaperone-like activity after preincubation at 100°C with almost unchanged surface hydrophobicity. The interaction between Hsp16.3 and dithiothreitol-treated insulin B chains was markedly weakened in the presence of NaCl but greatly enhanced by the addition of a low-polarity alcohol, accompanied by significantly increased and decreased surface hydrophobicity, respectively. A working model for Hsp16.3 binding to its substrate proteins is proposed.