Animals at the extremes: The desert environment

## 3.5 Summary of Section 3

### 4 Integrating across disciplines

# 4.1 Heat-shock proteins

Molecular biology provides further insights into the biochemical and physiological responses of vertebrates to extreme temperatures and aridity in the desert environment. Animals living in hot deserts are at risk of overheating, which in turn results in denaturation of enzymes and other essential proteins. Physiologists were puzzled for a long time about how desert reptiles such as the desert iguana (Dipsosaurus dorsalis) function normally at T b = 44–46°C. Such high temperatures would be expected to result in complete denaturation of enzymes and other important proteins such as haemoglobin. Yet we saw in Figure 36 (in Section 3.4) that ATPase of Dipsosaurus continues to function even at 43°C or 44°C.

Discovery of heat-shock proteins (Hsps) suggested an explanation. The name ‘heat-shock proteins’ is applied because levels of Hsps in cells rise rapidly after exposure to abnormally high temperatures, e.g. around 40°C for mammalian cells. Hsps comprise at least 12 families of related proteins that are found in cells of all animal and plant species so far investigated, so they are molecules that have been highly conserved in evolution, and are not specific to desert species. Nevertheless, Hsps are important for species at risk of exposure to high T a because of their role as chaperone proteins that ‘rescue’ proteins whose tertiary structure has been disrupted by overheating. Chaperone proteins bind to denatured regions of a protein and alter the misfolded structure so that the correct three-dimensional structure is regained.

Heat-shock proteins maintain the biologically active conformation of enzymes and other essential proteins in cells during heat stress. For example, exposure of the fruit fly, Drosophila, to high temperatures, results in rapid transcription of a heat-shock gene, Hsp70. To begin gene transcription, various transcription factors, then RNA polymerase bind to special DNA sequences known as promoters; the latter determine where RNA polymerase binds, e.g. TATA (Figure 41), and starts transcription. Each gene has its own promoter. Nucleotide sequences in promoters tend to be highly conserved and are therefore the same in many species.

Figure 41 Control of mRNA synthesis during transcription of Hsp70. (a) RNA polymerase II pauses after synthesising about 25 nucleotides of the transcript as CHBF binds to HSE. (b) After a heat shock that produces part-denatured proteins heat-shock transcription factor (HSTF) is converted from an inactive into an active DNA-binding form. This occurs in response to the presence of part-denatured proteins. Monomers of HSTF link to form trimers that enter the nucleus. (c) Binding of activated (trimerized) HSTF to the heat-shock regulatory element (HSE) of the promoter of the Hsp70 gene, releases the paused RNA polymerase II, leading to rapid transcription of the Hsp70 gene
Figure 41

Gene sequences that regulate the promoter may be located adjacent to the promoter, or upstream or downstream of the gene. Regulatory elements control the transcription of genes that are not active all the time. A heat-shock regulatory element (HSE) has been identified in the promoter region of a heat-shock gene. Heat-shock transcription factors (HSTFs) are transcription factors that control Hsp gene expression through interaction with HSEs. The complex interlinking functions of the components involved in control of transcription of heat-shock proteins are shown in Figure 41.

Given the number of stages involved in Hsp gene transcription, researchers were puzzled initially by the rapid response, just a few seconds, to heat shock. Research on Drosophila demonstrated that such a rapid response is possible because Hsp70 is normally partly transcribed by RNA polymerase II, producing an RNA transcript about 25 nucleotides long. Mechanisms that pause the transcription are not understood well, but appear to involve constitutive HSE binding factors (CHBF) that bind to the promoter and halt transcription of the Hsp gene (Figure 41).

To understand how transcription of Hsp is resumed, we must begin with the role of HSTF. Normally, before heat-induced activation, HSTF exists as monomers in the cytoplasm. Research on heat-shocked mammalian cell cultures suggests that the signal for activation of HSTF is contact with the hydrophobic domains of proteins that have been denatured (Figure 42). HSTF monomer extracted from unstressed mammalian cells is bound to Hsp molecules. As the Hsp molecules bind to denatured protein during heat stress, it is likely that as a consequence, HSTF monomer is released from Hsp molecules. The released HSTF monomers trimerise and translocate to the nucleus where they stimulate resumption of transcription of Hsp genes by binding to the HSEs in the promoter regions.

Figure 42 Regulation of transcription of heat-shock protein genes by heat-shock transcription factor
Figure 42
Pockley, G. (2001) Heat shock proteins in health and disease …, Expert Reviews in Molecular Medicine. Cambridge University Press; Pockley, G. (2001) Heat shock proteins in health and disease …, Expert Reviews in Molecular Medicine. Cambridge University Press;

Most work on Hsps has been carried out using cell lines and tissue cultures. Few studies have been carried out on vertebrates, and most of those have concentrated on fish. However, Zatsepina et al. (2000) studied the synthesis, properties and activation of Hsp70 in three species of desert lizard: Phrynocephalus interscapularis, a highly thermoresistant diurnal species, and Gymnodactylus caspius and Crossobamon eversmanni, both nocturnal species. All three species were captured in a sand desert in Turkestan. Studies on a temperate species, Lacerta vivipara, provided comparisons with the desert species. All lizards were acclimated for 2 weeks at 25°C. Heat-shock treatment involved one hour exposure of lizards of each species to a specific T a at or greater than 39°C. Following heat shock the animals were killed and cell extracts from the whole body were prepared. Samples of the extracts were mixed with 32P-labelled HSE and incubated at 20°C for 20 minutes, during which time any HSTF or CHBF present would complex with the 32P-HSE. The free 32P-HSE was separated from the 32P-HSE-HSTF and 32P-HSE-CHBF complexes by gel electrophoresis. The gels were dried and exposed to X-ray film. Figure 43 shows the results of the analysis of binding of 32P-HSE to HSTF (complex III) and to CHBF (complexes I and II), in lizards kept at T a 25°C and lizards heat shocked at 42, 45 and 49°C.

Figure 43 Analysis of heat-shock regulatory element (HSE) binding activity in lizard species from different ecological niches. Gel-mobility-shift analysis of whole-cell extracts from control (c) and heat-shocked animals. The extracts analysed by gel mobility shift were prepared from Gymnodactylus caspius (G) control animals (lane 1) and from individuals heated to 45°C for 60 min (lane 2), from Phrynocephalus interscapularis (P) control animals (lane 3) and from individuals heated to 42, 45 or 49°C for 60 min (lanes 4, 5 and 6, respectively), and from Lacerta vivipara control animals (lane 7) and from individuals heated to 42°C for 60 min (lane 8). The locations of the 32P-HSE-CHBF complexes (I and II) and heat-shock-induced 32P-HSE-HSTF complex (III) are indicated by arrows
Figure 43
Zatsepina et al. (2000) Thermotolerant desert lizards …, Journal of Experimental Biology, 203. Copyright © Company of Biologists Ltd Zatsepina et al. (2000) Thermotolerant desert lizards …, Journal of Experimental Biology, 203. Copyright © Company of Biologists Ltd

At T a 25°C for Lacerta (c, lane 7) the levels of complex II were high, but they were low for Gymnodactylus and Phrynocephalus. In contrast, levels of complex I were high for Gymnodactylus and Phrynocephalus but complex I was absent in Lacerta. Complex III, consisting of trimerised and therefore active HSTF bound to 32P-HSE, was present in both Gymnodactylus and Phrynocephalus, but not in Lacerta. So both desert species have activated HSTF even when acclimated to 25°C. Following heat shock, complex III, activated HSTF, was present in all three species (lanes 2, 4, 5, 6 and 8).

## Activity 16

What is the significance of the presence of complex III in cell extract?

## Answer

Complex III consists of HSTF bound to 32P-HSE which means that the HSTF was trimerised and therefore in a form that combines to HSE, and consequently activates transcription of Hsp genes.

Zatsepina et al. isolated mRNA from samples of cell extract prepared from control and heat-shocked lizards, and carried out hybridisation, probing with Xenopus laevis Hsp70, lizard HSTF1 and αβ-actin genes, the latter acting as a standard for comparison (Figure 44).

Figure 44 Expression of Hsp70 and HSTF in different lizard species. (a) Northern blot analysis of mRNA present in the cells of P. interscapularis (P) and L. vivipara (L) at 25°C (lanes 1 and 2) and after heat-shock treatment for 1 h at 42°C (lanes 3 and 4). HSTF, Hsp70 and actin are indicated by arrows. (b) Western blot analysis of proteins in G. caspius (G) (lanes 1 and 2), L. vivipara (L) (lanes 3 and 4) and P. interscapularis (P) (lanes 5 and 6). Lanes 1, 3 and 5, animals at 25°C, lanes 2, 4 and 6, individuals heat shocked at 42°C for 60 min. HSTF and Hsp70 are indicated by arrows
Figure 44
Zatsepina et al. (2000) Thermotolerant desert lizards …, Journal of Experimental Biology, 203. Copyright © Company of Biologists Ltd Zatsepina et al. (2000) Thermotolerant desert lizards …, Journal of Experimental Biology, 203. Copyright © Company of Biologists Ltd

## Activity 17

Compare the levels of HSTF mRNA and Hsp70 mRNA in Phrynocephalus and Lacerta, before and after heat shock.

## Answer

Prior to heat shock, levels of HSTF mRNA were lower in Phrynocephalus than in Lacerta; in contrast, constitutive levels of Hsp70 mRNA were higher in the thermoresistant species Phrynocephalus than in Lacerta. Following heat shock both species showed strong induction of Hsp70 mRNA, demonstrating increased transcription of Hsp70 genes (Figure 44a). Neither of the two species showed increased amounts of HSTF mRNA after heat shock, in contrast to the large increases in Hsp70 mRNA.

Zatsepina et al. interpret these data as showing that HSTF genes in lizards are not induced by heat shock. Western analysis using antibodies to human Hsp70 showed that levels of cellular Hsp70 are higher in both desert species than in Lacerta (Figure 44b). Earlier work had measured a three- to fivefold higher Hsp70 content in desert species than in L. vivipara kept at T a 25°C.

Comparison of Lacerta vivipara with the nocturnal desert species Crossobamon eversmanni showed that the latter has 2–3 times higher levels of Hsp in its cells. After heat shock, liver cells of Lacerta synthesised high levels of Hsps 68 and 85. In the desert species, normal synthesis of all proteins in the liver continued after heat shock at 39, 42 and 43°C, whereas in Lacerta, protein synthesis reduced after heat shock at 37°C and 39°C and almost ceased at 42°C.

High levels of Hsps in cells of desert reptiles may stabilise protein structure sufficiently in the absence of thermostable proteins, and enable continuation of normal rates of protein synthesis at T b up to 45°C. Zatsepina et al. demonstrated a high level of Hsp70 transcription (Figure 44a) linked to activated HSTF bound to HSE (Figure 43, complex III), in the desert species Phrynocephalus, even in animals kept at relatively low T a. Tight regulation of Hsp gene expression ensures a response appropriate for the level of heat stress and a subsequent repression of the response when the stress is over. As T a was increased for Phrynocephalus (Figure 43), relative amounts of complex III increased as complexes I and II decreased, suggesting removal of suppression of Hsp expression. Desert and temperate lizards differed in the quantity and state of HSTF and constitutive HSE-binding activity (CHBA), both under normal and heat-shock conditions.

In the desert species induction of Hsp synthesis at 3–7°C higher than in temperate forms may link to high constitutive levels of Hsps. Temperate lizards, in contrast, have low constitutive levels of Hsps but high levels of HSTF which probably expedite intense synthesis of Hsps after a brief exposure to heat shock. Severe heat shock is lethal for the temperate lizards but is tolerated by the desert species. Phrynocephalus maintains cellular Hsps, and therefore can continue to function normally even when T b rises to 45°C. This capacity is of great advantage to a diurnal desert reptile as foraging times can be prolonged. By foraging during the hottest parts of the day diurnal desert reptiles avoid predators.

As you have read in Section 2.3, many desert vertebrates avoid overheating by behavioural means, so you may conclude that Hsps are of no more importance in desert species than in temperate species. However, the need for behavioural thermoregulation at any one time may conflict with other needs, e.g. finding food or escaping from predators. A spurt of intense physical activity may raise T b sufficiently to initiate Hsp transcription. Species such as the camel that use relaxed homeothermy as a means of reducing TEWL experience T b as high as 41°C. Desert lizards that forage during the day may routinely experience T b values high enough to trigger a heat-shock response. Although desert animals are not unique in having Hsps, life in the desert environment, where animals are at risk of overheating, is probably facilitated by efficient functioning of Hsps.

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