Definiciones y descripciones fundamentales

Although the transcriptional activators of heat shock genes in both prokaryotes and eukaryotes are now being identified, the molecular mechanism which senses changes in temperature is presently unknown. Recent work suggests that a homeostatic mechanism involving the level of free HSPs in the cell provides a thermometer for detecting and reacting to temperature changes. Under normal growth conditions, HSPs may bind to HSF and repress its activity. These HSPs may include HSP70 and HSP90, binding of HSP90 to HSF having been recently demonstrated(Nadeau et a/., 1993).During heat shock competition with high levels of thermally damaged proteins for binding of HSPs may cause the dissociation of HSF-HSP complexes, causing either an increase in the DNA-binding affinity of the factor (Drosophila) or activation of the C-terminal domain of HSF (yeast). DNA-bound HSF may then direct increased HSP synthesis until levels are sufficiently high to result in the reassociation of HSPs with HSF and the re-establishment of the repressed state. Ensuring overproduction of HSPs will ensure a large free pool of these HSPs and may cause HSF to be inhibited in its action. Strains which synthesise low levels of HSPs might prematurely activate the heat shock response. In yeast, strains carrying deletions of two of the constitutively expressed HSP70 genes (SSAl and SSA2 ) express HSPs at high levels, even at 23°C (Craig and Jacobsen, 1984). However, it should be noted that HSP70 is essential for cell viability so reducing the levels of this protein may itself be stressful, causing activation of the heat shock response by a mechanism other than that described above. One of the early events of heat shock must be a dramatic drop in the pool of free HSPs. This is a feasible proposition given the increase in aberrant protein that will ensue, but it has only been reported for levels of free ubiquitin which decrease 75% (Rose and Warms, 1987). Recombinant Drosophila HSF produced in E.coli will bind to HSEs with high affinity in the absence of heat shock (Clos et a l, 1990). In contrast to this, the same HSF will not bind to HSEs if produced in Xenopus oocytes. This suggests that HSF may interact with one or more negative regulators found in eukaryotic cells, possibly eukaryotic HSPs. However, in this thesis (Chapter 3) it is demonstrated that the overproduction of HSP90 does not interfere with normal heat shock induction of HSP genes in S. cerevisiae.

Aberrant protein seems to be a recurring theme in studies to investigate the heat shock response trigger. Firstly, many of the conditions known to induce HSP genes are thought to cause dénaturation of intracellular proteins (Table 1.1). Denatured Lambda repressor can induce the heat shock response in E.coli (Parsell and Sauer, 1989) and injecting denatured protein (but not native protein ) into Xenopus oocytes has the same effect (Ananthan et al.,

mutant mouse cell line which cannot ubiquitinate proteins above a certain temperature exhibits abnormally high synthesis of HSPs at such temperatures (Finley et al, 1984). Finally, there is evidence from biophysical studies of cellular protein dénaturation in vivo

within the temperature range of HSP induction in both bacteria and mammalian cells (Lepock a/., 1988,1990).

It has been suggested that HSF itself is a cellular thermometer (Hightower, 1991). HSF from unshocked HeLa cells can be induced to bind HSEs in vitro by exposing nuclear extracts to elevated temperatures (Larson et a l, 1988) and other conditions that promote protein unfolding such as nonionic detergents and increasing concentrations of urea (Mosser et al, 1990). Such results, however, do not distinguish between models where HSF per se responds to environment by direct conformational change and models where it is the interaction of HSF with other proteins that is affected.

Heat shock has many effects on the cell besides the build up of aberrant protein. For example, intracellular pH falls and levels of calcium rise. These may also play a role in triggering the heat shock response. Evidence for this is the activation of HSF in vitro by decreasing the pH of buffers and by increasing levels of calcium (Mosser e ta l, 1990). There may even be different induction pathways triggered by different cellular events. Yuzawa has isolated two groups of E.coli mutants, one defective in responding to unfolded proteins as inducers but still heat inducible and the other unresponsive to both inducers. This suggests the existence of at least two distinct induction pathways (Hightower, 1991).

Finally, the regulation of the heat shock response in eukaryotes has taken a new twist. Morimoto has described the cloning and characterisation of two murine HSF genes {HSFl

and HSF2) whose products display heat inducible and constitutive HSE binding respectively (Hightower, 1991). This system may be widespread among higher eukaryotes. There are also two human HSFs, HSFl exhibiting heat inducible HSE binding (Rabindran e ta l, 1991), while the binding characteristics of HSF2 are unknown (Schuetz et al, 1991). It is well established that HeLa cells have two HSE binding activities, one found in unshocked extracts and the other in stressed cells (Kingston et al,

1987). The HSE protein complex in unstressed cells may correspond to DNA bound to HSF2. Different HSFs may have evolved to respond to different temperature thresholds or to chemical stress signals. This emerging data suggesting the existence of separable induction pathways, make regulation of the response to stress far more complicated than previously thought. Treatment with antibiotics known to inhibit various aspects of

ribosome function has implicated ribosomes as the sensors for both heat and cold shock in prokaryotes (VanBogelen and Neidhardt, 1990). Ribosomes may serve a similar role in eukaryotes, HSPs functioning to alter the translational capacity of the cell.