Molecular Biology Of The Eukaryotic Heat Shock Response
Introduction
Among the most common cellular defense mechanisms, is that of the Heat Shock Response. The Heat Shock Response mechanism (HSR) is the most highly evolutionarily conserved defense mechanism in both eukaryotes and prokaryotes (Mizera and Gambin, 2010). This exogenous cellular mechanism is also known as “Heat Stress” or “Hyperthermia” and enables the cells to adapt and survive in stress conditions, via heat induced proteins that are strictly regulated both in transcriptional and translational level (Shreedhar and Pardhasaradhi et al. , 1999)The study of HSR mechanisms dates back to the 1910s when it was discovered that elevated environmental temperatures may cause mutations to Drosophila (Plough, 1917) and it was not until the early 1960s that heat stress was correlated with transcriptional activation again in Drosophila (Ritossa, 1962) and the Heat Shock Proteins (HSPs) were discovered. Overall, it has been found that the transcription of more than one hundred genes including chaperons and encoding factors for protein degradation, transport and so on, are heat induced; with the most studied ones being HSP70 and HSP90 (Velichko and Markova et al. , 2013).
At the same time, the lack of some certain HSP genes, has been shown to cause cellular death like in the case of rat histiocytic cell line (Shreedhar and Pardhasaradhi et al., 1999). Ferruccio Ritossa first discovered in 1962 that high temperatures caused the induction of the Polytene Chromosomes in Drosophila’s salivary glands, which more than ten years later was found to be due to HSPs.
Cellular Level of Response
A common effect of abnormally high temperatures, is the fluidization of the membrane, which ultimately leads to its destabilization and that of its molecular components. This causes various disturbances to the cell, like changes to the membrane potential, seizure of intercellular interactions and of course morphological changes. The cells respond to the effects of hyperthermia via the production of HSPs which manage to restore membrane rigidity. Heat shock may also damage cytoplasmic organelles; it can cause mitochondrial swelling, fragmentation of the Golgi apparatus and the Endoplasmic Reticulum and nucleic disintegration. Cells tend to amend this critical damage through the formation of stress granules, also called Heat Shock Granules (HSGs), the exact function of which is not entirely clear. However, they seem to contain HSPs and to potentially protect different kinds of mRNA or even repair denatured proteins. Under stress conditions, tertiary protein structure is compromised and many proteins agitate, hence protein homeostasis is destabilized.
The cell restores protein homeostasis either by refolding or destroying agitated proteins through HSPs (Velichko and Markova et al. , 2013, Nover and Scharf et al. , 1989). Imminently, this agitation of cellular homeostasis can result in cell death due to irreversible damage. Extreme hyperthermia can initiate two possible modes of cell death, either “rapid” or “slow”. The first can be caused quite instantly, or within hours of exposure to heat, whereas the latter is caused due to long term effects of HS, like centromere or mitotic destruction. Additionally, it has also been shown that HS can commence unnatural cell apoptosis. The severity or even fatality of HS, depends on the cell type, the magnitude and duration of the HS and the cell cycle phase; cells undergoing their S phase, tend to be more susceptible due to DNA replication being affected (Velichko and Markova et al. , 2013).
Molecular Level of Response
As already mentioned, Ritossa in the early 1960s, showed that there are some heat induced genes in the salivary glands of Drosophila. High temperature activates the formation of chromosome puffs, which produce the mRNA that was later shown to regulate the production of HSPs (Ashburner and Bonner, 1979). There are two kinds of HS proteins, sorted based on their molecular weight; small HSPs which may weight up to 40,000 Da and bigger ones between 60 and 100 kDA. As expected, in many cases HSPs are in low concentrations when the cell is in normal conditions, whereas their levels rise under stress. Many HSPs are not only heat induced, in fact they may be activated under various “stressful” conditions such as extremely low temperatures or when their surroundings lack oxygen or even when they are exposed to certain metals or ethanol (Velichko and Markova et al. , 2013).
In eukaryotic cells, the production of HSPs is tightly regulated by a Heat Shock transcription Factor (HSF), which interacts and binds Heat Shock Elements (HSEs) in order to activate HS genes (Wu, 1995, Guertin and Lis, 2010). Among the first heat induced gene families that were discovered, were the HSP90 and HSP70 gene lines. HSP90 Gene LineHSP90 genes have been identified in many evolutionarily different organisms; besides fruit flies, they have been cloned and sequenced in mammals, yeast or even in prokaryotes. In fact, they are highly conserved between eukaryotes and prokaryotes. Also, they have been found to be in significant concentrations, despite the cell not being under stress, in normal temperatures. Their concentration is elevated further as temperature rises beyond normal. In Drosophila only one gene has been identified: HSP83, which is induced in the embryonic stage of ovogenesis.
Furthermore, in Saccharomyces Cerevisiae there has been discovered another gene of this family, besides HSP83; which is HSC83 and is somewhat less heat-induced than HSP83. Last but not least, HSP83 in S. Cerevisiae assists in yeast sporulation (Kurtz and Lindquist, 1984, Lindquist and Craig, 1988). It appears that HSP90s generally function as chaperons, which means that they assist in protein covalent folding or unfolding. Their exact function is still quite unclarified, however it has been proven so far, that they are certainly essential for eukaryotic survival, since they are also associated with protein signaling. Thanks to a series of enlightening crystallographic studies, it is now proven that HSP90s are ATP-dependent. Regarding HSP90 structure, they consist of three main domains: the N-terminal domain (or ATP-binding domain), an M (or middle) domain and a C-terminal dimerization domain.
When the N-terminal domain is not bound to ATP, the protein is in an “open” state, whereas the binding of ATP results in a conformational change (“closed” state); which is capable of clamping a particular set of proteins, called “client proteins”. These are transcription factor and protein kinases, involved in cell proliferation, differentiation or apoptosis (Terasawa and Minami et al. , 2005, Pearl and Prodromou, 2006). HSP70 gene lineHSP70s are also chaperones which are vital to eukaryotic survival. Similarly to HSP90s they are involved in protein folding; in particular, they are associated with the de novo folding (i. e. nascent chain protein folding) of polypeptides, the correction of misfolded proteins or even the degradation of proteins. They are also involved in protein translocation across membranes. HSP70s also have a similar structure to HSP90s; where they contain an N-terminal ATP-binding domain, a Substrate Binding Domain (SBD) which as its name implies can bind different amino acid residues thanks to its groove.
There is also a C-terminal domain which functions as a “lid” for the SBD. The protein switches from low substrate affinity to high, when N-terminal is either ATP bound or not respectively (Mayer and Sinning et al. , 2012). Multiple HSP70 genes have been sequenced and cloned in various eukaryotic organisms, including D. Melanogaster and S. Cerevisiae and prokaryotes like E. Coli (Lindquist and Craig, 1988). In addition, up to eleven HSP70 genes have also been identified in humans in chromosomes 1, 5, 9, 11, 14 and potentially 21 (Tavaria and Gabriele et al. , 1996).
Conclusion
In summation, Heat Shock Response mechanisms have been broadly studied for the past decades, both in eukaryotes and prokaryotes. These cell defense processes against hyperthermia and other “stressful” instances have been analyzed and covered in various scientific articles, however many of their aspects remain obscure and unclear. The criticality of HS and the significance of HSPs have been demonstrated in various scenarios, like that of cellular necrosis and apoptosis or in the way hyperthermia interferes with DNA replication. This indicates that HSR really helps in cell survival. In general, stress response mechanisms are a key component to cell adaptation in stressful scenarios, like temperature fluctuation, oxygen deprivation or in the presence of toxins. Consequently, stress response mechanisms overall should draw more the attention of the scientific community.
