Abstract
In contrast to the detrimental action of severe stress conditions, the beneficial effects of mild stress, known as hormesis, is increasingly discussed and studied. A variety of applications for hormesis in risk assessment processes, anti-ageing strategies and clinical therapies have been proposed. The molecular mechanisms underlying the phenomenon of hormesis, however, are not yet fully understood. A possible mechanism that has been proposed for hormesis, the homoeostasis overshoot hypothesis, assumes that an overshoot of repair- and self-recovery mechanisms in response to mild damage can be held responsible for the beneficial effects of hormesis. The present paper proposes ‘cellular quality control’ as a further explanation of the molecular mechanisms underlying the benefits observed after exposure to mild stress. The most important quality control mechanisms are outlined and their known and hypothesised actions in hormesis are discussed. As an example, different aspects of protein quality control will be described in more detail, which includes the reaction of the cell upon stress-induced protein damage and –aggregation. The regulation of Heat Shock Proteins and components from the ubiquitin proteasome system as part of cellular quality control is described in relation to its beneficial role in hormesis.
INTRODUCTION
The survival and long-term health of all organisms is linked to their ability to cope with stressful conditions. This ability depends on their capacity to respond and to adapt to internal and external disturbances, as well as on their ability to repair damage of cellular macromolecules including DNA, lipids and proteins. Traditionally, this intrinsic property of all living systems to counteract cellular disturbances and to maintain an internal equilibrium has been defined as ‘homeostasis'. More recently, the dynamic regulation of the internal cellular environment has been referred to as ‘allostasis’ (McEwen and Wingfield 2003) and ‘homeodynamics’ (Rattan 2012) in which the internal environment is not necessarily fixed. When cells are exposed to stressors, which can be defined as any signal which alters homeostasis, molecular pathways will be activated that counteract the disequilibrium in order to prevent (severe) damage. This ability of sensing cellular disturbances and responding accordingly is based on the presence of ‘Quality Control Systems', which refer to the mechanisms involved in cellular defense, maintenance and repair (Hurtley and Helenius 1989; Rorth 2008).
In the literature, different, and sometimes overlapping, parts of this general capacity of cells have been referred to with a wide variety of terms, amongst which ‘cell vitality’ (and vitagenes) (Calabrese et al. 2010), ‘allostasis’ (McEwen and Wingfield 2003), ‘homeodynamics’ (Rattan 2012), ‘resilience’ (Cuesta and Singer 2012), ‘programmed cell life’ and ‘adaptive response’ (Calabrese et al. 2007). However, to find a single term encompassing all beneficial mechanisms involved in cellular defence, maintenance and repair, is more difficult than it appears at first glance. In this paper, the term ‘cellular quality control mechanisms’ is therefore proposed to indicate this capacity. In addition, the term ‘homeostasis’ will be used in the remainder of this paper, even though at times ‘allostasis’ or ‘homeodynamics’ could have been used.
The term ‘quality control', although widely used, is poorly defined. It was originally coined in 1989, by Hurtley and Helenius, in order to explain the finding that only correctly folded, modified and assembled proteins were exported from the endoplasmic reticulum (ER) (Hurtley and Helenius 1989; Bergeron et al. 1994; Ellgaard and Helenius 2001). The term has been redefined and applied quite loosely in the years that followed. Ellgaard and Helenius later used quality control to refer to quality control systems for “practically every step that leads to the synthesis of DNA, RNA, and protein molecules” (Ellgaard and Helenius 2003). In a variety of other articles covering different fields of cellular biology, the term has been used to refer to other forms of cellular defence, repair and maintenance mechanisms.
The term ‘cellular quality control’ as used in this paper will refer to all the detection, repairing and elimination processes involved in the cell that ensure the quality of macromolecules and organelles. Hence, cellular quality control plays a crucial role in maintaining the optimal three-dimensional structure and function of a large variety of macromolecules within the cell. Depending on the mode of action, components from the quality control system can be classified as preventive defense mechanisms, which are permanently activated and ensure damage is not inflicted, and cellular repair mechanisms, which become active once homeostasis has been disturbed in order to reinstate the steady state of the system and prevent further damage (Hurtley and Helenius 1989, Bergeron et al. 1994). Finally, protein complexes indicated as proteasomes and the process of autophagy are involved in removal of irreversibly damaged proteins as well as of worn-out organelles respectively (Calabrese et al. 2010).
An important factor responsible for the continuous activation of the preventive and defensive component of cellular quality control includes antioxidants and other free radical scavengers. These molecules quench the free radicals and pro-oxidant compounds before they can damage cellular components, either by donating an electron or accepting an electron of the free radical. Preventive defence mechanisms against reactive oxidative species (ROS) can also be mediated by enzymes. ROS arise from the reduction of molecular oxygen and represent a threat for the cell because they can damage cellular components (Halliwell and Gutteridge 1999; Schrader and Fahimi 2006). The macromolecules DNA, proteins and lipids, which are essential for proper cell functioning, are susceptible to ROS damage. Limiting the ROS-induced damage is therefore essential for cell survival (Kohen and Nyska 2002). ROS that escape quenching can generate a variety of lesions and damage in DNA, proteins and lipids, thereby disturbing cellular homeostasis. Once damage has been inflicted and homeostasis is altered, cellular maintenance and repair mechanisms will be activated to repair or remove the damage before deleterious effects can occur. Depending on the macromolecule that has been affected or misfolded, their respective (DNA, protein or lipid) quality control systems will sense, and then repair or eliminate the aberrant molecule (Rorth 2008).
In order to perform their biological function, proteins must achieve and maintain their biologically active three-dimensional conformation. Consequently, cells have developed a protein quality control system consisting of stress-response signalling pathways that ensure proper protein assembly (Schröder and Kaufman 2005; Buchberger et al. 2010; Bar-Lavan et al. 2012). This protein quality control system consists of molecular chaperones that assist protein folding, assuring that they will be assembled into their active structure. In addition, chaperones are able to sense the aggregation of misfolded proteins in situations of cellular stress. If the concentration of misfolded protein increases, the quality control system activates the unfolded protein response (UPR), a signalling pathway that increases the folding and clearance capacity of the cell in order to counteract the disequilibrium induced by (partly) damaged or denatured proteins (Ellidson and Bottomley 2004). The UPR decreases protein synthesis and upregulates the synthesis of chaperones, foldases, and components from the proteasome degradation machinery (Ellidson and Bottomley 2004). When the damaged proteins cannot be repaired, the quality control system targets them for degradation in the proteasome in order to prevent sustained damage to the cell (Beedholm et al. 2004; Ciechanover 2012; Rattan 2004; Weissman et al. 2011). Infliction of mild damage doesn't necessarily cause a deleterious effect. The cell usually possesses a buffer capacity to impede that mild damage becomes noticeable and hence will not hamper normal metabolism. Chaperones and proteasomes are in place to cope with mild damage. Only when the damage exceeds the buffer capacity of the quality control system of the cell, a deleterious effect will be noticed which includes irreversible damage and denaturation of proteins, aggregate formation, etc.
GLOSSARY
An explanation for non-specialists of some frequently used terms
Protein folding and misfolding, hydrophobic patches, aggregation and proteotoxicity
A protein obtains its functional shape or conformation during protein folding, the physical process by which a protein folds into its functional three dimensional structure. The three dimensional structure of a protein is essential for its function. Various forms of molecular stress may cause mis-folding or denaturation of the protein in which the normal, native, structure is lost. The functional structure of a protein during its synthesis is usually obtained with the assistance of specific proteins often indicated as ‘chaperones'. During misfolding or stress-induced denaturation often some hydrophobic amino acids which are usually located in the interior, can become exposed on the outer surface of the protein. These hydrophobic patches make a protein particularly ‘sticky’ and prone to combine with other molecules, proteins or cellular structures by forcing the hydrophobic parts/patches away from the water environment. Random binding to other proteins will easily lead to the formation of aggregates. An accumulation of aggregated proteins (like beta-amyloids in Alzheimer disease) can impair the normal metabolic processes leading to deterioration of cellular functions. The cellular damage and toxicity caused by misfolded, denatured and aggregated proteins has been coined ‘proteotoxicity’ by Hightower (1991) in analogy of the term ‘genotoxicity'.
Chaperones
This class of proteins does not only play a crucial role in the folding of proteins, but also in the repair of misfolded, damaged and partly denatured proteins, These proteins are indicated with names like ‘foldases', ‘chaperones’ and stress proteins, which include the ‘heat shock proteins’ (HSPs). Molecular chaperoning is also indicated as accompanying partly folded proteins. As chaperones they have the capacity to bind to folding intermediates (partly folded, misfolded or partly denatured proteins) and prevent interactions with other proteins, which would otherwise lead to irreversible denaturation. The name ‘chaperone’ is derived from the social role an adult used to play in accompanying or supervising young people on social occasions in order to prevent interactions that were socially unacceptable in those days. In molecular biology a chaperone is a protein that assists the non-covalent folding/unfolding in molecular biology and which prevents them to engage in unwanted interactions like aggregate formation.
Proteasomes
These are protein complexes in the cytoplasm and in the nucleus whose main function is to degrade damaged and/or misfolded proteins. The enzymes that carry out the degradation of proteins are called ‘proteases'. Proteasomes in general produce small peptides of about 6–8 amino acids long, which, for purposes of recycling, can be further degraded into single amino acids and re-used in the synthesis of new proteins. Proteasomes are part of a mechanism by which cells regulate the concentration of specific proteins. Damaged and misfolded proteins or proteins that are not needed anymore are tagged for degradation and recycling with a small protein called ‘ubiquitin’; which represents the signal which is recognized by the proteasome. Tagged proteins are degraded by the protease activity in the proteasome.
HORMESIS AND SUGGESTED MECHANISMS
It has for long been proposed that hormesis, the brief exposure to periods of mild stress, which are damaging or detrimental at higher doses, can trigger an adaptive and beneficial effect by enhancing the cells resistance to similar or more severe stress (Calabrese and Baldwin 1997, 2001; Stebbing 2003a, 2003b; Agutter 2008).
Hormesis has been defined as “a stimulatory effect of subinhibitory concentrations of any toxic substance on any organism” (Southam and Ehrlich 1943). Mild oxidative stress, for instance, might have a stimulatory effect (e.g. longevity or increased stress resistance), whereas severe oxidative stress will have disadvantageous or even detrimental consequences for a cell. In a database compiled by Baldwin and Calabrese, 5600 examples of hormetic dose-response relationships have been gathered. These examples were found in the literature via a priori defined criteria (Calabrese and Baldwin 1997; Calabrese and Blain 2005). This database serves on the one hand to assess the frequency of hormesis within toxicological literature, and, on the other hand, to assess the generalizability of hormesis and to identify cases of hormesis (Calabrese and Blain 2005). What is particularly interesting about hormesis is that all the major groups of chemicals and even some physical agents can induce a hormetic effect. This implies that there is no single shared property of the chemicals or physical agents that can be responsible for the stimulatory effect at low doses. It is more likely that not the agent itself, but the biological response to the agent can be held accountable for the effect (Calabrese and Baldwin 2001; Stebbing 2003a, 2003b).
Even though the molecular mechanisms underlying hormesis remain elusive, its description as a dose-response phenomenon has been generally recognized. The hormetic dose-response model can be depicted as an inverted U-shaped or a J-shaped curve, in which cellular homeostasis becomes disrupted by a mild stressor (temperature, chemical, exercise or dietary restriction) leading to the onset of downstream molecular pathways that detect the damage and recruit factors involved in repairing the damage leading to reinstating cellular homeostasis or achieving allostasis with a new setpoint for optimal functioning. This process is adaptive in nature and confers a beneficial effect for the cell or organism as it bestows enhanced resistance to stress (Calabrese and Baldwin 2002; Calabrese et al. 2007; Mattson 2008a, 2008b, Stebbing 2003a, 2003b, 2009).
One theory that has been proposed assumes that mild stress inflicted on an organism results in a disturbance in homeostasis (Calabrese and Baldwin 2002, Stebbing 2003b). In response, the over-expression of genes is triggered that will support the development of stress-resistance at the molecular and cellular level, reestablishing the cells capacity to deal with stress and contributing to an explanation of the phenomenon of hormesis (Calabrese and Baldwin 2000; Calabrese et al. 2010; Mattson 2008a; Rattan 2008, 2012; van Wijk et al. 1994). These stress resistance genes include HSPs, chaperones and anti-oxidant enzymes. Moreover, it has been suggested that in its attempt to restore homeostasis the cell over-activates maintenance and repair mechanism, leading to a so-called over-correction, which can account for the benefits of hormesis (Stebbing 2003a, 2003b). Because of this over-activation of repair mechanisms, not only the damage inflicted by mild stress but also other, previously unnoticed damage or gradually accumulated damage might be removed (Sørensen et al. 2005). Hormesis is an adaptive response to low levels of stress or damage resulting in improved fitness for some physiological systems for a finite period. The duration of enhanced resistance is dependent on the time period of the enhanced expression of stress resistance genes, as well as on the half life of proteins responsible for stress resistance and quality control. In more specific terms, hormesis is defined as a modest overcompensation to a disruption in homeostasis. Therefore, it has been proposed that the key conceptual features of hormesis are the disruption of homeostasis, activation of corrective cellular pathways that lead to modest overcompensation, the reestablishment of homeostasis and the adaptive nature of the process upon future exposure to stressors (Calabrese and Baldwin 2002; Stebbing 2003b). The various mechanisms involved in detection and repair of damage are increasingly indicated with the overall term ‘cellular quality control'. Since hormesis is a response to mild stressors and the cell responds to stressful conditions by activating the ‘cellular quality control’ systems, it seems plausible to suggest that the signaling pathways activated by the ‘quality control systems’ represent the molecular mechanisms underlying the beneficial effects of hormesis.
According to this model, exposure to mild stress disturbs cellular homeostasis. In response, the cell strives to normalize the situation by up-regulating its defense, maintenance and repair mechanisms. An optimal response of the cell would be the induction of the quality control mechanisms in such way that there is enough of an up-regulation to counter exactly the amount of damage inflicted. However, it can be hypothesized that the quality control systems are not sensitive enough to determine the precise extent of the response, leading to an over-activation of repair mechanisms in order to deal with the mild-damage. Because of this over-activation of repair mechanisms, not only the damage inflicted by the hormetic substance but also other previously unnoticed damage will be removed. Furthermore, quality control mechanisms are not immediately inactivated once homeostasis has been restored. The over-activated repair mechanisms can more adequately deal with future and more severe forms of damage by either protecting the cell or by increasing the rate of repair of the aberrant molecules. This hypothesis explains why mild damage caused by one form of stress can protect organisms against subsequent forms of stress, since components of the quality control system are over-active conferring the cell with an increased capacity to deal with molecular damage that is generally inflicted by a variety of stress conditions (Sørensen et al. 2005; Stebbing 2003b).
This paper expands on the search for a molecular basis for hormesis. In the quest to explain the molecular mechanisms underlying hormesis, the following questions need to be addressed. What becomes disrupted upon exposure to stressors? How does the cell detect and respond to such disequilibrium? What molecular mechanisms underlie this response and how are they initiated and regulated? What mechanisms are involved in establishing a buffer capacity, reflected as cellular stress resistance? Moreover, this paper proposes the cellular quality control system as the molecular mechanism underlying the beneficial effects observed after exposure to mild stress. In order to elaborate on the molecular basis of hormesis, this paper will review the molecular pathways involved in recognition, repair and prevention of cellular stress, how these mechanisms are activated upon exposure to mild stress and how they can account for the benefits observed in hormesis. As an example this paper will focus mainly on protein quality control. The following section explores how the protein quality control system may represent the molecular mechanism for the beneficial outcome of hormesis. It is currently widely accepted that under normal and stressed conditions the protein quality control is responsible for the repair and degradation of proteins in order to assure cell viability. Moreover, it appears that exposure to mild stress increases the sensitivity of the protein quality control system by over-expressing molecules involved in protein detection, folding, repair and destruction. Consequently, further understanding on the effects on mild stress on the signaling pathways involved in the protein quality control system might aid to explain the importance of hormesis in the maintenance of cellular viability.
PROTEIN QUALITY CONTROL SYSTEMS
The attainment of a biologically active conformation is essential for proteins to perform their proper function. Nascent proteins interact with molecular chaperones and foldases present in the cytoplasm and in the endoplasmic reticulum, which assist the folding of the polypeptide which is being synthesized into its three dimensional conformation and prevent its aggregation (Schröder and Kaufman 2005). The cell is continuously exposed to natural stressors that can alter cellular homeostasis by hampering the folding capacity of the cell, leading to proteotoxicity (Hightower 1991; Morimoto 2008). Disruption in calcium homeostasis, enhanced levels of ROS, overexpression or mutations of certain genes can jeopardize the assembly of nascent proteins or denature mature folded proteins. Such aberrant proteins will aggregate via their surface exposed hydrophobic patches giving rise to aggregates that will accumulate within the cell triggering severe damage and disease (Bukau et al. 2006; Morimoto 2008). Consequently, stress response networks that detect, monitor, recruit and respond to hostile environmental changes are crucial for the viability of the cell. Different stress response networks within the cell have been identified that prevent protein aggregation (Table 1). In the endoplasmic reticulum (ER) two mechanisms involved in protein quality control have been identified, which are indicated as the ‘unfolded protein response’ (UPR) and the ‘endoplasmic reticulum associated degradation’ (ERAD). These two mechanisms together assure detection and clearance of misfolded protein within the ER in order to allow maintenance of ER homeostasis and counteract stress induced by protein aggregation in the ER (Chakrabarti et al. 2011; Guerriero and Brodsky 2012; Kim et al. 2008; Lin et al. 2008). The ERAD targets and translocates misfolded proteins unable to achieve their native conformation from the ER lumen to the cytoplasm where they are degraded via proteasome-mediated proteolysis (Yoshida 2007). If ER homeostasis is not restored, persistent aggregation of aberrant proteins leads to a state of chronic stress and the cell undergoes apoptosis (Guerriero and Brodsky 2012). In the cytoplasm and the nucleus, the heat shock response (HSR) upregulates chaperones and proteasomes upon protein aggregation (van Anken and Braakman 2005; Kim et al. 2008). In this paper a main focus is on the details of HSR regulation.
Elements involved in Protein Quality Control
The Heat Shock Response (HSR)
Under stressful situations for the cell that lead to the aggregation of toxic misfolded proteins in the cytoplasm or the nucleus, the heat shock response (HSR) initiates a signal transduction pathway that will lead to an increased expression, activation and nuclear translocation of heat shock proteins (HSP) (Rattan et al. 2004; Vabulas et al. 2010). Upon protein aggregation, HSPs serve as molecular chaperones involved in the regulation of protein refolding and clearance of damaged polypeptides that cannot achieve their native conformation (Gabai and Sherman 2002). An optimal HSR is essential for cell viability, since rapid induction of HSPs upon cellular stress will enhance the ability of the cell to refold non-native proteins, prevent proteotoxicity and overcome the harmful stress assault (Freedman and Morimoto 1996; Schumacher et al. 1996; van Wijk and Wiegant 2006).
Activation of HSPs
In response to hostile stress conditions, expression and activation of HSPs such as HSP72, HSP27, HSP40 and HSP90 is mediated by heat shock transcription factors (HSFs) (Gabai and Sherman 2002; Sandqvist et al. 2009). In unstressed cells, monomeric HSF1 is located in the cytoplasm and in the nucleus. Due to its association with HSPs, HSF1 is maintained as an inactive non-DNA binding complex with no transcriptional activity. It is widely assumed that the suppression of HSF1 is achieved by binding to the molecular chaperones HSP70 and HSP90 (Shi et al. 1998; Zou et al. 1998). These HSPs interact with the transactivation domain of HSF1 and form a stable complex that maintains the HSF1 monomeric conformation and prevents its trimerization and subsequent activation (Dai et al. 2003). Upon stress, an imbalance between protein synthesis and secretions leads to the accumulation of misfolded proteins. Increased levels of aberrant proteins recruit HSPs via their exposed hydrophobic patches to assist their refolding. Consequently, HSF1 dissociates from HSP70 and HSP90. Dissociated HSF1 can undergo transition from inactive monomer to a nuclear localized, phosphorylated and activated trimer. Active HSF1 is a transcription factor capable of binding to high-affinity Heat Shock Element (HSE) sequences to enhance the expression of HSP genes, further increasing the cells ability to cope with stress (Guo et al. 2001; Anckar and Sistonen 2007). The enhanced synthesis of HSPs serves as a negative feedback loop, as their increase decreases the presence of unfolded proteins after which the HSPs can bind HSF1 again and the cell returns to an unstressed state in which misfolded and damaged proteins have been repaired (Santoro 2000).
It has been suggested that HSPs are not the only modulators of HSF1 activation. Co-chaperone C-terminus of HSP70-interacting protein (CHIP) can act upon HSF1 as well. Although the exact mechanisms remain elusive, it has been proven that CHIP can either directly or indirectly activate HSF1 leading to an increased concentration of chaperones within the cell (Voellmy and Boellman 2007). It has been suggested that upon heat shock, increasing levels of CHIP bind with chaperones, disturbing the stable complex that represses HSF1 activation. The released HSF1 can then form an HSF1-CHIP heterocomplex that can bind to DNA and activate the transcription of HSP genes (Kim et al. 2005; Dai et al.2003).
Under conditions of cellular stress, HSPs have been shown to assist protein refolding, protect cellular systems against damage, dissolve protein aggregates, target irreversibly damaged proteins for degradation and interfere with the apoptotic program (Söti and Csermely 2000). In mammals, different HSP families have been identified with different mode of action and intracellular location. Members from the HSP60 and HSP70 families, helped by co-chaperones, bind to incorrectly folded proteins in the cytosol, mitochondria, and ER and provide an optimal environment for the protein to achieve its native conformation. The protein binding and release in both HSP60 and HSP70 is driven by ATP-hydrolysis, yet the mechanism differs among the chaperones. The HSP60 contains a central cavity ring in which proteins can fold or refold, in an ATP dependent manner, secluded from the environment. HSP60 interacts with the co-chaperone HSP10, which enhances its ability to interact with misfolded proteins, displace them into its cavity and release them once proteins had achieved their final conformation (Hartl and Hayer-Hartl 2002). HSP70 assists protein folding in an ATP-dependent manner by sequestering non-polar regions of the polypeptide, which could potentially aggregate and impede protein folding. The functional cycle of HSP70 is governed by energy dependent conformational changes that regulate substrate binding and release. When HSP70 is bound to ATP, the chaperone exhibits low affinity towards the substrates (Schröder and Kaufman 2005). Upon aggregation of aberrant proteins, co-chaperone HSP40 interacts with HSP70 and accelerates the ATPase activity triggering the hydrolysis of ATP into ADP. ADP-bound HSP70 undergoes a conformational change that increases its affinity for the misfolded proteins, which are then trapped in the chaperone. Then, the exchange of ADP for ATP triggers substrate release (Hartl and Hayer-Hartl 2002; Mayer and Bakau 2005; Marcinowski et al. 2011; Hartl et al. 2011).
Regulation of the HSR
As stated above, the main mechanism of induction of HSPs is through HSF1- mediated activation of transcription of HSPs genes. When aberrant proteins aggregate, they trigger the dissociation of HSP70 and HSP90 from a complex with HSF1, thus activating the latter. However, the activity of HSF1 can also be modulated via reversible phosphorylation at multiple sites. Several kinases activated by the HSR, are involved in the regulation of HSF1. For example, it has been suggested that activation of Akt, a protein kinase, increases HSF1 activity via the inhibition of glycogen synthase kinase-3, a negative regulator of HSF1 (Gabai and Sherman 2002). Moreover, activation of JNK after exposure to prolonged stress seems to decrease HSF1 activity by phosphorylation at distinct sites. Upon exposure to high doses of stress, the burden of aberrant proteins is so large that JNK pathway prevails, triggering apoptosis. However, if the stress is mild, activation of ERK and p38 MAP-Kinase induces the activation of HSF1 that, in turn, enhances HSPs activity leading to cell survival and increased tolerance to stress. It has been suggested that the reason why mild stress enhances survival is because exposure to moderate stress only affects newly synthesized polypeptides, accordingly activating HSF1 and HSPs moderately. Thus, the favorable outcome of low doses of stress may be explained by the detection of elevated levels of proteins that expose hydrophobic patches on their surfaces, inducing regulatory tran-scriptional factors to recruit more HSPs that play a crucial role in repair (Parsell and Lindquist 1993; Putics et al. 2008; Park et al. 2005; Gabai and Sherman 2002). Therefore, stress-activated signaling pathways seem to be involved in the regulation of HSP transcription and, consequently might be important factors in the regulation of balance between cell death and survival.
Protein degradation
When the cell is unable to repair damaged proteins, they are targeted for destruction. The majority of proteins are degraded by proteasome-mediated proteolysis, while the rest are degraded in the lysosomes or by certain HSPs with protease activity (Beedholm et al. 2004; Ciechanover 2012; Schwartz and Ciechanover 2009). After processing, the substrate proteins are cleaved into peptides consisting of approximately 3 to 20 amino acids, which can then be further degraded by downstream amino-peptidases and endo-peptidases (Mikecz 2006; Schwartz and Ciechanover 2009). The proteasome consists of several subunits, which can combine to form different complexes. The central processing unit of the proteasome, the 20S proteasome, can associate with the 11S activator or the 19S regulator. The 11S activator is capable of stimulating peptidase activity of the 20S proteasome, and has been implied to function as an adapter molecule between the 20S proteasome and cytosolic chaperones (Ciechanover 2012). The 20S proteasome can degrade oxidized proteins in an ATP-dependent manner. The 20S proteasome can also associate with the 19S regulator to form the 26S proteasome complex, capable of degrading proteins in an ATP-dependent manner.
In order to be degraded, misfolded proteins must be recognized and delivered to the proteasomes. Misfolded proteins present within the ER must translocate across the ER membrane to the cytosol before being targeted for degradation (Tsai and Weissman 2011; Guerriero and Brodsky 2012). Once the aberrant proteins are present in the cytosol, ubiquitylation, the covalent modification of proteins with ubiquitin chains occurs as an essential step in the targeting of aberrant proteins for proteasomal degradation (Vucic et al. 2011; Ciechanover 2012).
Ubiquitylation takes place in a three-step amplified cascade of reactions (Tsai and Weissman 2011; Weissman et al. 2011). During the ATP-dependent activation reaction, a stable thioester linkage is formed between the carboxyl terminus of ubiquitin and a cysteine residue of an ubiquitin-activating enzyme E1 enzyme. The activated ubiquitin will then be transferred in a conjugating reaction to an ubiquitin-conjugating enzyme (E2), which will carry the ubiquitin to one of the ubiquitin ligases (E3). Finally, E3 enzymes will catalyze the covalent ligation of ubiquitin to its specific substrates through stable isopeptide bonds between the carboxyl group of ubiquitin and the ∊-amino group of lysine residues (Vucic et al. 2011; Weissman et al. 2011). A polyubiquitin chain can be formed through the repetition of these ubiquitin activating, conjugating and ligating reactions. Then, the 19S regulator of the proteasome stimulates ubiquiting-dependent degradation. Proteins marked with a polyubiquiting chain can be recognized by the S5a subunit of the 19S particle of the proteasome, and can be degraded in an ATP-dependent manner (Ciechanover 2012).
Besides regulating HSF1 activation, CHIP has been identified to stimulate the ubiquitin-proteasome pathway. CHIP contains a conserved U-box domain via which proteins bound to chaperones can cooperate with E1, E2 and E3 enzymes in order to be ubiquitylated and targeted for degradation (Beere 2004; Hatakeyama et al. 2001; Connell et al. 2001). Moreover, CHIP exhibits E3 ligase activity itself and can selectively target abnormal proteins for degradation in the proteasomes. Because CHIP can interact with HSP70, this co-chaperone could provide the missing link between failure of repair mechanisms and, as consequence, elimination of the substrate. This connection between the repair and eliminations machinery assures cell viability: if the folding does not succeed, the denatured substrates associated with chaperones for a prolonged period of time will be degraded (Goldberg 2003). By steering peptides towards degradation, CHIP provides a means to dispose damaged proteins towards degradation (Dai et al. 2003). Moreover, CHIP's dual ability to simultaneously activate HSF1 and the protein degradation pathway suggests that this co-chaperone plays an important role in determining whether the predominant action of the quality control system results in protection or degradation (McClellan and Frydman 2001; Ravid and Hochstrasser 2008).
PROTEIN QUALITY CONTROL AND HORMESIS
Mild stress that causes minor cellular damage activates HSFs leading to the recruitment and production of extra HSPs that confers increased stress-resistance to the cell, which enhances cell viability and lifespan (Landry et al. 1982, Laszlo 1988, van Wijk et al. 1994; Wiegant et al. 2008, 2009; Frenkel and Wiegant 2011). Exposure to mild stress is suggested to activate the quality control system, which induces the up regulation of HSPs in moderation leading to increased tolerance to future stress and accounting for the benefits of hormesis. Increased resistance against subsequent, more severe stress is caused by an increased concentration of chaperones after the initial exposure to mild stress. Enhanced resistance even after homeostasis has been reinstated can be explained because chaperones will remain functional in the cell as long as their half-life permits them. Consequently, by activating the HSR through mild stress the cell exhibits an increased capacity to sense and clear damaged proteins leading to increased viability of cells and organisms (Morley and Morimoto 2004; Wiegant et al. 2009). In this respect, it has been shown that repeated mild heat shock (RMHS) treatment increase the basal level of a number of HSPs (Rattan 1998; Verbeke et al. 2001, 2002; Fonager et al. 2002). A significant increase in the basal level of HSP27 and HSP70 throughout aging was observed in RMHS treated cells, which was linked to an improved functionality and survival of the cells. In this respect, HSP27 has also been shown to offer enhanced resistance to harmful cytotoxic damage induced by heat shock and oxidative stress (Arrigo 2007). The augmented basal levels of HSPs is suggestive of an adaptive response of the RMHS exposed cells to increasing intracellular stress in the course of aging. RMHS treated cells thus reveal hormesis-like effects. The enhanced expression of HSPs under these conditions may explain an increased ability to cope with oxidative stress (Rattan 1998; Verbeke et al. 2001, 2002; Fonager et al. 2002). Further research should aim to elucidate whether an increase in tolerance results from a higher capacity or a higher need for cellular chaperones. The co-chaperone CHIP could also be an important player in the hormetic response to mild stress. CHIP is known to activate cytoprotective and antiapoptotic programs in stress situations. The significance of CHIP is well illustrated in CHIP deficient mice. Although these mice develop normally, they exhibit a decreased ability to initiate a proper response to stress. This implies that CHIP might be a mediator in the activation of the hormetic responses (Dai et al. 2003).
The damage-eliminating side of the quality control system, the proteasome-mediated degradation of denatured proteins, is also believed to be stimulated in the hormetic response in order to allow the cell to cope with increased amounts of damaged proteins. As a result, the proteasome may recognize and degrade not only the damaged proteins that resulted from the mild stress, but may also clear dysfunctional proteins that were already present before the stress exposure. This will obviously explain a net beneficial effect of mild stress following recovery. Results that confirm this hypothesis show that proteosomal activity is enhanced after exposure to mild stress. For instance, an increase has been observed in the expression of ubiquitin, which is necessary for targeting proteins for degradation (Kimura et al. 2009). Moreover, exposure to RMHS has been shown to up regulate the 20S and 11S proteasomal subunits contributing to an increased degradation capacity (Beedholm et al. 2004). Finally, it has been suggested that some HSPs synthesized in response to stress can enhance proteosomal degradation by accelerating the peptidase activity of the 20S proteasome when the 11S activator is bound to it, since the 11S activator acts as an adaptor molecule between the HSP and the proteasome (Beedholm et al. 2004).
CONCLUSION
The search for an underlying molecular mechanism that can explain the phenomenon of hormesis is ongoing. This review has proposed that the cellular quality control systems, activated when the cell is exposed to stress, could represent the underlying molecular mechanisms that account for the benefits of hormesis. Cellular quality control refers to the collective mechanisms aimed at preventing, detecting and repairing molecular damage. This paper has focused on the mechanisms involved in protein quality control. It has been described that mild stress enhances chaperone action in protein housekeeping and detection of protein damage by activating signaling pathways that lead to increased recruitment of HSPs. Accumulation of misfolded proteins within the cell or exposure to other stressors that disturb homeostasis triggers the activation of the UPR (unfolded protein response), the ERAD (the endoplasmic reticulum associated degradation) and the HSR (heat shock response). It has been observed that activation of the HSR after exposure to mild stress leads to elevated levels of HSPs, which decrease the burden of aberrant proteins by allowing proteins to refold to their native conformation. Furthermore, after exposure to mild stress, cells exhibit increased concentration of components from the ubiquitin-proteasome system and, thus have enhanced capacity to degrade proteins that cannot be refolded.
It may be stated that regulation of HSPs levels, overexpression of components of the ubiquitin-proteasome system are a credible molecular foundation for the beneficial effects of hormesis. The various elements of cellular quality control mechanisms may form a theoretical basis to explain the phenomenon of hormesis via underlying molecular mechanisms. In order to support this hypothesis more firmly, specific studies need to be designed that explores the role of the quality control system in the generation of beneficial effects in response to mild stress. In case these studies support this hypothesis, and clarify the mechanisms of hormesis, the application of hormesis in various fields of science would be more generally accepted. In addition, an improved knowledge with respect to hormesis could increase the current understanding of cellular quality control mechanisms. Additional research intended to unravel the molecular basis of hormesis is therefore of significant importance.