Investigating the Role of Hsp70 in Clearing out Damaged Proteins
Heat shock proteins such as the Hsp70 family are involved in the housekeeping processes that keep cells functioning well by destroying damaged proteins. They become active in response to stresses that cause a higher rate of damage to the protein machinery within cells, such as toxicity or heat - and hence the name. Many of the genetic alterations and other interventions shown to modestly slow aging in short-lived laboratory animals involve increased cellular maintenance in one way or another, so there is some interest in the research community in building therapies to artificially increase such maintenance activities. So far this hasn't resulted in useful approaches, however, and thus the only reliable way to improve these matters in your own life is still to exercise and practice calorie restriction - increased cellular maintenance is one of the ways in which these lifestyle choices make a difference to long-term health. There is no doubt value in going beyond this to seek much greater increases in cellular maintenance through medical science, but the large sunk costs and lack of results so far suggests that other, more direct means of repairing important forms of cell and tissue damage will be cheaper and more effective. Here I'm thinking of the SENS research proposals; the present state of natural repair processes would be sufficient given the existence of rejuvenation therapies capable of as-needed damage repair of the more critical issues.
One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. Members of the 70 kDa-heat shock protein (Hsp70) family are, in their function as molecular chaperones, involved in folding of newly synthesized proteins and refolding of damaged or misfolded proteins, as well as in assembly and disassembly of protein complexes. The role of Hsp70 in protection against oxidative stress-related damage has been widely accepted. However, to our knowledge, a possible function of Hsp70 in promoting the removal of oxidized proteins has not been investigated. In the current study, we are able to demonstrate not only the involvement of Hsp70 in protection against the oxidative stress-related accumulation of oxidized proteins, but also in their proteasomal degradation.
Hsp70 knockdown and prevention of Hsp70 induction during stress resulted in significantly increased levels of protein carbonyls after hydrogen peroxide treatment. Although heat shock proteins can refold mildly disordered proteins, it is clear that heat shock proteins are not able to repair covalently-modified oxidized proteins nor to reverse oxidative protein modifications. Thus, we suggested that Hsp70 must somehow be implicated in the removal of oxidized proteins. Moreover, Hsc70 deficiency did not lead to changes in protein carbonyl levels and, therefore, Hsc70 seems not to have a major role in this process. Albeit, Hsp70 and Hsc70 have a quite similar structure, it appears that their participation in (oxidative)-stress induced protein degradation is different. It is postulated that both proteins differ in their C-terminal regions, which may result in different cellular functions. Hsc70 is an important housekeeping protein, mostly responsible for the folding of newly synthesized proteins and involved in maintaining protein homeostasis in non-stressed conditions. In contrast, Hsp70 is mainly responsible for a rapidly inducible cell protection following stress situations. Relating to this, we have shown that Hsc70 expression is not affected by oxidative stress, while Hsp70 expression is induced about two-fold in our cellular model, which is comparable to results obtained in other cell lines. Since oxidative damage to proteins leads to their unfolding, the 'heat shock response' is activated and the expression of molecular chaperones is increased.
We demonstrated the ability of Hsp70 to bind oxidized proteins in vitro, as well as in our cell model and in vivo. Interestingly, these oxidized proteins bound to Hsp70 did not show a higher polyubiquitination, which further supports the widely accepted assumption that oxidized proteins are degraded by the 20S proteasome in an ubiquitin-independent way. It has been demonstrated that oxidized proteins are not preferentially ubiquitinated and that an intact ubiquitination system is not required for their degradation. Using various techniques, we demonstrated that Hsp70 interacts additionally with the 20S proteasome, confirming our hypothesis that Hsp70 seems to mediate the interaction between oxidized proteins and the 20S proteasome.
Taken together, the results presented in our current study demonstrate the involvement of the stress-inducible molecular chaperone Hsp70 in the 20S proteasomal degradation of oxidized proteins. We suggest that in the early phase after oxidative stress, Hsp70 binds to partially unfolded oxidized proteins and keeps them in a soluble, degradable form. Oxidized proteins bound to Hsp70 can then migrate to 20S proteasomes where they can be efficiently degraded. Thus, besides the direct recognition of oxidized protein substrates by the 20S proteasome, there seems to be another, Hsp70-mediated, way to catalyze the efficient degradation of oxidized proteins. Future studies should investigate the involvement of co-chaperones/interacting proteins and co-factors which may be involved in this process and which modulate the ability of Hsp70 to mediate shuttling of oxidized proteins to the 20S proteasome. Moreover possible interaction sites of Hsp70 on 20S proteasome subunits remain to be identified. Furthermore, there is increasing evidence that the stress-related inducibility of Hsp70 expression declines in aged cell models and organisms and that the chaperones are overloaded in aged cells due to increasing formation and accumulation of oxidized proteins and. Thus, modulating Hsp70 levels may be a possible pharmaceutical goal to maintain protein homeostasis and prevent the formation of toxic protein aggregates that can disrupt cellular function.