## 1 Stress Responses and the Chaperoning System
### Hsp60, One of the Most Ancient Anti-Stress Molecules  
Hsp60 belongs to one of the oldest and evolutionarily most conserved protein families of the chaperoning system [19, 20, 21]. These proteins are present in all living species, including plants, where they were first discovered [22, 23, 24], and, considering their unique molecular characteristics, they were named \"chaperonins\" to distinguish them from other chaperones [25]. The canonical classification divides chaperonins into two main groups. Group I chaperonins are found in bacteria, as well as inside eukaryotic organelles of endosymbiotic origin (mitochondria in animal cells, and chloroplasts in plant cells), and work together with a co-chaperonin which helps the closing of the folding cage. Group II chaperonins are found in the eukaryotic cytosol and in Archaea, and do not require a co-chaperonin since they have a built-in lid [26]. More recently, a third group (Group III chaperonins) has been discovered and it is now under characterization [27,28]. In humans, the Group I chaperonin is Hsp60 (or Cpn60, or HSPD1), and the Group II chaperin is CCT (chaperonin-containing TCP-1) or TRiC (T-complex protein Ring Complex). The former typically resides inside mitochondria but also occurs in various other locations intra- and extracellularly, while CCT is in the cytosol. Hsp60 and CCT form macromolecular double-ring complexes with a central internal cavity in which polypeptides in need of assistance for folding or refolding are encapsulated and assisted to achieve their functional final conformation (native state) via an ATP-dependent mechanism [29, 30, 31]. A considerable part of our knowledge about how human chaperonins assemble and work derives from the study of their bacterial homologues. The bacterial counterpart of human Hsp60 is the chaperonin GroEL [32], which, together with the co-chaperonin GroES (the bacterial counterpart of human the Hsp10 or Cpn10), forms a tubular, double-ring complex with a central cavity similar to that described above for the eukaryotic Hsp60 and CCT complexes, inside which protein folding occurs [33,34]. Human Hsp60 has been found in various conformations (monomer, single heptameric ring, double-ring tetradecamer), depending on their concentration, levels of ATP, and presence of Hsp10 and substrate [21, 35, 36, 37]. Early studies showed that human Hsp60 can assist in productive protein folding without forming a macro-double ring-complex [38,39]. However, more recent studies, based on transmission electron microscopy and X-ray crystallographic investigations, have provided strong evidence that it likely uses both double- and single-ring intermediates during its ATPase cycle [40, 41, 42]. Hsp60 has been found in extramitochondrial sites, such as cytoplasm and plasma-cell membrane, as well as in extracellular sites, inside extracellular vesicles (EVs), in circulation, and in body fluids [43, 44, 45, 46, 47, 48, 49]. Consequently, in addition to its canonical chaperoning function, Hsp60 also performs various other non-canonical activities, \"moonlighting functions\" unrelated to protein quality control. (Figure 1) [21, 49, 50, 51]. For instance, it has been observed that increased amounts of Hsp60 on the surface of cancer cells act as a signal to stimulate the immune system, leading to the activation and maturation of dendritic cells and the generation of an antitumor T-cell response [52, 53, 54]. A non-canonical function of human Hsp60 that is still under scrutiny is the regulation of cell apoptosis [55]. Some studies suggested a pro-apoptotic role, involving pro-caspase 3 proteolytic activation [56; 57], whereas other investigations support an anti-apoptotic role, involving various mechanisms: the sequestration of Bax-containing complexes [58; 59], the maintenance of mitochondria integrity and ATP generation [60], and the triggering of the IKK/NF-kB survival pathway [61].