*4.1. Molecular Chaperones for Folding*/*Refolding*

Molecular chaperones are present in all cell organelles and can be categorized as "folding helpers" and "holding types." A folding helper assists in polypeptide folding during translation and partially unfolds misfolded intermediates [59]. The holding type, on the other hand, guards an unfolded or misfolded protein from aggregation and degradation, and presents it to a folding helper [60]. They transiently interact with mutant proteins to protect them from interacting with normal proteins and buy time to refold them into their native conformation [27]. Most mutated proteins show a prolonged interaction with the chaperones compared with their corresponding correctly folded protein, because the chaperones need time to fold them properly [61]. However, the severity of the mutation determines whether the chaperones refold and rescue the proteins or direct them for degradation. Therefore, the ectopic expression of chaperones increases the chance that chaperones will interact with misfolded proteins and restore their active form, as observed in previous studies [62,63]. In other words, chaperones along with other members of PQC, play a decisive role in maintaining a pool of functional mutant proteins.

Molecular chaperones also help certain newly synthesized proteins to fold e fficiently and in a biologically relevant time frame. Chaperones such as HSP70/40 attach to a partially folded polypeptide during synthesis by a ribosome and stabilize it to its correct native conformation [64] (Figure 1). Most proteins fold to a native form in the cytosol and do not need any further assistance from chaperones, but certain proteins always need assistance from chaperones, such as HSP90 and the TCP-1 ring complex (TRiC) [65]. Likewise, chaperones such as HSP70 and HSP90 and some chaperonins (such as HSP60s) assist in the refolding of misfolded proteins through the ATP and co-factor binding and release

cycle. If a misfolded protein cannot be completely refolded, chaperones help target it for degradation to remove the toxic conformation. Co-chaperones such as the Bcl-2-associates-athanogene domain direct the interaction between a molecular chaperone and the protein degradation system [66,67].

**Figure 1.** Protein folding, misfolding, and degradation. Protein folding starts during the ribosomal translation process and attain the native conformation to execute cellular processes. The native folded proteins are often misfolded due to mutations and other environmental factors. Molecular chaperones catalyze the folding/refolding events, disaggregation of the protein aggregates, and targeting the protein for degradation. Aggregates are typically degraded by autophagy, whereas the ubiquitin proteasome system (UPS) degrades the destabilized/misfolded proteins by covalent attachment of a ubiquitin molecule assisted by E1-E2-E3 enzymes. The ubiquitinated proteins are recognized by the 26S proteasome and are degraded. However, the ubiquitin moiety is cleaved off by Deubiquitinating enzymes (DUBs) and the protein can be rescued from the degradation cycle.

Several classes of molecular chaperones are distinguished by molecular weight and mode of action, such as Hsp70, Hsp90, Hsp60, and Hsp40, and bind to the hydrophobic region of a non-native protein [64,68].
