The Removal of Misfolded Proteins

The removal of misfolded proteins from the ER is a consecutive process that is characterized by the interaction between many proteins in both the ER and the cytoplasm (Needham and Brodsky, 2013). To enter the ERAD pathway, the protein must be exported from the ER back into the cytosol in a process known as retro-translocation, also called dislocation. This process of retro-translocation takes place via the same translocon in which proteins used enter the ER initially – the Sec61 complex. This retro-translocation channel present in the ER membrane is made up of several multi-protein complexes (Zhang et al., 2015). Each complex functions around a ubiquitin ligase in the membrane, an enzyme required to recognize the targeted protein. Ubiquitin ligases are constituents of the ubiquitin proteasome system (UPS), a system which plays a critical role in ERAD. It is responsible for searching and destroying any damaged or faulty proteins or those simply surplus to requirements. The UPS uses a protein called ubiquitin to target these faulty proteins in a process called ubiquitination.

The process of ubiquitination is carried out by three different enzymes – ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligases (E3) and uses ATP as a source of energy/ E1 forms a high-energy thioester bond between a cysteine residue present in its active site and the C-terminus of ubiquitin. This activated ubiquitin is then transferred to E2. E3 binds to the misfolded protein and aligns it in such a way that the protein can bind with the ubiquitin that is attached to E2. An isopeptide bond forms between the C-terminal glycine of ubiquitin and lysine residues on the targeted protein. Several cycles of ubiquitination take place, forming a polyubiquitin chain (Nandi et al.). This polyubiquitin chain on the misfolded protein signals for its degradation via the 19S capping complexes of the multi-subunit 26S proteasome. The proteasome binds and removes the polyubiquitin chain, unfolding the protein into smaller peptides. The protein is then re-used for the synthesis of new proteins and the ubiquitin is recycled. Although the 26S proteasome recognizes poly-ubiquitinated proteins, the high specificity and selectivity of the UPS lies in different E3 enzymes, which are capable of recognizing a wide range of substrates (Wang and Maldonado).

In humans hundreds of E3s exists and are distinguished from one another by the presence of different domains - a RING-like (RING) or a HECT domain. RING domains transfer the ubiquitin protein from E2 directly to the targeted substrate while HECT domain E3s transfer the ubiquitin from E2 to E3 and then from E3 to the substrate (Berndsen and Wolberger, 2014). While a wide range of studies have been carried out on E3 enzymes, the mechanisms by which they work and further increase ubiquitin transfer are still to be considered (Berndsen and Wolberger, 2014). Research into how E3 ligases are regulated through interaction with different E2 enzymes and substrates may provide further insight into E3 specificity and reveal more about E3 ligase binding.