# Strategies for Post-Translational Control of Protein Expression and Their Applications
## 1 Introduction
Many techniques used for the post-translational control of protein expression rely on the ubiquitin-proteasome pathway, which achieves selective proteolysis. proteolytic substrates are modified with ubiquitin (Ub), a highly conserved 76-amino-acid polypeptide, which is conjugated to an internal lysine of the substrate through an isopeptide bond to the C-terminal glycine of Ub. This reaction is generally called ubiquitination and occurs sequentially through the three steps with activating (E1), conjugating (E2), and ligase (E3) enzymes (Figure 1a). Ub is initially activated in an ATP-consuming reaction by an E1 enzyme, to which Ub is conjugated by a high-energy thioester bond. Subsequently, the activated Ub is transferred to a cysteine residue of an E2 enzyme, which then catalyzes the transfer of (poly)Ub to the substrate bound to an E3 enzyme. There are two major classes of E3 enzymes, characterized by Really Interesting New Gene (RING) and Homologous to E6-AP Carboxy Terminus (HECT) domains. RING E3 enzymes serve as a hub for the assembly of the E2 enzyme and the substrate, and catalyze the transfer of Ub directly from the E2 enzyme to the substrate. In contrast, HECT E3 enzymes have a distinct function of Ub transfer. The thioester moiety of the complex of activated Ub and the E2 enzyme is reacted with a cysteine residue in the HECT domain of the E3 enzyme, and is finally transferred to the substrate. Seven lysine residues of Ub (K6, K11, K27, K29, K33, K48, and K63) allow the generation of Ub chains via an isopeptide bond between a lysine of one Ub and the C-terminal glycine of another Ub. The ubiquitinated substrate binds to internal Ub receptors in the 19S regulatory complex of the 26S proteasome, which is a multicatalytic protease. The substrate protein bound to the proteasome is unfolded by ATPases and the polyubiquitin chain is removed from the substrate by proteasome-associated deubiquitinating enzymes (DUBs) [19]. The unfolded protein is translocated into the central proteolytic chamber of 20S proteasome, where it is cleaved into short peptides. Certain types of DUBs are resident in the cytosol and act in the removal of the (poly)ubiquitin from substrates, which stabilizes the protein. In contrast to the ubiquitin-proteasome pathway, the autophagy-lysosome pathway is capable of targeting not only proteins, but also cellular organelles [20]. The substrates are enclosed by an isolation membrane (called phagophore) which associate with the microtubule-associated protein light chain 3 (LC3) on their surface (Figure 1b). After its subsequent maturation and closure, the autophagosome is formed. The outer membrane of the autophagosome fuses with the lysosome, and the internal materials are degraded in the autolysosome. Although these processes are called \"bulk\" degradation system, autophagy mediates the selective degradation of substrate proteins and/or organelles through autophagy receptors, such as p62. These receptor proteins contain the LC3-interacting region, which mediates their binding to nascent autophagosomes. The receptors  
Figure 1: Cellular proteolytic pathways. Eukaryotic cells have several protein degradation pathways: (**a**) the ubiquitin–proteasome pathway, (**b**) the autophagy–lysosomal pathway, and (**c**) the endocytosomal pathway. also recognize the ubiquitated substrates through an ubiquitin-binding domain and recruit them to the autophagome, resulting in selective degradation by the lysosomes. Furthermore, there is another kind of degradation machinery of lysosomes that can target extracellular and plasma membrane proteins via endocytosis (Figure 1c). After endocytosis, internalized extracellular and plasma membrane proteins enter the early endosome. The early endosome matures into the late endosome, which then fuses with the lysosome to form an endolysosome, within which the internal materials are degraded. The proteolytic pathways mentioned above have been utilized for various types of technologies of post-translational control of cellular protein expression.