Protein degradation is a highly regulated process that removes damaged, misfolded, or unneeded proteins, maintaining cellular homeostasis and controlling the levels of regulatory proteins. Two major systems accomplish intracellular protein degradation: the ubiquitin-proteasome system and the autophagy-lysosome pathway.
The Ubiquitin-Proteasome System
The ubiquitin-proteasome system is the primary pathway for selective protein degradation in eukaryotic cells. Proteins are targeted for degradation by covalent attachment of ubiquitin, a 76-amino acid protein, through an isopeptide bond between the C-terminal glycine of ubiquitin and a lysine residue on the target protein. Ubiquitination is reversible, and deubiquitinating enzymes remove ubiquitin from substrates.
Ubiquitination Cascade
Ubiquitination requires three enzymes acting sequentially. Ubiquitin-activating enzyme uses ATP to form a high-energy thioester bond between its active site cysteine and the C-terminus of ubiquitin. The activated ubiquitin is transferred to a ubiquitin-conjugating enzyme. A ubiquitin ligase then catalyzes the transfer of ubiquitin from the E2 to the target protein, forming an isopeptide bond. The human genome encodes about 35 E2 enzymes and over 600 E3 ligases, providing enormous substrate specificity.
Processive ubiquitination adds additional ubiquitin molecules to lysine 48 of the previous ubiquitin, forming a polyubiquitin chain. Chains of at least four ubiquitins linked through lysine 48 are the canonical signal for proteasomal degradation. Other linkage types, such as lysine 63 chains, signal for non-proteolytic functions including DNA repair and cell signaling.
The Proteasome
The 26S proteasome is a large protease complex consisting of a 20S core particle capped by one or two 19S regulatory particles. The 20S core is a barrel-shaped structure of four stacked rings, with the outer alpha rings forming a gated channel and the inner beta rings containing proteolytic active sites. Three types of catalytic subunits have chymotrypsin-like, trypsin-like, and caspase-like activities, cleaving after hydrophobic, basic, and acidic residues respectively.
The 19S regulatory particle recognizes ubiquitinated proteins, removes the ubiquitin chain through deubiquitinating enzymes, unfolds the substrate, and translocates it through the narrow channel into the 20S core. ATP hydrolysis by the regulatory particle’s ATPase subunits powers these processes. The proteasome degrades proteins into peptides of 7 to 9 amino acids, which are then further degraded by cytosolic peptidases or used for antigen presentation.
Autophagy
Autophagy delivers cytoplasmic material to the lysosome for degradation. In macroautophagy, a double-membrane structure called the phagophore elongates and engulfs a portion of cytoplasm, forming an autophagosome that fuses with the lysosome. The contents are degraded by lysosomal hydrolases, and the resulting amino acids and other metabolites are released back into the cytosol.
Autophagy is regulated by the mTOR signaling pathway, which inhibits autophagy when nutrients are abundant. Starvation, growth factor withdrawal, and cellular stress activate autophagy. Autophagy is essential for quality control, removing damaged organelles and protein aggregates. Defective autophagy contributes to neurodegenerative diseases, cancer, and aging.
The Ubiquitin-Proteasome System in Disease
The UPS is implicated in many diseases. In cancer, E3 ligases such as MDM2 that regulate p53 are often dysregulated, and components of the SCF complex are mutated in certain malignancies. Proteasome inhibitors such as bortezomib are effective treatments for multiple myeloma. Neurodegenerative diseases feature accumulation of ubiquitinated protein aggregates, suggesting impairment of the UPS. Angelman syndrome is caused by loss of function of the E3 ligase UBE3A.
The N-End Rule
The N-end rule relates the half-life of a protein to the identity of its N-terminal residue. Certain N-terminal amino acids, such as arginine, lysine, and leucine, are destabilizing and target proteins for rapid degradation through recognition by specific E3 ligases. The N-end rule pathway is important for regulating protein quality control, heme sensing, and chromosome segregation. N-terminal residues can be generated by proteolytic cleavage, providing a mechanism for regulated degradation of specific proteins.
