Ubiquitination, a proteasomal degradation process, is basedon covalent attachment of ubiquitin to a substrate lysine on a target protein,marking the protein for its degradation 1-7. This process renewsintracellular proteins balancing the rate of degradation with the rate ofprotein synthesis, resulting in homeostasis 4. Homeostasis is achieved byeliminating damaged proteins which typically result in disease as they competewith functional proteins for binding sites/partners 1. In addition tohomeostasis, the process also regulates cell cycle progression, gene transcription,DNA repair, apoptosis and receptor endocytosis, some of which require lysosomaldegradation 7. However, the UPS (ubiquitin proteasome system) differs fromthe UBL (ubiquitin like system) and from the lysosomal pathway (requiringautophagy for degradation) 3,7. The 76AA ubiquitin molecule contains 7 lysineresidues allowing the formation of isopeptide linked chains or Met1 chains(ubiquitin linked ubiquitin). The predominant linkage being Lys 48 due to itsdegradation role usually allows polyubiquitination to occur, with the Lys 63linkage known for its non-degradation role and subsequent activation ofpathways such as PKB/AKT 6. Following the covalent addition of the ubiquitinchain, the regulatory mechanism involves three enzymes in a cascade of activation,conjugation and ligation resulting in the degradation of the target protein bythe 26s proteasome 7.
Proteasomal degradation is an irreversible process oncethe target protein reaches the proteasome. It is comprised of at least one 20sregulatory particle (RP), for substrate recognition and a 19S hollow coreparticle (CP) 2 typically comprised of alpha and beta subunits, completingthe degradation of the unfolded protein 1. However, ubiquitination ispotentially reversible prior to this step. Initiation of the mechanism occurs through activationof ubiquitin in an ATP dependent manner by E1 3. A thioester bond resultsupon activation between the ubiquitin C terminus and an active cysteine on E1. E2,the ubiquitin-conjugating enzyme, then binds with E1, transferring theubiquitin to E2 at a catalytic cysteine residue 1. The final enzyme involvedin the process, E3, ubiquitin ligase, forms a complex with E2 through anisopeptide bond, facilitating the transfer of ubiquitin to the substrateprotein. Formation of this isopeptide bond occurs at the amino group of lysinein the substrate and the C terminal glycine residue of the ubiquitin molecule (Fig.
1). Considering E3 is the finalenzyme involved in the cascade, it determines specificity of the substrate 3.With a large number of substrates available, a large ligase family must alsoexist (>700 members). The E1 family, which typically lack specificity for E2or E3 only contain 2 members in humans, however the E2 family comprises of 40members as its main role determines which polyubiquitin chains are catalysed byE3.Classification of the E3 ligase family is crucial, impactingthe mechanism in which conjugation to the substrate occurs. Classificationvaries but was observed by FrancescaMorreale, University of Dundee, Scotland as a 3 member family including:RING (Really Interesting New Gene) and U-box (UFD2 homology), RBR (Ringin-Between Ring) and HECT (homologous to E6-associated protein C-terminus), eachwith a varying mechanism of action 5. The most prevalent being, RING, whichact as mediators, directly transferring ubiquitin from E2 to the substrate,never binding with ubiquitin itself but acting as a scaffold ensuring aflexible E2 orientation for the substrate 5. These ligases are comprised of azinc binding domain and possess the ability to act as monomers, homodimers orheterodimers.
Homodimer RING ligases allow the binding of an E2 per monomer,resulting in two E2’s bound. Similarly, U-box ligases contain a RING structurehowever lacking the zinc domain and potentially act as monomers and homodimershowever, their main role involves completing polyubiquitin elongation,previously begun by another ligase. RING ligases are often classified based ontheir multiple subunit composition such as cullin ring ligases (CRL) comprisedof a cullin scaffold or anaphase-promoting complex/cyclosome (APC/C) composedof 19 subunits, including a ring subunit (Apc11) and a cullin-like subunit(Apc2). HECT ligases function by a varying mechanism comprised of two steps.The ubiquitin forms an intermediate bond with the catalytic cysteine on E3prior to ubiquitin transfer to the target protein. This domain, positioned atthe C terminus of proteins contains an N-terminal lobe and C-terminal lobestructure, allowing catalysis (C-terminus) and specificity of the substrate(N-terminus). Subfamilies such as Nedd4 and HERC exist here due to varying Ntermini 3. The final group of ligases, RBR posses the same mechanism of actionas the HECT ligase family however, differ in structure.
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