Ubiquitination, a
proteasomal degradation process, is based on covalent attachment of ubiquitin
to a substrate lysine on a target protein, marking the protein for its
degradation 1-7. This process renews intracellular proteins balancing the
rate of degradation with the rate of protein synthesis, resulting in
homeostasis 4. Homeostasis is achieved by eliminating damaged proteins which
typically result in disease as they compete with functional proteins for
binding sites/partners 1. In addition to homeostasis, the process also
regulates cell cycle progression, gene transcription, DNA repair, apoptosis and
receptor endocytosis, some of which require lysosomal degradation 7. However,
the UPS (ubiquitin proteasome system) differs from the UBL (ubiquitin like
system) and from the lysosomal pathway (requiring autophagy for degradation) 3,7.
The 76AA ubiquitin molecule contains 7 lysine residues allowing the formation
of isopeptide linked chains or Met1 chains (ubiquitin linked ubiquitin). The
predominant linkage being Lys 48 due to its degradation role usually allows
polyubiquitination to occur, with the Lys 63 linkage known for its
non-degradation role and subsequent activation of pathways such as PKB/AKT 6.
Following the covalent addition of the ubiquitin chain, the regulatory
mechanism involves three enzymes in a cascade of activation, conjugation and
ligation resulting in the degradation of the target protein by the 26s
proteasome 7.

Initiation of the
mechanism which occurs through activation of ubiquitin by E1 is ATP dependent
resulting in the formation of a thioester bond 3. This bond forms upon activation
between the ubiquitin C terminus and an active cysteine on E1. E2, the
ubiquitin-conjugating enzyme, then binds with E1, transferring the ubiquitin to
E2 at a catalytic cysteine residue 1. The final enzyme involved in the
process, E3, ubiquitin ligase, results in the substrate protein obtaining the ubiquitin
by forming a complex with E2 through an isopeptide bond. Bond formation occurs
at the amino group of lysine in the substrate and the C terminal glycine
residue of the ubiquitin molecule (Fig.1).
Considering E3 is the final enzyme involved in the cascade, it determines
specificity of the substrate 3. With a large number of substrates available,
a large ligase family must also exist (>700 members). The E1 family, which
typically lack specificity for E2 or E3 only contain 2 members in humans,
however the E2 family comprises of 40 members as its main role determines which
polyubiquitin chains are catalysed by E3.

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Classification of the
E3 ligase family is crucial, impacting the mechanism in which conjugation to
the substrate occurs. Classification varies but was observed by Francesca Morreale, University of Dundee,
Scotland as a 3 member family including: RING (Really Interesting New Gene)
and U-box (UFD2 homology), RBR (Ring in-Between Ring) and HECT (homologous to
E6-associated protein C-terminus), each with a varying mechanism of action 5.
The most prevalent being, RING, which act as mediators, directly transferring
ubiquitin from E2 to the substrate, never binding with ubiquitin itself but
acting as a scaffold ensuring a flexible E2 orientation for the substrate 5. These
ligases are comprised of a zinc binding domain and possess the ability to act
as monomers, homodimers or heterodimers. Homodimer RING ligases allow the
binding of an E2 per monomer, resulting in two E2’s bound. Similarly, U-box
ligases contain a RING structure however lacking the zinc domain and potentially
act as monomers and homodimers however, their main role involves completing polyubiquitin
elongation, previously begun by another ligase. RING ligases are often
classified based on their multiple subunit composition such as cullin ring
ligases (CRL) comprised of a cullin scaffold or anaphase-promoting
complex/cyclosome (APC/C) composed of 19 subunits, including a ring subunit
(Apc11) and a cullin-like subunit (Apc2). HECT ligases function by a varying
mechanism comprised of two steps. Ubiquitin forms an intermediate bond with the
catalytic cysteine on E3 prior to its transfer to the target protein. This
domain, positioned at the C terminus of proteins contains an N-terminal lobe
and C-terminal lobe structure, allowing specificity of the substrate and catalysis
respectively. Subfamilies such as Nedd4 and HERC exist here due to varying N
termini 3. The final group of ligases, RBR posses the same mechanism of
action as the HECT ligase family however, differ in structure. RBR ligases are
comprised of two RING structures, one which recruits the E2 molecule (ubiquitin
charged) and the second containing the catalytic cysteine. Proteasomal
degradation is an irreversible process once the target protein reaches the
proteasome. It is comprised of at least one 20s regulatory particle (RP), for
substrate recognition and a 19S hollow core particle (CP) typically comprised
of alpha and beta subunits, completing the degradation of the unfolded protein
1,2. However, 

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