Ubiquitination, is an irreversible process once the target

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. 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) 2 typically comprised of alpha and beta subunits, completing
the degradation of the unfolded protein 1. However, ubiquitination is
potentially reversible prior to this step.

Initiation of the mechanism occurs through activation
of ubiquitin in an ATP dependent manner by E1 3. A thioester bond results
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, forms a complex with E2 through an
isopeptide bond, facilitating the transfer of ubiquitin to the substrate
protein. Formation of this isopeptide bond 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

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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.
The ubiquitin forms an intermediate bond with the catalytic cysteine on E3
prior to ubiquitin 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 catalysis (C-terminus) and specificity of the substrate
(N-terminus). 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 ubiquitin charged E2 molecule and the second containing the
catalytic cysteine.



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