First, an E1 ubiquitin-activating enzyme binds to inactive Ub in eukaryotic cells via a thioester bond and mobilises it in an ATP-dependent process.

Like other members of the RING-between-RING (RBR) family of E3 ligases, parkin possesses two RING finger domains and an in-between-RING (IBR) region.

Addition of a charged phosphate destabilises hydrophobic interactions between Ubl and neighbouring subregions, reducing autoinhibitory effects of this N-terminus domain.

[8] Parkin substrates include mitofusins Mfn1 and Mfn2, which are large GTPases that promote mitochondria fusion into dynamic, tubular complexes that maximise efficiency of oxidative phosphorylation.

During mitophagy, parkin targets VDAC1, a voltage-gated anion channel that undergoes a conformational change upon mitochondrial membrane depolarisation, exposing a cytosolic domain for ubiquitination.

[17] Silencing of VDAC1 expression in HeLa cells significantly reduced parkin recruitment to depolarised mitochondria and their subsequent clearance,[23] highlighting the critical role of VDAC1 as a selective marker of mitochondrial damage and instigator of mitophagy.

HOIP triggers assembly of linear Ub polymers on NF-κB essential modulator (NEMO), potentiating transcription of mitochondrial GTPase OPA1.

[27] Parkin further elevates cytosolic glutathione levels and protects against oxidative stress, characterising it as a critical tumour suppressor with anti-glycolytic and antioxidant capabilities.

In humans, loss-of-function mutations in parkin PARK2 gene have been implicated in 50% of inherited and 15% of juvenile-onset sporadic forms of Parkinson's disease (PD).

[9] While mitochondria are essential for ATP generation in any eukaryotic cell, catecholaminergic neurons are particularly reliant on their proper function for clearance of reactive oxygen species produced by dopamine metabolism, and to supply high energy requirements of catecholamine synthesis.

[17] Their susceptibility to oxidative damage and metabolic stress render catecholaminergic neurons vulnerable to neurotoxicity associated with aberrant regulation of mitochondrial activity, as is postulated to occur in both inherited and idiopathic PD.

For example, enhanced oxidative stress in neurons, skeletal muscle and platelets, corresponding with reduced activity of complex I in the electron transport chain were reported in PD patients,[31] while deletions in the mitochondrial genome were found in the SNpc.

[8] Such mutations may be hereditary or stochastic and are associated with structural instability, reduced catalytic efficiency and aberrant substrate binding and ubiquitination.

Protein aggregation triggers neuronal toxicity, whilst accounting for lack of ubiquitinated Lewy bodies in parkin-mutant PD.

Similarly, native parkin reduces death of SH-SY5Y neurons by ubiquitinating other Lewy body constituents, such as the p38 subunit of aminoacyl-tRNA synthetase complex[36] and far upstream element-binding protein 1[37] through addition of Lys48-linked poly-Ub chains and directing them towards proteasomal degradation.

Parkin also influences axonal transport and vesicle fusion through ubiquitination of tubulin and synaptotagmin XI (SYT11) respectively, giving it a modulatory role in synapse function.

[10] Similarly, chromosomal breaks in PARK2 suppressed expression of afadin scaffold protein in breast cancer, thereby comprising epithelial integrity, enhancing metastatic potential and worsening overall prognosis.

[40] Haploinsufficient PARK2 expression, either due to reduced copy number or DNA hypermethylation, was further detected in spontaneous colorectal cancer where it accelerated all stages of intestinal adenoma development in mouse models.