Паркин: Митохондриялық дисфункция және Паркинсон ауруымен байланысы

Parkin is a 465 amino acid residue E3 ubiquitin ligase, a protein that in humans and mice is encoded by the PARK2 gene. Parkin plays a critical role in ubiquitination – the process whereby molecules are covalently labelled with ubiquitin (Ub) and directed towards degradation in proteasomes or lysosomes. Ubiquitination involves the sequential action of three enzymes. 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. Ub is then transferred to an E2 ubiquitin conjugating enzyme before being conjugated to the target protein via an E3 ubiquitin ligase. There exists a multitude of E3 ligases, which differ in structure and substrate specificity to allow selective targeting of proteins to intracellular degradation. In particular, parkin recognises proteins on the outer membrane of mitochondria upon cellular insult and mediates the clearance of damaged mitochondria via autophagy and proteasomal mechanisms. Parkin also enhances cell survival by suppressing both mitochondria dependent and independent apoptosis. Mutations are associated with mitochondrial dysfunction, leading to neuronal death in Parkinson's disease and aberrant metabolism in tumourigenesis.

The precise function of parkin is unknown; however, the protein is a component of a multiprotein E3 ubiquitin ligase complex which in turn is part of the ubiquitin proteasome system that mediates the targeting of proteins for degradation. Mutations in this gene are known to cause a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinson's disease (AR JP). Moreover, parkin is described to be necessary for mitophagy (autophagy of mitochondria). However, how loss of function of the parkin protein leads to dopaminergic cell death in this disease is unclear. The prevailing hypothesis is that parkin helps degrade one or more proteins toxic to dopaminergic neurons. Putative substrates of parkin include synphilin 1, CDC rel1, cyclin E, p38 tRNA synthase, Pael R, synaptotagmin XI, sp22 and parkin itself (see also ubiquitin ligase). Additionally, parkin contains a C terminal motif that binds PDZ domains. Parkin has been shown to associate in a PDZ dependent manner with the PDZ domain containing proteins CASK and PICK1. 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. RING1 forms the binding site for E2 Ub conjugating enzyme while RING2 contains the catalytic cysteine residue (Cys431) that cleaves Ub off E2 and transiently binds it to E3 via a thioester bond. Together these form the catalytic triad, whose assembly is required for parkin activation. Parkin also contains an N terminal Ub like domain (Ubl) for specific substrate recognition, a unique RING0 domain and a repressor (REP) region that tonically suppresses ligase activity. Under resting conditions, the tightly coiled conformation of parkin renders it inactive, as access to the catalytic RING2 residue is sterically blocked by RING0, while the E2 binding domain on RING1 is occluded by Ubl and REP. Ser65Ala missense mutations were found to ablate Ub parkin binding whilst inhibiting parkin recruitment to damaged mitochondria. PINK1 also phosphorylates Ub at Ser65, accelerating its discharge from E2 and enhancing its affinity for parkin. Considering that RING0 is unique to parkin and that its hydrophobic interface with RING1 buries Cys431 in inactive parkin, Mitophagy is the elimination of damaged mitochondria in autophagosomes, and is dependent on a positive feedback cycle involving synergistic action of parkin and PINK1. Following severe cellular insult, rundown of mitochondrial membrane potential prevents import of PINK1 into the mitochondrial matrix and causes it to aggregate on the outer mitochondrial membrane (OMM). Parkin is recruited to mitochondria following depolarisation and phosphorylated by PINK1, which simultaneously phosphorylates Ub pre conjugated to mitochondrial membrane proteins. PINK1 and Ub phosphorylation facilitate parkin activation and further assembly of mono and poly Ub chains. However, upon mitochondrial damage, degradation of fusion proteins is necessary to separate them from the network via mitochondrial fission and prevent the corruption of healthy mitochondria. Parkin is therefore required before mitophagy as it ubiquinates Mfn1/2, labelling it for proteasomal degradation. Proteomic studies identified additional OMM proteins as parkin substrates, including fission protein FIS, its adaptor TBC1D15 and translocase TOMM20 and TOMM70 that facilitate movement of proteins such as PINK1 across OMM. Miro (or RHOT1/RHOT2) is an OMM protein critical for axonal transport, and may be ubiquitinated and targeted towards proteasomal degradation by parkin. Miro breakdown produced a marked decrease in migration of compromised mitochondria along axons of mouse hippocampal neurons, reinforcing the importance of parkin in segregating defective mitochondria from their functioning counterparts and limiting the spatial spread of mitochondrial dysfunction, prior to autophagy. 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. highlighting the critical role of VDAC1 as a selective marker of mitochondrial damage and instigator of mitophagy. Following Ub conjugation, parkin recruits autophagy receptors such as p62, TAX1BP1 and CALCOCO2, facilitating assembly of autophagosomes that digest defective mitochondria. Increased OPA1 translation maintains cristae structure and reduces cytochrome C release from mitochondria, inhibiting caspase mediated apoptosis. Importantly, parkin activates HOIP with greater potency than other LUBAC associated factors HOIL 1 and sharpin, meaning that parkin mobilisation significantly enhances tolerance to moderate stressors. Parkin possesses DNA binding affinity and produces a dose dependent reduction in transcription and activity of pro apoptotic factor p53. Transfection of p53 promoter with truncated versions of parkin into SH SY5Y neurons revealed that parkin directly binds to the p53 promoter via its RING1 domain. Conversely, parkin may be a transcriptional target of p53 in H460 lung cells, where it mediates the tumour suppressor action of p53. Parkin further elevates cytosolic glutathione levels and protects against oxidative stress, characterising it as a critical tumour suppressor with anti glycolytic and antioxidant capabilities. Patients with an autosomal recessive family history of parkinsonism are much more likely to carry parkin mutations if age at onset is less than 20 (80% vs. 28% with onset over age 40). Patients with parkin mutations (PARK2) do not have Lewy bodies. Such patients develop a syndrome that closely resembles the sporadic form of PD; however, they tend to develop symptoms at a much younger age. 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). However, its symptoms resembles those of idiopathic PD, with patients presenting with resting tremors, postural instability and bradykinesia. while deletions in the mitochondrial genome were found in the SNpc. In accordance with its critical role in mitochondrial quality control, more than 120 pathogenic, PD inducing mutations have been characterised on parkin. Finally, Cys431Phe and Gly430Asp mutations impair ligase activity at the catalytic site and significantly reduce parkin function. Parkin might promote aggregation of alpha synuclein and synphilin 1 into Lewy bodies, which are conjugated to Lys63 linked poly Ub chains and directed towards autophagic degradation. Parkin mutations therefore inhibit this mechanism, leading to toxic accumulation of soluble proteins that overloads the proteasome. 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 and far upstream element binding protein 1 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. Parkin mutant PD patients also exhibit a four fold elevation in p53 immunoreactivity, Parkin deficiency further diminished disease free survival in infrared irradiated mice without increasing tumour incidence rate, suggesting that parkin deficiencies increase susceptibility to tumour promoting events, rather than initiating tumour formation. 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. Parkin is therefore a potent modulator of tumour progression, without directly instigating tumourigenesis.