Parkin is a 465-
amino acidresidueE3 ubiquitin ligase, a
protein that in humans and mice is encoded by the PARK2gene.[5][6] 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.[7] 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.[8] 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[9] and aberrant
metabolism in
tumourigenesis.[10]
Structure
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.[citation needed] 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
dopaminergiccell death in this disease is unclear. The prevailing
hypothesis is that parkin helps degrade one or more proteins toxic to dopaminergic neurons.[citation needed] 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.
A. Schematic diagram delineating arrangement of parkin functional domains. B. Cartoon representation of parkin in its autoinhibited state, with the catalytic cysteine in RING2 occluded by RING0 while Ubl and REP linker prevents E2 from binding to RING1. RING0, RING1, IBR and RING2 each coordinate two Zn ions (approximate location denoted by grey circles) for structural stability, leading to a
stoichiometry of 8 Zn2+/parkin.
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.[8] Ub transfer is aided by neighbouring residues
histidine His433, which accepts a proton from Cys431 to activate it, and
glutamate Glu444, which is involved in autoubiquitination.[11] Together these form the
catalytic triad, whose assembly is required for parkin activation.[12] 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.[8] Activating stimuli disrupt these interdomain interactions and induce parkin to collapse along the RING1-RING0 interface.[12] The active site of RING2 is drawn towards E2-Ub bound to RING1, facilitating formation of the Ub-thioester intermediate. Parkin activation requires
phosphorylation of
serine Ser65 in Ubl by
serine/threonine kinase,
PINK1. Addition of a charged
phosphate destabilises
hydrophobic interactions between Ubl and neighbouring subregions, reducing autoinhibitory effects of this N-terminus domain.[13] Ser65Ala
missense mutations were found to ablate Ub-parkin binding whilst inhibiting parkin recruitment to damaged mitochondria.[14] PINK1 also phosphorylates Ub at Ser65, accelerating its discharge from E2 and enhancing its
affinity for parkin.[13]
Although structural changes following phosphorylation are uncertain,
crystallisation of parkin revealed a cationic pocket in RING0 formed by
lysine and
arginine residues Lys161, Arg163 and Lys211 that forms a putative phosphate binding site.[15] Considering that RING0 is unique to parkin and that its hydrophobic interface with RING1 buries Cys431 in inactive parkin,[14] targeting of phosphorylated Ub and/or Ubl towards this binding niche might be critical in dismantling autoinhibitory complexes during parkin activation.
Function
Mitophagy
Parkin plays a crucial role in
mitophagy and clearance of
reactive oxygen species.[16] 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).[17] 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.[13] Considering the proximity of these chains to PINK1, further phosphorylation of Ub at Ser65 is likely, potentiating parkin mobilisation and substrate ubiquitination in a
self-reinforcing cycle.[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.[18] 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.[19] 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
translocaseTOMM20 and TOMM70 that facilitate movement of proteins such as PINK1 across OMM.[20]Miro (or
RHOT1/
RHOT2) is an OMM protein critical for
axonal transport, and may be ubiquitinated and targeted towards proteasomal degradation by parkin.[21] Miro breakdown produced a marked decrease in migration of compromised mitochondria along
axons of mouse
hippocampalneurons,[22] 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.[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. Following Ub conjugation, parkin recruits autophagy receptors such as p62,
TAX1BP1 and
CALCOCO2, facilitating assembly of autophagosomes that digest defective mitochondria.[20]
Cell survival
Through activation of
NF-κB signalling, parkin enhances survival and protects cells from stress-induced apoptosis. Upon cellular insult, parkin activates the catalytic HOIP
subunit of another E3 ligase LUBAC. HOIP triggers assembly of linear Ub
polymers on NF-κB essential modulator (NEMO), potentiating
transcription of mitochondrial GTPase
OPA1.[24] 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,[25] meaning that parkin mobilisation significantly enhances tolerance to moderate
stressors.
Parkin possesses
DNAbinding affinity and produces a
dose-dependent reduction in transcription and activity of
pro-apoptotic factor
p53.
Transfection of p53promoter with truncated versions of parkin into
SH-SY5Y neurons revealed that parkin directly binds to the p53 promoter via its RING1 domain.[26] Conversely, parkin may be a transcriptional target of p53 in H460 lung cells, where it mediates the
tumour suppressor action of p53.[10] Considering its role in mitochondrial
homeostasis, parkin aids p53 in maintaining mitochondrial
respiration while limiting glucose uptake and
lactate production, thus preventing onset of the
Warburg effect during tumourigenesis.[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.[10]
Clinical significance
Parkinson's disease
PARK2 (
OMIM*602544) is the parkin gene that may cause a form of autosomal recessive juvenile Parkinson disease (
OMIM600116) due to a mutation in the parkin protein. This form of genetic mutation may be one of the most common known genetic causes of early-onset
Parkinson disease. In one study of patients with onset of Parkinson disease prior to age 40 (10% of all PD patients), 18% had parkin mutations, with 5%
homozygous mutations.[28] 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).[29]
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.[32]
In accordance with its critical role in mitochondrial quality control, more than 120 pathogenic, PD-inducing mutations have been characterised on parkin.[8] Such mutations may be hereditary or stochastic and are associated with structural instability, reduced catalytic efficiency and aberrant substrate binding and ubiquitination.[9] Mutations can generally be categorised into three groups, depending on their location. Firstly, those clustered around Zn-coordinating residues on RING and IBR might compromise structural integrity and impair
catalysis.[12] A second class of mutations, including Thr240Arg, affect residues in and around the E2 binding site and alter autoinhibition of RING1 by REP.[33] Finally, Cys431Phe and Gly430Asp mutations impair
ligase activity at the
catalytic site and significantly reduce parkin function.[8]
The discovery of numerous non-mitochondrial parkin substrates reinforces the importance parkin in neuronal homeostasis, beyond its role in mitochondrial regulation. Potent
neuroprotective abilities of parkin in attenuating dopaminergic neurotoxicity, mitochondrial swelling and
excitotoxicity were demonstrated in cell cultures over-expressing parkin,[9] although the existence of such mechanisms at physiological parkin levels in vivo is yet unconfirmed. Another parkin substrate, synphilin-1 (encoded by SNCAIP), is an alpha-synuclein interacting protein that is enriched in the core of Lewy bodies and ubiquitinated by parkin in a manner abolished by familial PD-associated mutations.[34] 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.[35] 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[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.[9]
Finally, parkin protects dopaminergic neurons from
cytotoxicity induced by PD-mimetic
6-OHDA, mediated by suppression of neuronal p53 expression and its downstream activation of the apoptotic cascade.[26] Several PD-associated parkin mutations are localised to RING1 and might impair its ability to bind and downregulate the p53 promoter, leading to enhanced p53 expression.[38] Parkin-mutant PD patients also exhibit a four-fold elevation in p53
immunoreactivity,[26] insinuating that failure of parkin-mediated anti-apoptosis might be involved in etiology of PD.
Tumourigenesis
Consistent with parkin's potent anti-tumourigenic abilities, negative mutations and deletions have been reported in various tumours. For example, PARK2copy number was reduced in 85% of
glioblastoma samples while
lung cancers were associated with
heterozygous deletion of PARK2 at 6q25-q27 locus.[39] 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.[10] Similarly, chromosomal breaks in PARK2 suppressed expression of
afadinscaffold protein in
breast cancer, thereby comprising
epithelial integrity, enhancing
metastatic potential and worsening overall
prognosis.[40]HaploinsufficientPARK2 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.[41] Parkin is therefore a potent modulator of tumour progression, without directly instigating tumourigenesis.
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