Parkinson's disease is a common neurodegenerative disease with complex clinical features. Autosomal recessive juvenile parkinsonism (AR-JP) maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253; the former is deleted in one Japanese AR-JP patient. By positional cloning within this microdeletion, a complementary DNA clone has been isolated of 2,960 base pairs with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3-7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and the protein product has therefore been named 'Parkin' (Kitada, 1998).
Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson's disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. PARK2, mutations of which cause AR-JP, has been cloned but the function of the gene product, parkin, remains unknown. Parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7; mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. These findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. These findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson's disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer's disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis (Shimura, 2000).
To provide insight into the function of parkin, its intracellular distribution was examined in cultured cells. Parkin is localized in the trans-Golgi network and the secretory vesicles in U-373MG or SH-SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, it has been shown that parkin copurifies with synaptic vesicles (SVs) when low ionic conditions are used throughout the procedure. An immunoelectromicroscopic analysis indicates that parkin is present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non-ionic detergent. To elucidate its responsible region for membrane association, green fluorescent protein-tagged deletion mutants of parkin were transfected into COS-1 cells followed by subcellular fractionation. The ability of parkin to bind to the membranes resides in a broad region excluding the ubiquitin-like domain (Kubo, 2001).
Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson's disease (PD). It was hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Affinity-purified parkin antibodies label classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections has revealed that approximately 90% of anti-alpha S-reactive LBs are also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, are present in affinity-isolated LBs from DLB cortex. AlphaS and parkin co-localize within brainstem and cortical LBs. parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. It is concluded that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation (Schlossmacher, 2002).
Parkin is a ubiquitin-protein isopeptide ligase (E3) involved in ubiquitin/proteasome-mediated protein degradation. Mutations in the parkin gene cause a loss-of-function and/or alter protein levels of parkin. As a result, the toxic build-up of parkin substrates is thought to lead to autosomal recessive juvenile parkinsonism. To identify a role for the ubiquitin-like domain (ULD) of parkin, a number of hemagglutinin (HA)-tagged parkin constructs were created using mutational and structural information. Western blotting and immunocytochemistry show a much stronger expression level for HA-parkin residues 77-465 (without ULD) than HA-parkin full-length (with ULD). The deletion of ULD in Drosophila parkin also causes a sharp increase in expression of the truncated form, suggesting that the function of the ULD of parkin is conserved across species. By progressive deletion analysis of parkin ULD, it was found that residues 1-6 of human parkin play a crucial role in controlling the expression levels of this gene. HA-parkin residues 77-465 show ubiquitination in vivo, demonstrating that the ULD is not critical for parkin auto-ubiquitination; ubiquitination seemed to cluster on the central domain of parkin (residues 77-313). These effects are specific for the ULD of parkin and not transfection-, toxic-, epitope tag-, and/or vector-dependent. Taken together, these data suggest that the 76 most NH(2)-terminal residues (ULD) dramatically regulate the protein levels of parkin (Finney, 2003).
Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, the three-dimensional structure has been determined of the Ubl domain of parkin by NMR, in particular by extensive use of backbone (15)N-(1)H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data shows that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. These findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP (Sakata, 2003).
Mutations of the parkin gene are the most frequent cause of early onset autosomal recessive parkinsonism. Inactivation of the parkin gene in mice results in motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission. The levels of dopamine are increased in the limbic brain areas of parkin mutant mice and there is a shift towards increased metabolism of dopamine by MAO. Although there was no evidence for a reduction of nigrostriatal dopamine neurons in the parkin mutant mice, the level of dopamine transporter protein was reduced in these animals, suggesting a decreased density of dopamine terminals, or adaptative changes in the nigrostriatal dopamine system. GSH levels were increased in the striatum and fetal mesencephalic neurons from parkin mutant mice, suggesting that a compensatory mechanism may protect dopamine neurons from neuronal death. These parkin mutant mice provide a valuable tool to better understand the preclinical deficits observed in patients with PD and to characterize the mechanisms leading to the degeneration of dopamine neurons that could provide new strategies for neuroprotection (Itier, 2003).
Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, a mouse model was generated bearing a germline disruption in parkin. Parkin–/– mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin–/– mice. Intracellular recordings of medium-sized striatal spiny neurons show that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin–/– mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin–/– mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and alpha-synuclein, which have all been identified as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin–/– brains. Together these findings provide the first evidence of a novel role for parkin in dopamine regulation and nigrostriatal function, and a non-essential role for parkin in the survival of nigral neurons in mice (Goldberg, 2003).
Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkin gene. Parkin protein is characterized by a ubiquitin-like domain at its NH(2)-terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING). Parkin is a RING-type E3 ubiquitin-protein ligase that binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, unfolded protein stress induces up-regulation of both the mRNA and protein level of Parkin. Furthermore, overexpression of Parkin, but not a set of mutants without the E3 activity, specifically suppresses unfolded protein stress-induced cell death. These findings demonstrate that Parkin is an E3 enzyme and suggest that it is involved in the ubiquitination pathway for misfolded proteins derived from endoplasmic reticulum and contributes to protection from neurotoxicity induced by unfolded protein stresses (Imai, 2000).
Parkin gene mutations have been implicated in autosomal-recessive early-onset parkinsonism and lead to specific degeneration of dopaminergic neurons in midbrain. To investigate the role of Parkin in neuronal cell death, this protein was overproduced in PC12 cells in an inducible manner. In this cell line, neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide, but not by a variety of other cell death inducers (H(2)O(2), 4-hydroxynonenal, rotenone, 6-OHDA, tunicamycin, 2-mercaptoethanol and staurosporine). Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of Parkin. Interestingly, Parkin acted by delaying mitochondrial swelling and subsequent cytochrome c release and caspase-3 activation observed in ceramide-mediated cell death. Subcellular fractionation demonstrated enrichment of Parkin in the mitochondrial fraction and its association with the outer mitochondrial membrane. Together, these results suggest that Parkin may promote the degradation of substrates localized in mitochondria and involved in the late mitochondrial phase of ceramide-mediated cell death. Loss of this function may underlie the degeneration of nigral dopaminergic neurons in patients with Parkin mutations (Darios, 2003).
A putative G protein-coupled transmembrane polypeptide, named Pael receptor, has been identified as an interacting protein with Parkin, a gene product responsible for autosomal recessive juvenile parkinsonism (AR-JP). When overexpressed in cells, this receptor tends to become unfolded, insoluble, and ubiquitinated in vivo. The insoluble Pael receptor leads to unfolded protein-induced cell death. Parkin specifically ubiquitinates this receptor in the presence of ubiquitin-conjugating enzymes resident in the endoplasmic reticulum and promotes the degradation of insoluble Pael receptor, resulting in suppression of the cell death induced by Pael receptor overexpression. Moreover, the insoluble form of Pael receptor accumulates in the brains of AR-JP patients. The unfolded Pael receptor is a substrate of Parkin, the accumulation of which may cause selective neuronal death in AR-JP (Imai, 2001).
Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. CHIP is a cytosolic U-box protein that interacts with Hsc70 through a set of tetratricorepeat motifs. The U-box represents a modified form of the ring-finger motif that is found in ubiquitin ligases and that defines the E4 family of polyubiquitination factors. The amount of CHIP in the complex is increased during ER stress. CHIP promotes the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediates in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhances the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity (Imai, 2002).
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. It was hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. A protein complex in normal human brain has been identified that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD fails to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulates in a non-ubiquitinated form in parkin-deficient PD brains. It is concluded that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD (Shimura, 2001).
One hypothesis for the etiology of Parkinson's disease (PD) is that subsets of neurons are vulnerable to a failure in proteasome-mediated protein turnover. Overexpression of mutant alpha-synuclein increases sensitivity to proteasome inhibitors by decreasing proteasome function. Overexpression of parkin decreases sensitivity to proteasome inhibitors in a manner dependent on parkin's ubiquitin-protein E3 ligase activity, and antisense knockdown of parkin increases sensitivity to proteasome inhibitors. Mutant alpha-synuclein also causes selective toxicity to catecholaminergic neurons in primary midbrain cultures, an effect that can be mimicked by the application of proteasome inhibitors. Parkin is capable of rescuing the toxic effects of mutant alpha-synuclein or proteasome inhibition in these cells. Therefore, parkin and alpha-synuclein are linked by common effects on a pathway associated with selective cell death in catecholaminergic neurons (Petrucelli, 2002).
Parkinson's disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson's disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. parkin physically interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. Familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1 (Chung, 2002).
In addition to inhibiting the mitochondrial respiratory chain, toxins known to cause Parkinson's disease (PD), such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone, also strongly depolymerize microtubules and increase tubulin degradation. Microtubules are polymers of tubulin alpha/beta heterodimers, whose correct folding requires coordinated actions of cellular chaperonins and cofactors. Misfolded tubulin monomers are highly toxic and quickly degraded through a hitherto unknown mechanism. Parkin, a protein-ubiquitin E3 ligase linked to PD, tightly binds to microtubules in taxol-mediated microtubule coassembly assays. In lysates from the rat brain or transfected human embryonic kidney (HEK) 293 cells, alpha-tubulin and beta-tubulin strongly coimmunoprecipitate with parkin in the presence of colchicine, a condition in which tubulin exits as alpha/beta heterodimers. At the subcellular level, parkin exhibits punctate immunostaining along microtubules in rat brain sections, cultured primary neurons, glial cells, and cell lines. This pattern of subcellular localization is abolished in cells treated with the microtubule-depolymerizing drug colchicine. The binding between parkin and tubulin apparently leads to increased ubiquitination and accelerated degradation of alpha- and beta-tubulins in HEK293 cells. Similarly ubiquitinated tubulins are also observed in rat brain lysates. Furthermore, parkin mutants found in PD patients do not ubiquitinate or degrade either tubulin. Taken together, these results show that parkin is a novel tubulin-binding protein, as well as a microtubule-associated protein. Its ability to enhance the ubiquitination and degradation of misfolded tubulins may play a significant role in protecting neurons from toxins that cause PD (Ren, 2003).
Mutations of parkin, a protein-ubiquitin isopeptide ligase (E3), appear to be the most frequent cause of familial Parkinson's disease (PD). Parkin binds strongly to alpha/beta tubulin heterodimers and microtubules. The strong binding between parkin and tubulin, as well as that between parkin and microtubules, is mediated by three independent domains: linker, RING1, and RING2. These redundant strong interactions made it virtually impossible to separate parkin from microtubules by high concentrations of salt (3.8 m) or urea (0.5 m). Parkin co-purified with tubulin and was found in a highly purified tubulin preparation. Expression of either full-length parkin or any of its three microtubule-binding domains significantly attenuates colchicine-induced microtubule depolymerization. The abilities of parkin to bind to and stabilize microtubules are not affected by PD-linked mutations that abrogate its E3 ligase activity. Thus, the tubulin/microtubule-binding activity of parkin and its E3 ligase activity are independent. The strong binding between parkin and tubulin/microtubules through three redundant interaction domains may not only stabilize microtubules but also guarantee the anchorage of this E3 ligase on microtubules. Because many misfolded proteins are transported on microtubules, the localization of parkin on microtubules may provide an important environment for its E3 ligase activity toward misfolded substrates (Yang, 2005).
Mutations in parkin, which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis (Staropoli, 2003).
Parkin binds to the E2 ubiquitin-conjugating human enzyme 8 (UbcH8) through its C-terminal ring-finger. Parkin has ubiquitin-protein ligase activity in the presence of UbcH8. Parkin also ubiquitinates itself and promotes its own degradation. The synaptic vesicle-associated protein, CDCrel-1, interacts with Parkin through its ring-finger domains. Furthermore, Parkin ubiquitinates and promotes the degradation of CDCrel-1. Familial-linked mutations disrupt the ubiquitin-protein ligase function of Parkin and impair Parkin and CDCrel-1 degradation. These results suggest that Parkin functions as an E3 ubiquitin-protein ligase through its ring domains and that it may control protein levels via ubiquitination. The loss of Parkin's ubiquitin-protein ligase function in familial-linked mutations suggests that this may be the cause of familial autosomal recessive Parkinson's disease (Zhang, 2000).
Parkin, the most commonly mutated gene in familial Parkinson's disease, encodes an E3 ubiquitin ligase. A number of candidate substrates have been identified for parkin ubiquitin ligase action including CDCrel-1, o-glycosylated alpha-synuclein, Pael-R and synphilin-1. Parkin promotes the ubiquitination and degradation of an expanded polyglutamine protein. Overexpression of parkin reduces aggregation and cytotoxicity of an expanded polyglutamine ataxin-3 fragment. Parkin reduces proteasome impairment and caspase-12 activation induced by an expanded polyglutamine protein. Parkin forms a complex with the expanded polyglutamine protein, heat shock protein 70 (Hsp70) and the proteasome that may be important for the elimination of the expanded polyglutamine protein. Hsp70 enhances parkin binding and ubiquitination of expanded polyglutamine protein in vitro, suggesting that Hsp70 may help to recruit misfolded proteins as substrates for parkin E3 ubiquitin ligase activity. It is speculated that parkin may function to relieve ER stress by preserving proteasome activity in the presence of misfolded proteins. Loss of parkin function and the resulting proteasomal impairment may contribute to the accumulation of toxic aberrant proteins in neurodegenerative diseases including Parkinson's Disease (Tsai, 2003).
Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage and the major cleavage site is after Asp 126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK293 cells identified caspase-1, -3 and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9 and -11 do not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8 but not by caspase-3. In anti-FAS treated Jurkat cells, parkin cleavage is inhibited by caspase inhibitors hFlip and CrmA but not by XIAP indicating that caspase-8 but not caspase-3 is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3 deficient MCF7 cells, either by caspase-1 or -8 overexpression or by TNF-alpha treatment leads to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin, and suggest that death receptor activation and inflammatory stress can cause loss of parkins ubiquitin ligase activity and thus cause accumulation of toxic parkin substrates and trigger dopaminergic cell death (Kahns, 2003).
In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25-q27, a contig containing eight known genes, including the complete genomic structure of the Parkin gene, was constructed. Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including two marker loci. Both loci exhibited the highest frequencies of loss of homozygosity in this study and are each located within the Parkin genomic structure. Whereas mutation analysis revealed no missense substitutions, expression of the Parkin gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined. In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in two lung adenocarcinoma cell lines, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of Parkin in these samples. These data suggest that the loss of homozygosity observed at chromosome 6q25-q26 may contribute to the initiation and/or progression of cancer by inactivating or reducing the expression of the Parkin gene. Because Parkin maps to one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene located at a common fragile site (Cesari, 2003).
Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. A locus for a rare familial form of PD has been mapped chromosome 1p36 (PARK6). Mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. Two homozygous mutations have been identified affecting the PINK1 kinase domain in three consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD (Valente, 2004).
Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser/Thr protein kinase targeted to mitochondria. Investigated in this study were the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. Two point mutations were examined: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. Kinase activity was confirmed and artificial 'kinase-dead' mutants were produced that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. The localization of PINK1 in transfected mammalian cells was examined by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the N terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria (Beilina, 2005).
Parkinson's disease (PD) is a progressive neurodegenerative illness associated with a selective loss of dopaminergic neurons in the nigrostriatal pathway of the brain. Despite the overall rarity of the familial forms of PD, the identification of single genes linked to the disease has yielded crucial insights into possible mechanisms of neurodegeneration. Recently, a putative mitochondrial kinase, PINK1, has been found mutated in an inherited form of parkinsonism. PINK1 mutations confer different autophosphorylation activity, which is regulated by the C-terminal portion of the protein. The mitochondrial localization of both wild-type and mutant PINK1 proteins has been unequivocally demonstrated and it has been proven that a short N-terminal part of PINK1 is sufficient for its mitochondrial targeting (Silvestri, 2005).
Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease. To investigate the role of PINK1 in neurodegeneration, human and murine neuronal cell lines were designed expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. Under basal and staurosporine-induced conditions, the number of TUNEL-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. Overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogate the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations (Petit, 2005).
The Parkinson's disease (PD) causative PINK1 gene encodes a mitochondrial protein kinase called PTEN-induced kinase 1 (PINK1). The autosomal recessive pattern of inheritance of PINK1 mutations suggests that PINK1 is neuroprotective and therefore loss of PINK1 function causes PD. Indeed, overexpression of PINK1 protects neuroblastoma cells from undergoing neurotoxin-induced apoptosis. As a protein kinase, PINK1 presumably exerts its neuroprotective effect by phosphorylating specific mitochondrial proteins and in turn modulating their functions. Towards elucidation of the neuroprotective mechanism of PINK1, the baculovirus-infected insect cell system was used to express the recombinant protein consisting of the PINK1 kinase domain either alone [PINK1(KD)] or with the PINK1 C-terminal tail [PINK1(KD+T)]. Both recombinant enzymes preferentially phosphorylate the artificial substrate histone H1 exclusively at serine and threonine residues, demonstrating that PINK1 is indeed a protein serine/threonine kinase. Introduction of the PD-associated mutations, G386A and G409V significantly reduces PINK1(KD) kinase activity. Since Gly-386 and Gly-409 reside in the conserved activation segment of the kinase domain, the results suggest that the activation segment is a regulatory switch governing PINK1 kinase activity. PINK1(KD+T) is approximately 6-fold more active than PINK1(KD). Thus, in addition to the activation segment, the C-terminal tail also contains regulatory motifs capable of governing PINK1 kinase activity. Finally, the availability of active recombinant PINK1 proteins permits future studies to search for mitochondrial proteins that are preferentially phosphorylated by PINK1. As these proteins are likely physiological substrates of PINK1, their identification will shed light on the mechanism of pathogenesis of PD (Sim, 2006).
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