Phylogenetic and in silico structural analysis of the Parkinson disease-related kinase PINK1

Parkinson disease (PD) is the second most common neurodegenerative disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra. Mutations in PINK1 were shown to cause recessive familial PD, and today are proposed to be associated with the disease via mitochondrial dysfunction and oxidative damage. The PINK1 gene comprises eight exons, which encode a ubiquitously expressed 581 amino acid protein that contains an N-terminal mitochondrial targeting domain and a serine/threonine protein kinase. To better understand the relationship between PINK1 and PD we have first analyzed the evolutionary history of the gene showing its late emergence in evolution. In addition, we have modeled the three-dimensional structure of PINK1 and found some evidences that help to explain the effect of some PD-related mutations in this protein's function.     

PINK1 cleavage at position A103 by the mitochondrial protease PARL

Mutations in PTEN-induced kinase 1 (PINK1) cause early onset autosomal recessive Parkinson's disease (PD). PINK1 is a 63 kDa protein kinase, which exerts a neuroprotective function and is known to localize to mitochondria. Upon entry into the organelle, PINK1 is cleaved to produce a ～53 kDa protein (ΔN-PINK1). In this paper, we show that PINK1 is cleaved between amino acids Ala-103 and Phe-104 to generate ΔN-PINK1. We demonstrate that a reduced ability to cleave PINK1, and the consequent accumulation of full-length protein, results in mitochondrial abnormalities reminiscent of those observed in PINK1 knockout cells, including disruption of the mitochondrial network and a reduction in mitochondrial mass. Notably, we assessed three N-terminal PD-associated PINK1 mutations located close to the cleavage site and, while these do not prevent PINK1 cleavage, they alter the ratio of full-length to ΔN-PINK1 protein in cells, resulting in an altered mitochondrial phenotype. Finally, we show that PINK1 interacts with the mitochondrial protease presenilin-associated rhomboid-like protein (PARL) and that loss of PARL results in aberrant PINK1 cleavage in mammalian cells. These combined results suggest that PINK1 cleavage is important for basal mitochondrial health and that PARL cleaves PINK1 to produce the ΔN-PINK1 fragment.     

Mitochondrial translation initiation factor 3 gene polymorphism associated with Parkinson's disease

Mitochondrial dysfunction occurs early in late-onset sporadic Parkinson's disease (PD), but the mitochondrial protein network mediating PD pathogenesis is largely unknown. Mutations in the mitochondrial serine-threonine kinase PINK1 have recently been shown to cause the early-onset autosomal recessive PARK6 variant of PD. We have now tested a candidate interactor protein of PINK1, the mitochondrial translation initiation factor 3 (MTIF3) for involvement in PD pathogenesis. In two independent case-control collectives, the c.798C>T polymorphism of the MTIF3 gene showed allelic association with PD, with a maximal significance of p=0.0073. An altered function of variant MTIF3 may affect the availability of mitochondrial encoded proteins, lead to oxidative stress and create vulnerability for PD.     

Analysis of the regulatory and catalytic domains of PTEN-induced kinase-1 (PINK1)

Mutations of the phosphatase and tensin homolog (PTEN)‐induced kinase 1 (PINK1) gene can cause early‐onset familial Parkinson disease (PD). PINK1 encodes a neuroprotective protein kinase localized at the mitochondria, and its involvement in regulating mitochondrial dynamics, trafficking, structure, and function is well documented. Owing to the lack of information on structure and biochemical properties for PINK1, exactly how PINK1 exerts its neuroprotective function and how the PD‐causative mutations impact on PINK1 structure and function remain unclear. As an approach to address these questions, we conducted bioinformatic analyses of the mitochondrial targeting, the transmembrane, and kinase domains of PINK1 to predict the motifs governing its regulation and function. Our report sheds light on how PINK1 is targeted to the mitochondria and how PINK1 is cleaved by mitochondrial peptidases. Moreover, it includes a potential optimal phosphorylation sequence preferred by the PINK1 kinase domain. On the basis of the results of our analyses, we predict how the PD‐causative mutations affect processing of PINK1 in the mitochondria, PINK1 kinase activity, and substrate specificity. In summary, our results provide a conceptual framework for future investigation of the structural and biochemical basis of regulation and the neuroprotective mechanism of PINK1. Hum Mutat 33:1408–1422, 2012. © 2012 Wiley Periodicals, Inc.     

The PINK1/Parkin pathway regulates mitochondrial dynamics and function in mammalian hippocampal and dopaminergic neurons

PTEN-induced putative kinase 1 (PINK1) and Parkin act in a common pathway to regulate mitochondrial dynamics, the involvement of which in the pathogenesis of Parkinson's disease (PD) is increasingly being appreciated. However, how the PINK1/Parkin pathway influences mitochondrial function is not well understood, and the exact role of this pathway in controlling mitochondrial dynamics remains controversial. Here we used mammalian primary neurons to examine the function of the PINK1/Parkin pathway in regulating mitochondrial dynamics and function. In rat hippocampal neurons, PINK1 or Parkin overexpression resulted in increased mitochondrial number, smaller mitochondrial size and reduced mitochondrial occupancy of neuronal processes, suggesting that the balance of mitochondrial fission/fusion dynamics is tipped toward more fission. Conversely, inactivation of PINK1 resulted in elongated mitochondria, indicating that the balance of mitochondrial fission/fusion dynamics is tipped toward more fusion. Furthermore, overexpression of the fission protein Drp1 (dynamin-related protein 1) or knocking down of the fusion protein OPA1 (optical atrophy 1) suppressed PINK1 RNAi-induced mitochondrial morphological defect, and overexpression of PINK1 or Parkin suppressed the elongated mitochondria phenotype caused by Drp1 RNAi. Functionally, PINK1 knockdown and overexpression had opposite effects on dendritic spine formation and neuronal vulnerability to excitotoxicity. Finally, we found that PINK1/Parkin similarly influenced mitochondrial dynamics in rat midbrain dopaminergic neurons. These results, together with previous findings in Drosophila dopaminergic neurons, indicate that the PINK1/Parkin pathway plays conserved roles in regulating neuronal mitochondrial dynamics and function.     

C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1

The Parkinson's disease (PD) causative PINK1 gene encodes amitochondrial protein kinase called PTEN-induced kinase 1 (PINK1).The autosomal recessive pattern of inheritance of PINK1 mutationssuggests that PINK1 is neuroprotective and therefore loss ofPINK1 function causes PD. Indeed, overexpression of PINK1 protectsneuroblastoma cells from undergoing neurotoxin-induced apoptosis.As a protein kinase, PINK1 presumably exerts its neuroprotectiveeffect by phosphorylating specific mitochondrial proteins andin turn modulating their functions. Towards elucidation of theneuroprotective mechanism of PINK1, we employed the baculovirus-infectedinsect cell system to express the recombinant protein consistingof the PINK1 kinase domain either alone [PINK1(KD)] or withthe PINK1 C-terminal tail [PINK1(KD+T)]. Both recombinant enzymespreferentially phosphorylate the artificial substrate histoneH1 exclusively at serine and threonine residues, demonstratingthat PINK1 is indeed a protein serine/threonine kinase. Introductionof the PD-associated mutations, G386A and G409V significantlyreduces PINK1(KD) kinase activity. Since Gly-386 and Gly-409reside in the conserved activation segment of the kinase domain,the results suggest that the activation segment is a regulatoryswitch governing PINK1 kinase activity. We also demonstratethat PINK1(KD+T) is     

Kinase signaling dysfunction in Parkinson's disease: a reverse genetic approach in Drosophila

Drosophila genetics is one of the most powerful tools in modern biology. For many years, the "forward genetic" approach using Drosophila has been extraordinarily successful in elucidating the molecular pathways of many physiological processes and behaviors. Recently, the "reverse genetic" approach in Drosophila is increasingly being developed as a major tool for research in biology, especially in the study of human diseases. Parkinson's disease (PD) is the second most common neurodegenerative disease. Kinase signaling has been directly implicated in PD pathogenesis. Mutations in PTEN-induced kinase 1 (PINK1) cause PARK6 type PD, in which mitochondrial deficits are at the center of pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most prevalent genetic cause of both familial (PARK8 type with autosomal dominant inheritance) and sporadic PD. To understand the mechanism of PINK1- and LRRK2- mediated pathogenesis, reverse-engineered Drosophila models have been critical tools. Here the authors will discuss the usage of Drosophila models in their and other laboratories, and share scientific insights that originate from these studies, and discuss their experimental results of the effect of PINK1 on proteasome function. The authors will also comment on the different approaches taken in these lines of investigation.     

Bioenergetics of neurons inhibit the translocation response of Parkin following rapid mitochondrial depolarization

Recent studies delineate a pathway involving familial Parkinson's disease (PD)-related proteins PINK1 and Parkin, in which PINK1-dependent mitochondrial accumulation of Parkin targets depolarized mitochondria towards degradation through mitophagy. The pathway has been primarily characterized in cells less dependent on mitochondria for energy production than neurons. Here we report that in neurons, unlike other cells, mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone did not induce Parkin translocation to mitochondria or mitophagy. PINK1 overexpression increased basal Parkin accumulation on neuronal mitochondria, but did not sensitize them to depolarization-induced Parkin translocation. Our data suggest that bioenergetic differences between neurons and cultured cell lines contribute to these different responses. In HeLa cells utilizing usual glycolytic metabolism, mitochondrial depolarization robustly triggered Parkin-mitochondrial translocation, but this did not occur in HeLa cells forced into dependence on mitochondrial respiration. Declining ATP levels after mitochondrial depolarization correlated with the absence of induced Parkin-mitochondrial translocation in both HeLa cells and neurons. However, intervention allowing neurons to maintain ATP levels after mitochondrial depolarization only modestly increased Parkin recruitment to mitochondria, without evidence of increased mitophagy. These data suggest that changes in ATP levels are not the sole determinant of the different responses between neurons and other cell types, and imply that additional mechanisms regulate mitophagy in neurons. Since the Parkin-mitophagy pathway is heavily dependent on bioenergetic status, the unique metabolic properties of neurons likely influence the function of this pathway in the pathogenesis of PD.     

PINK1 gene knockdown leads to increased binding of parkin with actin filament

Mutations in the PINK1 gene are known to cause early onset familial Parkinson's disease (PD). Genetic fruit fly model studies and rescue experiments with parkin overexpression suggest that PINK1 and parkin are associated via an unidentified mechanism. To gain additional insight into this interaction, we have investigated the impact of PINK1 deficiency on the biological function of parkin using actin filament dynamics. Actin is known to be associated with parkin and is a key regulator of eukaryotic cell death. PINK1 gene knockdown (KD) significantly increased actin aggregation and its binding with parkin. Known PD-related pathological conditions, such as oxidative stress and mitochondrial dysfunction, also increased actin aggregation and parkin binding. PINK1 KD resulted in the increased phosphorylation of cofilin, a protein important for the remodeling of actin filament and neurodegeneration. These results suggest that altered actin dynamics and increased association of parkin with actin filament might underlie the pathological conditions resulting from PINK1 deficiency.     

Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1) and LRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q(10), rapamycin, or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in this neurodegenerative disease.     

Kinase Signaling Dysfunction in Parkinson's Disease: A Reverse Genetic Approach in Drosophila

Abstract: Drosophila genetics is one of the most powerful tools in modern biology. For many years, the “forward genetic” approach using Drosophila has been extraordinarily successful in elucidating the molecular pathways of many physiological processes and behaviors. Recently, the “reverse genetic” approach in Drosophila is increasingly being developed as a major tool for research in biology, especially in the study of human diseases. Parkinson's disease (PD) is the second most common neurodegenerative disease. Kinase signaling has been directly implicated in PD pathogenesis. Mutations in PTEN-induced kinase 1 (PINK1) cause PARK6 type PD, in which mitochondrial deficits are at the center of pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most prevalent genetic cause of both familial (PARK8 type with autosomal dominant inheritance) and sporadic PD. To understand the mechanism of PINK1- and LRRK2- mediated pathogenesis, reverse-engineered Drosophila models have been critical tools. Here the authors will discuss the usage of Drosophila models in their and other laboratories, and share scientific insights that originate from these studies, and discuss their experimental results of the effect of PINK1 on proteasome function. The authors will also comment on the different approaches taken in these lines of investigation.     

Functional alteration of PARL contributes to mitochondrial dysregulation in Parkinson's disease

Molecular genetics has linked mitochondrial dysfunction to the pathogenesis of Parkinson's disease by the discovery of rare, inherited mutations in gene products that associate with the mitochondria. Mutations in PTEN-induced kinase-1 (PINK1), which encodes a mitochondrial kinase, and PARKIN, encoding an E3 ubiquitin ligase, are the most frequent causes of recessive Parkinson's disease. Recent functional studies have revealed that PINK1 recruits PARKIN to mitochondria to initiate mitophagy, an important autophagic quality control mechanism that rids the cell of damaged mitochondria. PINK1 is post-translationally processed into a cleaved form whose levels are tightly regulated, although the significance of this processing is unknown. Here we demonstrate that the mitochondrial protease presenilin-associated rhomboid-like (PARL) can affect the proteolytic processing of PINK1 and that normal PINK1 localization and stability requires PARL's catalytic activity. PARL deficiency impairs PARKIN recruitment to mitochondria, suggesting PINK1's processing and localization are important in determining its interaction with PARKIN. We sequenced the PARL gene in Parkinson's disease patients and discovered a novel missense mutation in a functional domain of PARL's N-terminus. This PARL mutant is not sufficient to rescue PARKIN recruitment, suggesting that impaired mitophagy may be an underlying mechanism of disease pathogenesis in patients with PARL mutations.     

Regulation of parkin and PINK1 by neddylation

Neddylation is a posttranslational modification that plays important roles in regulating protein structure and function by covalently conjugating NEDD8, an ubiquitin-like small molecule, to the substrate. Here, we report that Parkinson's disease (PD)-related parkin and PINK1 are NEDD8 conjugated. Neddylation of parkin and PINK1 results in increased E3 ligase activity of parkin and selective stabilization of the 55 kDa PINK1 fragment. Expression of dAPP-BP1, a NEDD8 activation enzyme subunit, in Drosophila suppresses abnormalities induced by dPINK1 RNAi. PD neurotoxin MPP(+) inhibits neddylation of both parkin and PINK1. NEDD8 immunoreactivity is associated with Lewy bodies in midbrain dopaminergic neurons of PD patients. Together, these results suggest that parkin and PINK1 are regulated by neddylation and that impaired NEDD8 modification of these proteins likely contributes to PD pathogenesis.     

Cytoplasmic localization and proteasomal degradation of N-terminally cleaved form of PINK1

Mutations in PTEN-induced putative kinase 1 (PINK1) gene have been linked to an autosomal recessive form of familial Parkinson's disease. PINK1 encodes a predicted mitochondrial protein kinase. Although the mitochondrial localization of PINK1 has been suggested, the exact subcellular compartment in which PINK1 exerts its cytoprotective function is elusive. Thus, we studied the subcellular distribution and metabolism of PINK1 in cultured cells. Immunocytochemical analysis showed that PINK1 resides in cytoplasm in addition to mitochondria, and that the mitochondrial localization is dependent on its N-terminal sequence. Cellular expression of PINK1 yielded several N-terminally cleaved fragments as well as the full-length protein, among which the 54 kDa fragment (DeltaN 54 kDa) was highly accumulated in the presence of proteasome inhibitors. Endogenous PINK1 was detected dominantly in the form of DeltaN 54 kDa upon proteasome inhibition. Rapid turnover of DeltaN 54 kDa further supported its higher susceptibility to proteasomal degradation compared with that of full-length protein. These results indicate that DeltaN 54 kDa PINK1 undergoes constitutive degradation by proteasome, and underscore the significance of its localization in cytoplasm, especially in the N-terminally processed form.     

The three ‘P’s of mitophagy: PARKIN,PINK1, and post-translational modifications

Two Parkinson''s disease (PD)-associated proteins, the mitochondrial kinase PINK1 and the E3-ubiquitin (Ub) ligase PARKIN, are central to mitochondrial quality control. In this pathway, PINK1 accumulates on defective mitochondria, eliciting the translocation of PARKIN from the cytosol to mediate the clearance of damaged mitochondria via autophagy (mitophagy). Throughout the different stages of mitophagy, post-translational modifications (PTMs) are critical for the regulation of PINK1 and PARKIN activity and function. Indeed, activation and recruitment of PARKIN onto damaged mitochondria involves PINK1-mediated phosphorylation of both PARKIN and Ub. Through a stepwise cascade, PARKIN is converted from an autoinhibited enzyme into an active phospho-Ub-dependent E3 ligase. Upon activation, PARKIN ubiquitinates itself in concert with many different mitochondrial substrates. The Ub conjugates attached to these substrates can in turn be phosphorylated by PINK1, which triggers further cycles of PARKIN recruitment and activation. This feed-forward amplification loop regulates both PARKIN activity and mitophagy. However, the precise steps and sequence of PTMs in this cascade are only now being uncovered. For instance, the Ub conjugates assembled by PARKIN consist predominantly of noncanonical K6-linked Ub chains. Moreover, these modifications are reversible and can be disassembled by deubiquitinating enzymes (DUBs), including Ub-specific protease 8 (USP8), USP15, and USP30. However, PINK1-mediated phosphorylation of Ub can impede the activity of these DUBs, adding a new layer of complexity to the regulation of PARKIN-mediated mitophagy by PTMs. It is therefore evident that further insight into how PTMs regulate the PINK1–PARKIN pathway will be critical for our understanding of mitochondrial quality control.     

Enhanced vulnerability of PARK6 patient skin fibroblasts to apoptosis induced by proteasomal stress

Proteasomal dysfunction and apoptosis are major hallmarks in the pathophysiology of Parkinson's disease (PD). PARK6 which is caused by mutations in the mitochondrial protein kinase PINK1 is a rare autosomal-recessively inherited disorder mimicking the clinical picture of PD. To investigate the cytoprotective physiological function of PINK1, we used primary fibroblasts from three patients homozygous for G309D-PINK1 as well as SHEP neuroblastoma cells stably overexpressing GFP-tagged wild type (wt) PINK1. Here we demonstrate that overexpression of wt PINK1 inhibits activation of Bax and release of cytochrome c, thereby diminishing caspase 9 processing and effector caspase activity after induction of proteasomal stress with the proteasome inhibitor (PI) MG132 in SHEP cells. Conversely, effector caspase activation induced by PIs, but not by the unrelated apoptotic stimulus staurosporine was potently enhanced in primary fibroblasts from homozygous PARK6 patients in comparison to those of heterozygous carriers or unaffected siblings. SHEP cells overexpressing wt PINK1 showed an elevated expression of the cytoprotective gene parkin, whereas PARK6 fibroblasts displayed significantly decreased expression of parkin in comparison to wild type control cells. Interestingly, overexpressed GFP-PINK1 was exclusively localized in the mitochondria of SHEP cells, but was redistributed to the cytoplasm under conditions of proteasomal stress. Our data indicate that PINK1 plays an important and specific physiological role in protecting cells from proteasomal stress, and suggest that PINK1 might exert its cytoprotective effects upstream of mitochondria engagement.     

p62/SQSTM1 cooperates with Parkin for perinuclear clustering of depolarized mitochondria

PINK1 and Parkin were first identified as the causal genes responsible for familial forms of early‐onset Parkinson’s disease (PD), a prevalent neurodegenerative disorder. PINK1 encodes a mitochondrial serine/threonine protein kinase, whereas Parkin encodes an ubiquitin‐protein ligase. PINK1 and Parkin cooperate to maintain mitochondrial integrity; however, the detailed molecular mechanism of how Parkin‐catalyzed ubiquitylation results in mitochondrial integrity remains an enigma. In this study, we show that Parkin‐catalyzed K63‐linked polyubiquitylation of depolarized mitochondria resulted in ubiquitylated mitochondria being transported along microtubules to cluster in the perinuclear region, which was interfered by pathogenic mutations of Parkin. In addition, p62/SQSTM1 (hereafter referred to as p62) was recruited to depolarized mitochondria after Parkin‐directed ubiquitylation. Intriguingly, deletion of p62 in mouse embryonic fibroblasts resulted in a gross loss of mitochondrial perinuclear clustering but did not hinder mitochondrial degradation. Thus, p62 is required for ubiquitylation‐dependent clustering of damaged mitochondria, which resembles p62‐mediated ‘aggresome’ formation of misfolded/unfolded proteins after ubiquitylation.     

Assessment of PINK1 (PARK6) polymorphisms in Finnish PD

Recent data has demonstrated that mutations in PINK1, encoding PTEN-induced kinase 1, are a cause of early onset recessive parkinsonism (PARK6 locus). Common variability in genes implicated in hereditary forms of parkinsonism may be a predisposing factor in sporadic Parkinson's disease (PD). We analyzed whether six different genetic variants within and surrounding PINK1 contribute to the risk of sporadic PD in a Finnish case-control series. Our results indicate that this gene does not play a major role in the genetic predisposition to PD in this population.     

Association of PINK1 and DJ-1 confers digenic inheritance of early-onset Parkinson's disease

Mutations in genes encoding both DJ-1 and pten-induced kinase 1 (PINK1) are independently linked to autosomal recessive early-onset familial forms of Parkinson's disease (PD). We here report identification of a family with PD patients harboring novel heterozygous missense mutations in both PINK1 and DJ-1 genes encoding DJ-1A39S and PINK1P399L, respectively. In transfected cells, DJ-1 interacts with PINK1. PINK1P399L is less stable than the wild-type protein and is degraded via the ubiquitin-mediated proteasomal pathway. Expression of wild-type DJ-1 increased steady-state levels of PINK1, whereas expression of DJ-1A39S reduced steady-state levels of PINK1. Furthermore, co-expression of wild-type DJ-1 and PINK1 suppresses neurotoxin 1-methyl-4-phenylpyridinium (MPP(+))-induced death of dopaminergic SH-SY5Y cells. In contrast, co-expression of PD-associated DJ-1A39S and PINK1P399L significantly potentiated susceptibility of SH-SY5Y cells to MPP(+)-induced cell death. This study reports the first case of autosomal recessive PD with digenic inheritance and demonstrates that DJ-1 and PINK1 physically associate and collaborate to protect cells against stress via complex formation.     

Analysis of the PINK1 gene in a cohort of patients with sporadic early-onset parkinsonism in Taiwan

Mutations in the PINK1 gene have been shown to cause autosomal recessive Parkinson's disease (PD) and/or early onset sporadic PD in Italy, Spain, North America, Ireland, and Asia. However, there are limited data on PINK1 mutations in sporadic early onset Asian PD patients. To determine the prevalence of PINK1 mutation in Taiwanese population, we conducted genetic analysis of PINK1 mutation in 73 early onset sporadic PD and 94 normal control subjects. We only identified a novel single heterozygous mutation R 407Q mutation in exon 6 of this gene in one patient at the age onset of 54. Overall, these data indicate that PINK1 mutations are rare in our population. Based on our results, unless common mutational hotspots are identified, routine testing for this mutation at least in our population may not be cost-effective.