Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism

Silvestri, Laura; Caputo, Viviana; Bellacchio, Emanuele; Atorino, Luigia; Dallapiccola, Bruno; Valente, Enza Maria; Casari, Giorgio

doi:10.1093/hmg/ddi377

Cited by 423 publications

(405 citation statements)

References 42 publications

(34 reference statements)

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“…We next asked where the interaction between PINK1 and Beclin1 could take place within the cell. By performing confocal microscopy experiments with organelle-specific markers, we confirmed that PINK1 localizes mainly to the mitochondria, with only a small proportion of it localizing to other compartments 12,28 ( Figure 4c and Supplementary Figure 2a). Mitochondrial localization did not significantly change for PINK1 , PINK1 W437X and PINK1 G309D , whereas PINK1 112-581 presented diffuse cytoplasmic localization, as expected (Figure 4e).…”

Section: Resultsmentioning

confidence: 69%

“…We selected two PINK1 pathogenic mutations (p.W437X and p.G309D), 2 with similar stability and subcellular localization in comparison with the wild-type protein. 28 Mutant PINK1 W437X lacks the C-terminus and part of the kinase domain, yet it partly maintains the ability to phosphorylate specific substrates such as TRAP1. Conversely, kinase activity is lost by the missense mutant PINK1 G309D .…”

Section: Resultsmentioning

confidence: 99%

“…The constructs PINK1-FL, PINK1 W437X and PINK1 G309D have been described previously. 28 The PINK1 and PINK1 112-581 constructs were generated from PINK1-FL cDNA by introducing, respectively, an EcoRI restriction site at position 497 and a start codon at position 112 using the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The two fragments, 1-496 and 112-581, were then subcloned in pcDNA 3.1( þ ) (Invitrogen, Carlsbad, CA, USA) and HA-tagged at the C-terminus as described.…”

Section: Methodsmentioning

confidence: 99%

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The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

Michiorri

Gelmetti

Giarda³

et al. 2010

Cell Death Differ

225

176

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Mutations in the PINK1 gene cause autosomal recessive Parkinson's disease. The PINK1 gene encodes a protein kinase that is mitochondrially cleaved to generate two mature isoforms. In addition to its protective role against mitochondrial dysfunction and apoptosis, PINK1 is also known to regulate mitochondrial dynamics acting upstream of the PD-related protein Parkin. Recent data showed that mitochondrial Parkin promotes the autophagic degradation of dysfunctional mitochondria, and that stable PINK1 silencing may have an indirect role in mitophagy activation. Here we report a new interaction between PINK1 and Beclin1, a key pro-autophagic protein already implicated in the pathogenesis of Alzheimer's and Huntington's diseases. Both PINK1 N-and C-terminal are required for the interaction, suggesting that full-length PINK1, and not its cleaved isoforms, interacts with Beclin1. We also demonstrate that PINK1 significantly enhances basal and starvation-induced autophagy, which is reduced by knocking down Beclin1 expression or by inhibiting the Beclin1 partner Vps34. A mutant, PINK1 W437X , interaction of which with Beclin1 is largely impaired, lacks the ability to enhance autophagy, whereas this is not observed for PINK1 G309D , a mutant with defective kinase activity but unaltered ability to bind Beclin1. These findings identify a new function of PINK1 and further strengthen the link between autophagy and proteins implicated in the neurodegenerative process. Parkinson's disease (PD) is a frequent neurodegenerative disorder resulting from massive degeneration of the dopaminergic neurons in the substantia nigra. Although most cases are sporadic, several genes are known to cause familial PD, especially with early onset. 1 Mutations in the PINK1 gene are the second most frequent cause of autosomal recessive PD after those in the Parkin gene. 2,3 The PINK1 gene encodes a serine-threonine kinase with an N-terminal mitochondrial import sequence, first characterized as a protein aimed at maintaining mitochondrial integrity and preventing apoptosis in response to cellular stressors. 2,[4][5][6][7][8] This neuroprotective role is partly exerted through phosphorylation of the mitochondrial chaperon, TRAP1, although cytoplasm-restricted PINK1 was also shown to protect against MPTP damage. 9,10 The full-length PINK1 (PINK1-FL) is processed within mitochondria to generate two mature proteins; 4,11 all three isoforms localize both to the mitochondria and cytosol, their relative ratio being regulated by several factors. [10][11][12][13] Increasing data have demonstrated that absence of functional PINK1 induces abnormalities of mitochondrial morphology. 6,14,15 In several studies (mostly in Drosophila), PINK1 was shown to promote fission acting upstream of the Fis1-Drp1 machinery, and the mitochondrial phenotype observed in PINK1 knockout flies or silenced cells was associated to reduced fission. 16,17 Subsequent studies in mammalian cell systems contradicted these results, demonstrating that mutant or silenced PINK1 resulted in incre...

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Section: Resultsmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

Michiorri

Gelmetti

Giarda³

et al. 2010

Cell Death Differ

225

176

View full text Add to dashboard Cite

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“…PINK1 is a cytosolic serine/threonine kinase mainly localized at mitochondria via an N-terminal mitochondrial-targeting sequence (Silvestri et al, 2005;Valente et al, 2004). Cells isolated from patients with a PINK1 mutation exhibit reduced complex I activity and increased oxidative damage compared with controls (Hoepken et al, 2007).…”

Section: Mitochondrial Kinase Pink1 Is Critical For Maintaining Mitocmentioning

confidence: 99%

PINK1 as a Molecular Checkpoint in the Maintenance of Mitochondrial Function and Integrity

Koh

Chung

2012

Molecules and Cells

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Parkinson's disease (PD), the most prevalent neurodegenerative movement disorder, is characterized by an agedependent selective loss of dopaminergic (DA) neurons. Although most PD cases are sporadic, more than 20 responsible genes in familial cases were identified recently. Genetic studies using Drosophila models demonstrate that PINK1, a mitochondrial kinase encoded by a PD-linked gene PINK1, is critical for maintaining mitochondrial function and integrity. This suggests that mitochondrial dysfunction is the main cause of PD pathogenesis. Further genetic and cell biological studies revealed that PINK1 recruits Parkin, an E3 ubiquitin ligase encoded by another PD-linked gene parkin, to mitochondria and regulates the mitochondrial remodeling process via the Parkin-mediated ubiquitination of various mitochondrial proteins. PINK1 also directly phosphorylates the mitochondrial proteins Miro and TRAP1, subsequently inhibiting mitochondrial transport and mitochondrial oxidative damage, respectively. Moreover, recent Drosophila genetic analyses demonstrate that the neuroprotective molecules Sir2 and FOXO specifically complement mitochondrial dysfunction and DA neuron loss in PINK1 null mutants, suggesting that Sir2 and FOXO protect mitochondria and DA neurons downstream of PINK1. Collectively, these recent results suggest that PINK1 plays multiple roles in mitochondrial quality control by regulating its mitochondrial, cytosolic, and nuclear targets.

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“…3 PINK1 encodes a 63 kDa mitochondrial protein kinase, which is processed by mitochondrial proteases to generate two smaller isoforms. [4][5][6][7] We and others have shown that PINK1 acts as a key neuroprotective protein, aimed at preventing mitochondrial dysfunction and apoptotic cell death in response to multiple stress conditions. [8][9][10] This pro-survival activity is exerted through several mechanisms, including phosphorylation of the mitochondrial proteins TRAP1 and Omi/HtrA2, and regulation of mitochondrial calcium buffering.…”

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confidence: 99%

PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage

et al. 2013

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Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson's disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1-Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer. Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, with prevalence of 1% in the population older than 60 years. 1 Several biochemical abnormalities, including mitochondrial dysfunction, oxidative stress and misfolded protein damage, have been implicated in PD pathogenesis. 2 Although the majority of late-onset cases are sporadic, early-onset PD is frequently caused by mutations in genes with autosomal recessive inheritance, mainly Parkin (GeneID: 5071) and PINK1 (GeneID: 65018). 3 PINK1 encodes a 63 kDa mitochondrial protein kinase, which is processed by mitochondrial proteases to generate two smaller isoforms. [4][5][6][7] We and others have shown that PINK1 acts as a key neuroprotective protein, aimed at preventing mitochondrial dysfunction and apoptotic cell death in response to multiple stress conditions. [8][9][10] This pro-survival activity is exerted through several mechanisms, including phosphorylation of the mitochondrial proteins TRAP1 and Omi/HtrA2, and regulation of mitochondrial calcium buffering. [11][12][13][14] Increasing data now indicate that PINK1 acts upstream of Parkin in an evolutionary conserved pathway implicated in regulating mitochondrial biogenesis, trafficking and fusion/ fission events, to maintain mitochondrial network health. 15 In particular, upon mitochondrial depolarization, PINK1 processing is impaired, determining a marked accumulation of the full-length protein on the surface of dysfunctional mitochondria, where it recruits Parkin. This process results in the phosphorylation and/or ubiquitination of several mitochondrial substrates, leading to the selective quarantine of damaged mitochondria and their degradation through mitophagy. [16][17][18][19] In line with this, we reported that coexpression of mutant, but not wild-type (wt) PINK1, with mutant alpha-synuclein resulted in the formation of enlarged autophagosomes surrounding abnormal mitoch...

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Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism

Cited by 423 publications

References 42 publications

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy

PINK1 as a Molecular Checkpoint in the Maintenance of Mitochondrial Function and Integrity

PINK1 protects against cell death induced by mitochondrial depolarization, by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage

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