Yuzuru Imai

A Serine Protease, HtrA2, Is Released from the Mitochondria and Interacts with XIAP, Inducing Cell Death

X chromosome-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspase-3, -7, and -9. Smac/DIABLO, an inhibitor of XIAP, is released from mitochondria upon receiving apoptotic stimuli and binds to the BIR2 and BIR3 domains of XIAP, thereby inhibiting its caspase-inhibitory activity. Here we report that a serine protease called HtrA2/Omi is released from mitochondria and inhibits the function of XIAP by direct binding in a similar way to Smac. Moreover, when overexpressed extramitochondrially, HtrA2 induces atypical cell death, which is neither accompanied by a significant increase in caspase activity nor inhibited by caspase inhibitors, including XIAP. A catalytically inactive mutant of HtrA2, however, does not induce cell death. In short, HtrA2 is a Smac-like inhibitor of IAP activity with a serine protease-dependent cell death-inducing activity.

An Unfolded Putative Transmembrane Polypeptide, which Can Lead to Endoplasmic Reticulum Stress, Is a Substrate of Parkin

A putative G protein-coupled transmembrane polypeptide, named Pael receptor, was 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. Here, we show that the unfolded Pael receptor is a substrate of Parkin, the accumulation of which may cause selective neuronal death in AR-JP.

Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity

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 NH2-terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING). Here, we show that Parkin is a RING-type E3 ubiquitin-protein ligase which binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, we found that 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 suppressed 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.

CHIP Is Associated with Parkin, a Gene Responsible for Familial Parkinson's Disease, and Enhances Its Ubiquitin Ligase Activity

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. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced 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.

Phosphorylation of 4E-BP by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila

Dominant mutations in leucine‐rich repeat kinase 2 (LRRK2) are the most frequent molecular lesions so far found in Parkinsons disease (PD), an age‐dependent neurodegenerative disorder affecting dopaminergic (DA) neuron. The molecular mechanisms by which mutations in LRRK2 cause DA degeneration in PD are not understood. Here, we show that both human LRRK2 and the Drosophila orthologue of LRRK2 phosphorylate eukaryotic initiation factor 4E (eIF4E)‐binding protein (4E‐BP), a negative regulator of eIF4E‐mediated protein translation and a key mediator of various stress responses. Although modulation of the eIF4E/4E‐BP pathway by LRRK2 stimulates eIF4E‐mediated protein translation both in vivo and in vitro, it attenuates resistance to oxidative stress and survival of DA neuron in Drosophila. Our results suggest that chronic inactivation of 4E‐BP by LRRK2 with pathogenic mutations deregulates protein translation, eventually resulting in age‐dependent loss of DA neurons.

Parkin Suppresses Dopaminergic Neuron-Selective Neurotoxicity Induced by Pael-R in Drosophila

Parkin, an E3 ubiquitin ligase that degrades proteins with aberrant conformations, is associated with autosomal recessive juvenile Parkinsonism (AR-JP). The molecular basis of selective neuronal death in AR-JP is unknown. Here we show in an organismal system that panneuronal expression of Parkin substrate Pael-R causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. Coexpression of Parkin degrades Pael-R and suppresses its toxicity, whereas interfering with endogenous Drosophila Parkin function promotes Pael-R accumulation and augments its toxicity. Furthermore, overexpression of Parkin can mitigate alpha-Synuclein-induced neuritic pathology and suppress its toxicity. Our study implicates Parkin as a central player in the molecular pathway of Parkinsons disease (PD) and suggests that manipulating Parkin expression may provide a novel avenue of PD therapy.

Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression

Gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial as well as sporadic Parkinson’s disease characterized by age-dependent degeneration of dopaminergic neurons. The molecular mechanism of LRRK2 action is not known. Here we show that LRRK2 interacts with the microRNA (miRNA) pathway to regulate protein synthesis. Drosophila e2f1 and dp messenger RNAs are translationally repressed by let-7 and miR-184*, respectively. Pathogenic LRRK2 antagonizes these miRNAs, leading to the overproduction of E2F1/DP, previously implicated in cell cycle and survival control and shown here to be critical for LRRK2 pathogenesis. Genetic deletion of let-7, antagomir-mediated blockage of let-7 and miR-184* action, transgenic expression of dp target protector, or replacement of endogenous dp with a dp transgene non-responsive to let-7 each had toxic effects similar to those of pathogenic LRRK2. Conversely, increasing the level of let-7 or miR-184* attenuated pathogenic LRRK2 effects. LRRK2 associated with Drosophila Argonaute-1 (dAgo1) or human Argonaute-2 (hAgo2) of the RNA-induced silencing complex (RISC). In aged fly brain, dAgo1 protein level was negatively regulated by LRRK2. Further, pathogenic LRRK2 promoted the association of phospho-4E-BP1 with hAgo2. Our results implicate deregulated synthesis of E2F1/DP caused by the miRNA pathway impairment as a key event in LRRK2 pathogenesis and suggest novel miRNA-based therapeutic strategies.

PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy

Parkinsons disease genes PINK1 and parkin encode kinase and ubiquitin ligase, respectively. The gene products PINK1 and Parkin are implicated in mitochondrial autophagy, or mitophagy. Upon the loss of mitochondrial membrane potential (ΔΨm), cytosolic Parkin is recruited to the mitochondria by PINK1 through an uncharacterised mechanism – an initial step triggering sequential events in mitophagy. This study reports that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarisation of ΔΨm. The introduction of mutations at Ser65 suggests that phosphorylation of Ser65 is required not only for the efficient translocation of Parkin, but also for the degradation of mitochondrial proteins in mitophagy. Phosphorylation analysis of Parkin pathogenic mutants also suggests Ser65 phosphorylation is not sufficient for Parkin translocation. Our study partly uncovers the molecular mechanism underlying the PINK1-dependent mitochondrial translocation and activation of Parkin as an initial step of mitophagy.

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.

Parkinson's Disease-Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinsons disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.