Hiroyoshi Ariga

DJ‐1 has a role in antioxidative stress to prevent cell death

Deletion and point (L166P) mutations of DJ‐1 have recently been shown to be responsible for the onset of familial Parkinsons disease (PD, PARK7). The aim of this study was to determine the role of DJ‐1 in PD. We first found that DJ‐1 eliminated hydrogen peroxide in vitro by oxidizing itself. We then found that DJ‐1 knockdown by short interfering RNA rendered SH‐SY5Y neuroblastoma cells susceptible to hydrogen peroxide‐, MPP+‐ or 6‐hydroxydopamine‐induced cell death and that cells harbouring mutant forms of DJ‐1, including L166P, became susceptible to death in parallel with the loss of oxidized forms of DJ‐1. These results clearly showed that DJ‐1 has a role in the antioxidative stress reaction and that mutations of DJ‐1 lead to cell death, which is observed in PD.

Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Previous studies have demonstrated that chronic systemic exposure of Lewis rats to rotenone produced many features of PD, and cerebral tauopathy was also detected in the case of severe weight loss. The present study was designed to assess the neurotoxicity of rotenone after daily oral administration for 28 days at several doses in C57BL/6 mice. In addition, we examined the protective effects of 4‐phenylbutyrate (4‐PBA) on nigral dopamine (DA) neurons in rotenone‐treated mice. 4‐PBA was injected intraperitoneally daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone at high doses induced specific nigrostriatal DA neurodegeneration, motor deficits and the up‐regulation of α‐synuclein in the surviving DA neurons. In contrast to the Lewis rat model, cerebral tauopathy was not detected in this mouse model. 4‐PBA inhibited rotenone‐induced neuronal death and decreased the protein level of α‐synuclein. These results suggest that this rotenone mouse model may be useful for understanding the mechanism of DA neurodegeneration in PD, and that 4‐PBA has a neuroprotective effect in the treatment of PD.

Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress

DJ-1 is a novel oncogene and causative gene for familial form of the Parkinsons disease (PD). DJ-1 has been shown to play a role in anti-oxidative stress by eliminating reactive oxygen species (ROS). The onset of PD is thought to be caused by oxidative stress and mitochondrial injury, which leads to protein aggregation that results in neuronal cell death. However, the mechanism by which DJ-1 triggers the onset of PD is still not clear. In this study, we analyzed association and localization of DJ-1 and its mutants with various chaperones. The results showed that DJ-1 and its mutants were associated with Hsp70, CHIP and mtHsp70/Grp75, a mitochondria-resident Hsp70, and that L166P and M26I mutants found in PD patients were strongly associated with Hsp70 and CHIP compared to wild-type and other DJ-1 mutants. DJ-1 and its mutants were colocalized with Hsp70 and CHIP in cells. Furthermore, association and colocalization of wildtype DJ-1 with mtHsp70 in mitochondria were found to be enhanced by treatment of cells with H2O2. These results suggest that translocation of DJ-1 to mitochondria after oxidative stress is carried out in association with chaperones.

Neuroprotective function of DJ-1 in Parkinson's disease.

Parkinsons disease (PD) is caused by dopaminergic neuronal death in the substantia nigra, resulting in a reduced level of dopamine in the striatum. Oxidative stress and mitochondrial dysfunction are thought to be major causes of neurodegeneration in PD. Although genetic and environmental factors are thought to affect the onset of PD, precise mechanisms at the molecular level have not been elucidated. The DJ-1 gene is a causative gene for familial PD (park7) and also an oncogene. DJ-1 has various functions, including transcriptional regulation, antioxidative stress reaction, and chaperone, protease, and mitochondrial regulation, and its activity is regulated by its oxidative status, especially that of cysteine 106 (C106) of DJ-1. Excess oxidation of DJ-1, which renders DJ-1 inactive, has been observed in patients with sporadic PD and Alzheimers disease, suggesting that DJ-1 also participates in the onset and pathogenesis of sporadic PD as well as familial PD. DJ-1 is also a stress sensor and its expression is increased upon various stresses, including oxidative stress. In this review, we describe functions of DJ-1 against oxidative stress and possible roles of DJ-1 in the pathogenesis of PD.

Proper SUMO-1 conjugation is essential to DJ-1 to exert its full activities

DJ-1 is a multifunctional protein that plays roles in transcriptional regulation and antioxidative stress, and loss of its function is thought to result in the onset of Parkinsons disease (PD). Here, we report that DJ-1 was sumoylated on a lysine residue at amino-acid number 130 (K130) by PIASxα or PIASy. The K130 mutation abrogated all of the functions of DJ-1, including ras-dependent transformation, cell growth promotion and anti-UV-induced apoptosis activities. Sumoylation of DJ-1 was increased after UV irradiation concomitant with a pI shift to an acidic point of DJ-1. Furthermore, L166P, a mutant DJ-1 found in PD patients, and K130RX, an artificial mutant containing four mutations in DJ-1, were improperly sumoylated, and they became insoluble, partly localized in the mitochondria and degraded by the proteasome system. Both L166P-expressing cells and DJ-1-knockdown cells were found to be highly susceptible to UV-induced cell apoptosis.

A Role for the Cleaved Cytoplasmic Domain of E-cadherin in the Nucleus

Cell-cell contacts play a vital role in intracellular signaling, although the molecular mechanisms of these signaling pathways are not fully understood. E-cadherin, an important mediator of cell-cell adhesions, has been shown to be cleaved by γ-secretase. This cleavage releases a fragment of E-cadherin, E-cadherin C-terminal fragment 2 (E-cad/CTF2), into the cytosol. Here, we study the fate and function of this fragment. First, we show that coexpression of the cadherin-binding protein, p120 catenin (p120), enhances the nuclear translocation of E-cad/CTF2. By knocking down p120 with short interfering RNA, we also demonstrate that p120 is necessary for the nuclear localization of E-cad/CTF2. Furthermore, p120 enhances and is required for the specific binding of E-cad/CTF2 to DNA. Finally, we show that E-cad/CTF2 can regulate the p120-Kaiso-mediated signaling pathway in the nucleus. These data indicate a novel role for cleaved E-cadherin in the nucleus.

Identification of heterochromatin protein 1 (HP1) as a phosphorylation target by Pim-1 kinase and the effect of phosphorylation on the transcriptional repression function of HP11

Pim‐1, a protooncogene product, is a serine/threonine kinase and is thought to play a role in signal transduction in blood cells. Few phosphorylated target proteins for Pim‐1, however, have been identified. In the present study, two‐hybrid screening to clone cDNAs encoding proteins binding to Pim‐1 was carried out, and a cDNA for heterochromatin protein 1γ (HP1γ) was obtained. Binding assays both in yeast and in vitro pull‐down using the purified HP1γ and Pim‐1 expressed in Escherichia coli showed that Pim‐1 directly bound to the chromo shadow domain of HP1γ. HP1γ was also associated with Pim‐1 in human HeLa cells and the serine clusters located at the center of HP1γ were phosphorylated by Pim‐1 in vitro. Furthermore, a transcription repression activity of HP1γ was further stimulated by the deletion of the serine clusters targeted by Pim‐1. These results suggest that Pim‐1 affects the structure or silencing of chromatin by phosphorylating HP1.

DJ-1 binds to mitochondrial complex I and maintains its activity

Parkinsons disease (PD) is caused by neuronal cell death, and oxidative stress and mitochondrial dysfunction are thought to be responsible for onset of PD. DJ-1, a causative gene product of a familial form of Parkinsons disease, PARK7, plays roles in transcriptional regulation and anti-oxidative stress. The possible mitochondrial function of DJ-1 has been proposed, but its exact function remains unclear. In this study, we found that DJ-1 directly bound to NDUFA4 and ND1, nuclear and mitochondrial DNA-encoding subunits of mitochondrial complex I, respectively, and was colocalized with complex I and that complex I activity was reduced in DJ-1-knockdown NIH3T3 and HEK293 cells. These findings suggest that DJ-1 is an integral mitochondrial protein and that DJ-1 plays a role in maintenance of mitochondrial complex I activity.

The Actin-Binding Domain of Slac2-a/Melanophilin Is Required for Melanosome Distribution in Melanocytes

ABSTRACT Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (ΔABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

MM-1, a novel c-Myc-associating protein that represses transcriptional activity of c-Myc.

We have isolated the cDNA encoding a novel c-Myc-binding protein, MM-1, by the yeast two-hybrid screening of a human HeLa cell cDNA library. The protein deduced from the cDNA comprises 167 amino acids and was localized in the nucleus of introduced COS-I cells. The MM-1 mRNA was highly expressed in human pancreas and skeletal muscle and moderately in other tissues. As for the c-Myc binding, glutathione S-transferase MM-1 expressed in Escherichia coli bound in vitro to c-Myc translated in reticulocyte lysate, and almost whole, the MM-1 molecule was necessary for the binding in the yeast two-hybrid system. The mammalian two-hybrid assays in hamster CHO cells revealed that MM-1 interacts in vivo with the N-terminal domain covering themyc box 2, a transcription-activating domain, of c-Myc. Furthermore, MM-1 repressed the activation of E-box-dependent transcription by c-Myc.