Isolated RING2 domain of parkin is sufficient for E2-dependent E3 ligase activity.

The E3 ubiquitin ligase activity of the parkin protein is implicated in playing a protective role against neurodegenerative disorders including Parkinson's, Huntington's, and Alzheimer's diseases. Parkin has four zinc-containing domains: RING0, RING1, IBR (in-between ring), and RING2. Mutational analysis of full-length parkin suggests that the C-terminal RING2 domain contains the catalytic core. Here, a catalytically competent recombinant RING2 containing an N-terminal GB1 solubility peptide is described. In cell-free in vitro ubiqitination reactions, the RING2 construct catalyzes the transfer of ubiquitin from the E2 enzyme UbcH7 to the attached GB1 tag. This intramolecular autoubiquitination reaction indicates that (a) ubiquitination by RING2 can occur in the absence of other parkin domains and (b) UbcH7 can interact directly with RING2 to transfer its bound ubiquitin. Mass spectrometry identified sites of mono- and diubiquitin attachment to two surface-exposed lysine residues (Lys24 and Lys39) on the GB1 peptide. The sites of diubiquitination involved Lys11 and Lys48 linkages, which have been identified as general signals for proteasome degradation. Cleaving the linker between the GB1 tag and RING2 resulted in loss of ubiquitination activity, indicating that the substrate must be tethered to RING2 for proper presentation to the active site. Atomic absorption spectrometry and selective mutation of zinc ligands indicated that only one of the two zinc binding sites on RING2, the N-terminal site, needs to be occupied by zinc for expression of ubiquitination activity. This is consistent with the hypothesis that the second, C-terminal, zinc binding site on RING2 has a regulatory rather than a catalytic function.

Infectious prion protein alters manganese transport and neurotoxicity in a cell culture model of prion disease.

Protein misfolding and aggregation are considered key features of many neurodegenerative diseases, but biochemical mechanisms underlying protein misfolding and the propagation of protein aggregates are not well understood. Prion disease is a classical neurodegenerative disorder resulting from the misfolding of endogenously expressed normal cellular prion protein (PrP(C)). Although the exact function of PrP(C) has not been fully elucidated, studies have suggested that it can function as a metal binding protein. Interestingly, increased brain manganese (Mn) levels have been reported in various prion diseases indicating divalent metals also may play a role in the disease process. Recently, we reported that PrP(C) protects against Mn-induced cytotoxicity in a neural cell culture model. To further understand the role of Mn in prion diseases, we examined Mn neurotoxicity in an infectious cell culture model of prion disease. Our results show CAD5 scrapie-infected cells were more resistant to Mn neurotoxicity as compared to uninfected cells (EC(50)=428.8 µM for CAD5 infected cells vs. 211.6 µM for uninfected cells). Additionally, treatment with 300 µM Mn in persistently infected CAD5 cells showed a reduction in mitochondrial impairment, caspase-3 activation, and DNA fragmentation when compared to uninfected cells. Scrapie-infected cells also showed significantly reduced Mn uptake as measured by inductively coupled plasma-mass spectrometry (ICP-MS), and altered expression of metal transporting proteins DMT1 and transferrin. Together, our data indicate that conversion of PrP to the pathogenic isoform enhances its ability to regulate Mn homeostasis, and suggest that understanding the interaction of metals with disease-specific proteins may provide further insight to protein aggregation in neurodegenerative diseases.

Selenium-modified TiO2 and its impact on photocatalysis.

This work describes the preparation of a selenium-modified TiO(2) photocatalyst and a preliminary evaluation of its photocatalytic activity. Se-TiO(2) displayed greater visible absorption than undoped TiO(2) and was still capable of degrading quinoline at a slightly faster rate than undoped TiO(2) under UV light. Se-TiO(2) was also able to degrade organic molecules under purely visible light by a single electron transfer pathway. Irradiation with >435 nm light showed no evidence of efficient production of HO•-like species. Se-TiO(2) was also examined under hypoxic conditions, where the Se atoms were capable of trapping photogenerated electrons as evidenced by XPS.

Vanadium induces dopaminergic neurotoxicity via protein kinase Cdelta dependent oxidative signaling mechanisms: relevance to etiopathogenesis of Parkinson's disease.

Environmental exposure to neurotoxic metals through various sources including exposure to welding fumes has been linked to an increased incidence of Parkinson's disease (PD). Welding fumes contain many different metals including vanadium typically present as particulates containing vanadium pentoxide (V2O5). However, possible neurotoxic effects of this metal oxide on dopaminergic neuronal cells are not well studied. In the present study, we characterized vanadium-induced oxidative stress-dependent cellular events in cell culture models of PD. V2O5 was neurotoxic to dopaminergic neuronal cells including primary nigral dopaminergic neurons and the EC50 was determined to be 37 microM in N27 dopaminergic neuronal cell model. The neurotoxic effect was accompanied by a time-dependent uptake of vanadium and upregulation of metal transporter proteins Tf and DMT1 in N27 cells. Additionally, vanadium resulted in a threefold increase in reactive oxygen species generation, followed by release of mitochondrial cytochrome c into cytoplasm and subsequent activation of caspase-9 (>fourfold) and caspase-3 (>ninefold). Interestingly, vanadium exposure induced proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield a 41 kDa catalytically active fragment resulting in a persistent increase in PKCdelta kinase activity. Co-treatment with pan-caspase inhibitor Z-VAD-FMK significantly blocked vanadium-induced PKCdelta proteolytic activation, indicating that caspases mediate PKCdelta cleavage. Also, co-treatment with Z-VAD-FMK almost completely inhibited V2O5-induced DNA fragmentation. Furthermore, PKCdelta knockdown using siRNA protected N27 cells from V2O5-induced apoptotic cell death. Collectively, these results demonstrate that vanadium can exert neurotoxic effects in dopaminergic neuronal cells via caspase-3-dependent PKCdelta cleavage, suggesting that metal exposure may promote nigral dopaminergic degeneration.