Glutathione decreases in dopaminergic PC12 cells interfere
with the ubiquitin protein degradation pathway: relevance 
for Parkinson's disease?

Abstract

Parkinson's disease (PD) is characterized by the presence of proteinaceous neuronal inclusions called Lewy bodies in susceptible dopaminergic midbrain neurons. Inhibition of the ubiquitin-proteasome protein degradation pathway may contribute to protein build-up and subsequent cell death. Ubiquitin is normally activated for transfer to substrate proteins by interaction with the E1 ubiquitin ligase enzyme via a thiol ester bond. Parkinson's disease is also characterized by decreases in midbrain levels of total glutathione which could impact on E1 enzyme activity via oxidation of the active site sulfhydryl. We have demonstrated that increasing reductions in total glutathione in dopaminergic PC12 cells results in corresponding decreases in ubiquitin-protein conjugate levels suggesting that ubiquitination of proteins is inhibited in a glutathione-dependent fashion. Decreased ubiquitinated protein levels appears to be due to inhibition of E1 activity as demonstrated by reductions in endogenous E1-ubiquitin conjugate levels as well as decreases in the production of de novo E1-ubiquitin conjugates when glutathione is depleted. This is a reversible process as E1 activity increases upon glutathione restoration. Our data suggests that decreases in cellular glutathione in dopaminergic cells results in decreased E1 activity and subsequent disruption of the ubiquitin pathway. This may have implications for neuronal degeneration in PD. Ubiquitin is a highly conserved, 8.5 kDa, 76 residue protein found in all eukaryotes. It is present either in a free state or covalently bound to a variety of cytoplasmic, nuclear and integral membrane proteins, e.g. cell cycle regulators, transcription factors, tumor suppressors and oncoproteins. It is primarily known for its involvement in a pathway that regulates the bulk of intracellular protein turnover (Ciechanover and Schwartz 1994; Harshko and Ciechanover 1998). Ubiquitin acts as a covalent tag to mark damaged or shortlived proteins for degradation by the ATP-dependent multiprotease 26S proteasome complex (Wilkinson 1997(Wilkinson , 1999. Ubiquitin is first activated by conjugation of its carboxy terminal glycine residue to the thiol group of a cysteine residue on the E1 activating enzyme via an ester bond . It is then transferred to a cysteine thiol group on one of the several substrate-specific E2 conjugating enzymes and finally either conjugated directly to the side chain group of a lysine residue on an acceptor protein substrate or indirectly via an E3 ligating enzyme (Hershko et al. 1983;Pickart and Rose 1985;Haas and Bright 1988). Ubiquitin forms self-linked polyubiquitin chains on the protein substrate via isopeptide linkages between its lysine 48 residues. These polyubiquitin adducts act as the preferred substrates for rapid proteolysis by the 26S proteasome complex, which degrades the tagged protein to small peptides (Ciechanover and Schwartz 1994). If the ubiquitin pathway is not functioning properly, this can result in the build-up of proteins in the cytoplasm ultimate...

Histone deacetylase inhibitors prevent oxidative neuronal death independent of expanded polyglutamine repeats via an Sp1-dependent pathway

“…Although neuronal death associated with decreased glutathione content cannot be blocked by competitive or noncompetitive glutamate receptor antagonists, it can be effectively circumvented by treatment with the antioxidants vitamin E and idebenone (14). Glutathione depletion and hypofunction of glutathione-dependent antioxidant enzymes have been linked directly to the pathogenesis of stroke (15), Huntington's disease (16,17), and Parkinson's disease (18)(19)(20)(21) in studies of rodent models as well as human autopsy tissue.…”

Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome

“…Polyubiquitin is added to proteins targeted for UPS degradation by a series of ligases (E1, E2, and E3 ligases). Dithiocarbamates can lead to GSH oxidation (27), and GSH depletion results in the loss of E1 ligase activity (14). Ziram does not appear to act by lowering GSH, because depleting cellular GSH using buthionine sulfoximine (1 mM) had no effect on UPS activity, and ziram did not alter the amount of reduced GSH in the cells at concentrations up to 50 M (data not shown).…”

“…Ziram-treated SK-N-MC cells were washed with phosphate-buffered saline and then lysed in a thiol stabilizing buffer using the method of Jha et al (14). The samples were sonicated for 10 s, centrifuged for 15 min at 13,000 ϫ g, mixed with 2 parts thiol gel buffer (33 mM Tris-HCl, pH 6.8, 2.7 M urea, 2.5% sodium dodecyl sulfate, and 13% glycerol), and boiled for 2 min, and 10 g of protein/ lane was loaded on a 12% SDS-PAGE gel (15).…”

“…The carbon disulfide progenitor enzone (12) was also inactive. Environmental degradation converts ethylenebis(dithiocarbamate) fungicides to ethylenethiourea (13) and sodium methyldithiocarbamate (14) to methyl isothiocyanate (15), all of which were inactive. Thus, the dialkyldithiocarbamate moiety is required to alter UPS activity.…”

The scientific and clinical basis for the treatment of Parkinson disease (2009)

“…It is possible that cell death results from an interactive network of pathogenic factors in which no one component is essential and where the initiating pathogenic factor may vary in different individuals (figure 6). Indeed, laboratory studies demonstrate that oxidative stress can damage mitochondria and proteasomes, [152][153][154] mitochondrial dysfunction leads to oxidative stress and proteasomal damage, 155,156 and proteasomal dysfunction causes oxidative stress and mitochondrial dysfunction. 155,157,158 Furthermore, it has been shown that oxidative stress and proteasome inhibition act synergistically to promote protein misfolding.…”