Redox Control of 20S Proteasome Gating

Article (PDF Available)inAntioxidants & Redox Signaling 16(11):1183-94 · March 2012with57 Reads
DOI: 10.1089/ars.2011.4210 · Source: PubMed
Abstract
The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin- and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities. The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation. Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal α5 subunit of yeast cells grown to the stationary phase in glucose-containing medium. We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity. S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.
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    • "was determined according to the previously described method (Silva et al. 2012). Cells and brain tissue in each treatment group were lysed in RIPA buffer, except for the absence of protease inhibitor cocktail. "
    [Show abstract] [Hide abstract] ABSTRACT: Protein aggregation is an important feature of neurodegenerative disorders. In Alzheimer's disease (AD) protein aggregates are composed of hyperphosphorylated Tau and amyloid beta peptide (Aβ). Despite the involvement and identification of the molecular composition of these aggregates, their role in AD pathophysiology is not fully understood. However, depositions of these insoluble aggregates are typically reported as pathogenic and toxic for cell homeostasis. New evidences suggest that the deposition of these aggregates is a protective mechanism that preserves cell from toxic insults associated with the early stages of neurodegenerative diseases. To better understand the biological role of the protein aggregation with regard its effects in cellular homeostasis, the present study investigated the role of insoluble Tau and Tau aggregates on crucial cellular parameters such as redox homeostasis, proteasome activity and autophagy in hippocampal cell cultures and hippocampus of aged Lewis rats using a rotenone-induced aggregation model. Neurons were exposed to rotenone in different concentrations and exposure time aiming to determine the interval required for Tau aggregation. Our experimental design allowed us to demonstrate that rotenone exposure induces Tau hyperphosphorylation and aggregation in a concentration and time-dependent manner. Oxidative stress triggered by rotenone exposure was observed with the absence of Tau aggregates and was reduced or absent when Tau aggregates were present. This reduction of oxidative stress along with the presence of insoluble Tau was independent of alterations in antioxidant enzymes activities or cell death. In addition, rotenone induced oxidative stress was mainly associated with decrease in proteasome activity and autophagy flux. Conversely, when insoluble Tau appeared, autophagy turns to be overactivated while proteasome activity remained low. Our studies significantly advance the understanding that Tau aggregation might exert protective cellular effects, at least briefly, when neurons are facing neurodegeneration stimulus. We believe that our data add more complexity for the understanding of protein aggregation role in AD etiology.
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    • "S-glutathionylation of α5-C221 and –C76 residues as observed in extracts from yeast cells grown to stationary phase in glucoserich medium, degraded oxidized proteins at an increased rate [30]. "
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