Protein : Protein aggregation induced by protein oxidation

Department of Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA.
Journal of Chromatography B (Impact Factor: 2.73). 10/2008; 873(1):8-14. DOI: 10.1016/j.jchromb.2008.04.025
Source: PubMed


When the level of reactive oxygen species (ROS) in cells exceeds a genetically coded defense capacity, the cells experience damage to vital components such as DNA, proteins and lipids that leads to non-specific interactions and the production of a series of high molecular weight protein aggregates. The dynamics of oxidative stress induced aggregation were studied here using model proteins and yeast. Model proteins were oxidized at increasing ROS concentrations and analyzed using size exclusion chromatography (SEC). Changes in the SEC elution profile showed that aggregation happens in stages and protein fragments produced as a result of oxidation also give rise to aggregates. Yeast cells were stressed with hydrogen peroxide to investigate in vivo aggregation. Equal amounts from control and oxidized lysates were chromatographed on a size exclusion column and proteins of molecular weight exceeding 700 kDa were collected from both samples which were then differentially labeled using light and heavy isotope coded N-acetoxysuccinamide and mixed in a 1:1 ratio. The coded mixture was analyzed using LC/MS and peptides that appeared as singlets representing the proteins that aggregated with higher molecular mass protein complexes were identified. Twenty-five proteins were identified to be of this type. Fifteen members in this group were found to have been carbonylated. These proteins are part of the proteome known as the aggresome. The protein content of the aggresome may provide vital information for mechanistic studies targeting disease and aging.

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    • "Free radical-mediated protein modification could be an alternative approach to modify the properties of protein films. Generation of free radicals during processing or storage can alter the molecular weight of biopolymers (Farahnaky, Gray, Mitchell, & Hill, 2003), by either polymerisation or fragmentation (Kocha, Yamaguchi, Ohtaki, Fukuda, & Aoyagi, 1997; Mirzaei & Regnier, 2008). Several chemicals have been known to induce protein oxidation and fragmentation . "

    • "Protein aggregate formation affects cellular function and has been implicated in various aging related disorders [12] and complications as reviewed by Talaei [13]. Indeed, cells from WS patients show high levels of oxidatively modified proteins [14], which might contribute to an accumulation of reactive oxygen species (ROS) [15] [16], in turn likely negatively affecting DNA stability [17] and possibly contributing to aggregation of oxidized proteins [18]. Moreover, while in normal cells oxidative damage of DNA leads to inhibition of proliferation, this process seems impaired in WS [19]. "
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    ABSTRACT: Werner syndrome (WS) protein is involved in DNA repair and its truncation causes Werner syndrome, an autosomal recessive genetic disorder with a premature ageing phenotype. WRN protein mutation is currently known as the primary cause of WS. In cultured WS fibroblasts, we found an increase in cytosolic aggregates and hypothesized that the phenotype is indirectly related to an excess activation of the mTOR (mammalian target of rapamycin) pathway, leading to the formation of protein aggregates in the cytosol with increasing levels of oxidative stress. As we found that the expression levels of the two main H2S producing enzymes, cystathionine beta synthase and cystathionine gamma lyase, were lower in WS cells compared to normal, we investigated the effect of administration of H2S as NaHS (50μM). NaHS treatment blocked mTOR activity, abrogated protein aggregation and normalized the phenotype of WS cells. Similar results were obtained by treatment with the mTOR inhibitor rapamycin. This is the first report suggesting that hydrogen sulfide administered as NaHS restores proteostasis and cellular morphological phenotype of WS cells and hints to the importance of transsulfuration pathway in WS.
    Pharmacological Research 05/2013; 74. DOI:10.1016/j.phrs.2013.04.011 · 4.41 Impact Factor
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    • "Carbonylation is known to cause inappropriate inter- and intra-protein cross-links as well as protein misfolding, which in turn results in the formation of high-molecular-mass aggregates (Grune et al., 1997; Mirzaei and Regnier, 2008). As these aggregates get larger they precipitate, become resistant to proteolytic degradation and reduce cell viability (Nyström, 2005; Maisonneuve et al., 2008a). "
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    ABSTRACT: Previous work from our laboratory implicated protein carbonylation in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Subsequent in vitro studies revealed that the accumulation of protein carbonyls, triggered by glutathione deficiency or proteasome inhibition, leads to protein aggregation and neuronal cell death. These findings prompted us to investigate if their association can be also established in vivo. In this study, we characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice. The results show that protein carbonyls accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. We also show a temporal correlation between protein carbonylation (but not oxidative stress) and apoptosis. Furthermore, carbonyl levels are significantly higher in apoptotic cells than in live cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are present during the course of EAE. The LC3-II/LC3-I ratio is significantly reduced in both acute and chronic EAE indicating reduced autophagy and explaining why aggresomes accumulate in this disorder. Altogether, our data suggest a link between protein oxidation and neuronal/glial cell death in vivo and also demonstrate impaired proteostasis in this widely used murine model of MS.
    ASN Neuro 03/2013; 5(2). DOI:10.1042/AN20120088 · 4.02 Impact Factor
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