Protein : Protein aggregation induced by protein oxidation
ABSTRACT 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 . "
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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). "
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 · 4.44 Impact Factor
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- "It has been shown that carbonylation causes 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., 2008). "
ABSTRACT: While the build-up of oxidized proteins within cells is believed to be toxic, there is currently no evidence linking protein carbonylation and cell death. In the present study, we show that incubation of nPC12 (neuron-like PC12) cells with 50 μM DEM (diethyl maleate) leads to a partial and transient depletion of glutathione (GSH). Concomitant with GSH disappearance there is increased accumulation of PCOs (protein carbonyls) and cell death (both by necrosis and apoptosis). Immunocytochemical studies also revealed a temporal/spatial relationship between carbonylation and cellular apoptosis. In addition, the extent of all three, PCO accumulation, protein aggregation and cell death, augments if oxidized proteins are not removed by proteasomal degradation. Furthermore, the effectiveness of the carbonyl scavengers hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies PCOs as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. While the study focused mostly on nPC12 cells, experiments in primary neuronal cultures yielded the same results. The findings are also not restricted to DEM-induced cell death, since a similar relationship between carbonylation and apoptosis was found in staurosporine- and buthionine sulfoximine-treated nPC12 cells. In sum, the above results show for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. To the best of our knowledge, this is the first study to place direct (oxidative) protein carbonylation within the apoptotic pathway.ASN Neuro 02/2012; 4(3). DOI:10.1042/AN20110064 · 4.44 Impact Factor