Carbonylated Proteins Are Detectable Only in a Degradation-Resistant Aggregate State in Escherichia coli

Abstract

Carbonylation is currently used as a marker for irreversible protein oxidative damage. Several studies indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts. In this study, we observed that in Escherichia coli, more than 95% of the total carbonyl content consisted of insoluble protein and most were cytosolic proteins. We thereby demonstrate that, in vivo, carbonylated proteins are detectable mainly in an aggregate state. Finally, we show that detectable carbonylated proteins are not degraded in vivo. Here we propose that some carbonylated proteins escape degradation in vivo by forming carbonylated protein aggregates and thus becoming nondegradable. In light of these findings, we provide evidence that the accumulation of nondegradable carbonylated protein presented in an aggregate state contributes to the increases in carbonyl content observed during senescence.Proteins can become modified by a large number of reactions involving reactive oxygen species. Among these modifications, carbonylation has attracted a great deal of attention due to its irreversible and irreparable nature. Carbonyl derivatives are formed by a direct metal-catalyzed oxidative attack on the amino acid side chains of proline, arginine, lysine, and threonine (2). With the development of sensitive immunochemical methods for the detection of protein carbonyls, the presence of such groups has been extensively used as a marker of reactive oxygen species-mediated protein oxidation (17) and associated with a large number of age-related disorders, including Parkinson's disease, Alzheimer's disease, and cancer (5, 17). While carbonylated proteins are considered soluble in healthy cells, a decrease in proteolysis has been suggested to provoke increases in levels of carbonylated protein which may form aggregates during aging or disease (5,(12)(13)(14). Interestingly, in starvation, aging, or disease states, only some proteins appear more prone to carbonylation (3,11,17,24). Finally, in vivo studies using exponentially grown Escherichia coli cells or other organisms indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts (10, 14-16, 18, 21). Moreover, several groups have postulated that carbonylation may act as a tag for degradation (10,15,21).Here, contrary to observations made previously by Dukan et al. (10) and other groups, we show, using E. coli exponentialor stationary-phase cells, that carbonylated proteins are mainly cytosolic and that most of them are detectable in an aggregate state that does not degrade with time. As a consequence, we propose that increases in carbonyl content observed during bacterial senescence could be due at least in part to the accumulation of nondegradable carbonylated proteins presented in an aggregate state. MATERIALS AND METHODSBacterial strain and medium. E. coli MG1655 was grown aerobically or anaerobically in liquid Luria-Bertani (LB) medium in a rotary shaker at 37°C and 200 rpm.Protein preparation. Exponential (optical...

Protein aggregation in bacteria: the thin boundary between functionality and toxicity

“…Furthermore, treating E. coli cells with streptomycin led to aggregation of metabolic proteins and an increase in the level of protein carbonylation, which is a marker for irreversible protein oxidation. The accumulation of such non-degradable carbonylated proteins in an aggregate state contributes to the increase in carbonyl content observed during bacterial senescence (Dukan & Nyström, 1998;Maisonneuve et al, 2008b). An extensive proteome analysis of oxidized protein upon streptomycin treatment showed that streptomycin-resistant strains overexpress peroxiredoxin AhpCF, a primary scavenger of free radicals (Ling et al, 2012).…”

YajL, Prokaryotic Homolog of Parkinsonism-associated Protein DJ-1, Functions as a Covalent Chaperone for Thiol Proteome

“…Most of the YajL substrates belong to the thiol proteome, a set of thiol-or disulfide-containing proteins that are retained on activated thiol-Sepharose (shown in boldface characters in Table 1) (21). Several of these proteins form disulfides after menadione treatment of bacteria (underlined in Table 1), whereas others (S4, L14, AdhE, GAPDH, and Tpx (22)) harbor H 2 O 2 -mediated thiol modifications or are heavily oxidized and accumulate in proteolysis-deficient bacteria (AceE, RpoB, EF-Tu, and DnaK (38)). …”

“…Proteins printed in boldface belong to the thiol proteome (i.e. thiol-and disulfidecontaining proteins isolated on activated thiol-Sepharose (21)); underlined proteins contain disulfide(s) after menadione treatment (21); proteins in italics possess cysteines essential for metal or FeS cluster binding; proteins with asterisks are heavily oxidized upon oxidative stress (22,38); and shaded ribosomal proteins form aggregates in the yajL mutant (7). FIGURE 5.…”

Cardiovascular Redox and Ox Stress Proteomics

“…For example, protein carbonyls are initially formed under metal-catalyzed oxidations, can undergo further modification, act as a signal for protein degradation, and have also been suggested as being a reversible enzymatic modification. Thus, the levels of carbonyls measured may vary depending on their further reactivity, cellular antioxidant status and detoxification capacity, and presence of degradative enzymes (177). These factors would indicate that the carbonyl content may be in constant flux, and, therefore, would not reflect the total oxidative state of the cell.…”