Identification of protein carbonyls after two-dimensional electrophoresis

Conrad, Craig C.; Choi, Joungil; Malakowsky, Christina A.; Talent, John M.; Dai, Rong; Marshall, Pam L.; Gracy, Robert W.

doi:10.1002/1615-9861(200107)1:7<829::aid-prot829>3.0.co;2-r

Cited by 80 publications

(37 citation statements)

References 14 publications

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“…Protein samples (100 g) in rehydration buffer containing 7 M urea, 2 M thiourea, 4% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 100 mM dithiothreitol, 0.2% (vol/vol) ampholyte 3-10 (Bio-Rad), and 0.01% (wt/vol) bromophenol blue were adsorbed onto 17-cm immobilized pH gradient strips (linear pH 3 to 10 gradient). After isoelectric focusing, the strips were (i) subjected to equilibration for 20 min in 60 mM Tris base containing 2.3% (wt/vol) sodium dodecyl sulfate (SDS), 10% (vol/vol) glycerol, 5% (vol/vol) ␤-mercaptoethanol, and 0.1% (wt/ vol) bromophenol blue or (ii) first derivatized with 2,4-dinitrophenylhydrazone (6). Separation based on molecular weight was performed with a 9% acrylamide gel using the Protean II XL SDS-polyacrylamide gel electrophoresis (PAGE) multicell slab gel system (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Existence of Abnormal Protein Aggregates in Healthy Escherichia coli Cells

Maisonneuve

Fraysse

Moinier³

et al. 2008

J Bacteriol

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Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and "healthy" Escherichia coli cells. We observed overrepresentation of "aberrant proteins," as well as substrates of the major conserved chaperone DnaK (Hsp70) and the protease ClpXP (a serine protease), in the aggregates. In addition, the protein aggregates appeared to interact with chaperones known to be involved in the aggregate repair pathway, including ClpB, GroEL, GroES, and DnaK. Finally, we showed that the levels of reactive oxygen species and unfolded or misfolded proteins determine the levels of protein aggregates. Our results led us to speculate that protein aggregates may function as a temporary "trash organelle" for cellular detoxification.Formation or accumulation of aggregated proteins, which are present in all organisms, has become an important area of intensive research, mainly due to observations that there is a link with many disorders, including aging or neurodegenerative diseases (16).Major substrates for aggregation are unfolded or misfolded proteins. Indeed, misfolded proteins inappropriately expose hydrophobic surfaces normally buried in the protein's interior, leading to nonnative conformations able to interact and form aggregates. In cells, protein folding may fail because of amino acid misincorporation. Moreover, DnaK/Hsp70 and GroEL/ Hsp60 chaperone protein substrates, as well as partially degraded proteins, are more prone to unfolding and thus to aggregation (15).A common theory is that protein aggregation may be the consequence of a failed quality control mechanism normally charged with repairing or removing the misfolded or unfolded proteins. Indeed, to maintain functional proteins, the cells contain chaperones or proteases that assist proper protein folding and disaggregation of aggregates (17). For instance, Hsp104/ClpB has the capacity to rescue unfolded or misfolded proteins from an aggregated state in cooperation with the Hsp70/DnaK chaperone system (9). However, as recently shown in Huntington's disease, protein aggregates could serve as "temporary storage zones" within cells in order to maintain their function and integrity for prolonged periods of time (1, 13).A common opinion is that in healthy cells, the levels of formation and accumulation of protein aggregates are extremely low, although protein unfolding occurs constantly. Indeed, several groups have observed a virtual absence of protein aggregates in healthy cells (2, 5, 15), although it is possible that insufficient material was used for detection.Here we isolated and characterized protein aggregates in exponentially growing "healthy" Escherichia coli cells using classical procedures to extract E. coli insoluble cell fractions and protein aggregates. Our results led us to speculate that the...

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Section: Methodsmentioning

confidence: 99%

Existence of Abnormal Protein Aggregates in Healthy Escherichia coli Cells

Maisonneuve

Fraysse

Moinier³

et al. 2008

J Bacteriol

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“…Following isoelectric focusing, the strips were subjected to in-strip dinitrophenyl (DNP) derivatization by incubating (15 minutes at 22°C) in 2 N HCl/10 mmol/L 2,4-dinitrophenyl-hydrazine (Sigma). 34,35 Strips were washed with 2 mol/L Tris-base/30% glycerol (15 minutes); equilibrated in 50 mmol/L Tris-HCl (pH 8.8) containing 6 mol/L urea, 2% sodium dodecyl sulfate, 30% glycerol, and 1% dithiothreitol (15 minutes); then reequilibrated in the same buffer containing 2.5% iodoacetamide instead of dithiothreitol (15 minutes). The strips were then subjected to a second dimension gradient gel SDS-PAGE, transferred to polyvinylidene difluoride membranes, then immunoblotted with goat anti-DNP antibody (Bethyl Laboratories, Inc., Montgomery, TX).…”

Section: Methodsmentioning

confidence: 99%

Keratin mutation primes mouse liver to oxidative injury†

Zhou

Chen

et al. 2005

Hepatology

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Mutation of the cytoskeletal intermediate filament proteins keratin 8 and keratin 18 (K8/K18) is associated with cirrhosis in humans, whereas transgenic mice that overexpress K18 Arg893 Cys (R89C) have significant predisposition to liver injury. To study the mechanism of keratinassociated predisposition to liver injury, we used mouse microarrays to examine genetic changes associated with hepatocyte keratin mutation and assessed the consequences of such changes. Liver gene expression was compared in R89C versus nontransgenic or wild-type K18-overexpressing mice. Microarray-defined genetic changes were confirmed by quantitative polymerase chain reaction. Nineteen genes had a more than two-fold altered expression (nine downregulated, 10 upregulated). Upregulated genes in keratin-mutant hepatocytes included the oxidative metabolism genes cytochrome P450, S-adenosylhomocysteine (SAH) hydrolase, cysteine sulfinic acid decarboxylase, and oxidation-reduction pathway genes. Downregulated genes included fatty acid binding protein 5, cyclin D1, and some signaling molecules. Several methionine metabolism-related and glutathione synthetic pathway intermediates, including S-adenosylmethionine (SAMe) and SAH, were modulated in R89C versus control mice. R89C livers had higher lipid and protein oxidation by-products as reflected by increased malondialdehyde and oxidized albumin. In conclusion, K18 point mutation in transgenic mice modulates several hepatocyte oxidative stress-related genes and leads to lipid and protein oxidative by-products. Mutationassociated decreases in SAH and SAMe could compromise needed cysteine availability to generate glutathione during oxidative stress. Hence keratin mutations may prime hepatocytes to oxidative injury, which provides a new potential mechanism for how keratin mutations may predispose patients to cirrhosis. (HEPATOLOGY 2005;41:517-525.)

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“…Two-dimensional (2-D) SDS-PAGE was performed on NuPAGE 2-D gels (Invitrogen, Carlsbad, CA), with modifications for protein carbonyl identification. 15 Briefly, following sample in-strip rehydration and isoelectric focusing (IEF) on the 7-cm IPG strips with a linear pH 3-10 gradient, for 2,000 volt-hours they were placed in 15-ml test tubes and incubated for 15 min in 2 N HCl with 10 mM 2,4-dinitrophenylhydrasin (DNPH, Sigma) at 258C. After the reaction, samples were washed with 2 M Tris-Base/30% glycerol (Plusone, Uppsala, Sweden) for 15 min.…”

Section: Biochemical Methodsmentioning

confidence: 99%

Protein Oxidation by Chronic Pulmonary Diseases in Children

Starosta

Griese

2005

Pediatric Pulmonology

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Summary. The oxidation of proteins may play an important role in the pathogenesis of chronic inflammatory lung diseases, and may contribute to lung damage. However, the extent of oxidation and the distribution among proteins are not known for most pediatric lung diseases. In this work, protein oxidation was assessed as protein carbonyls. Bronchoalveolar lavages (BAL) from children with chronic lung diseases were investigated by dot-blot assay for content and for pattern of distribution of oxidized proteins by two-dimensional (2D) electrophoresis and Western blotting. Significantly higher levels of protein oxidation than in healthy controls were determined in groups of patients with interstitial lung disease, gastro-esophageal reflux disease, and pulmonary alveolar proteinosis. The proteins most sensitive to oxidation were serum albumin, surfactant protein A, and a1-antitrypsin. Our data show increased oxidative stress in lungs of children with chronic pulmonary diseases, with significant interindividual variations. The extent of protein oxidation was proportional to the count of neutrophilic granulocytes in BAL fluid. These findings strongly support the concept that an abundance of reactive oxygen species produced during neutrophilic inflammation may be a deleterious factor, leading to pulmonary damage in these patients.

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Identification of protein carbonyls after two-dimensional electrophoresis

Cited by 80 publications

References 14 publications

Existence of Abnormal Protein Aggregates in Healthy Escherichia coli Cells

Existence of Abnormal Protein Aggregates in Healthy Escherichia coli Cells

Keratin mutation primes mouse liver to oxidative injury†

Protein Oxidation by Chronic Pulmonary Diseases in Children

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