June 2013

Protein carbonylation

Not just another -ation in the acylation nation

The advent of high-sensitivity mass spectrometers has allowed for the identification of numerous covalent additions to amino acid side chains and has heightened awareness of the role of intermediary metabolism and oxidative stress and major effects of protein structure and function. Indeed, protein propionylation, malonylation, butyrlation and succinylation are but a few of the most recent additions to the acylation nation (1, 2). Linking lipid metabolism and oxidative stress to the covalent modification spectrum is protein carbonylation.
 
Protein carbonylation is a generic term used to describe the covalent adduction of lipid aldehydes, often six, nine or 12 carbons, to the side chains of lysine, histidine and cysteine residues (3). Lipid aldehydes are produced from hydroperoxidation of polyunsaturated fatty acyl groups followed by nonenzymatic Hock cleavage. The resultant aldehydes can undergo Schiff-base formation with lysine residues but more commonly are subject to Michael addition reactions that produce a lipid acyl group containing a free carbonyl – hence the nomenclature. Such carbonyl groups are capable of secondary Schiff-base formation with an adjacent amine or cyclization, but in many cases the free aldehyde remains unmodified, thereby allowing for detection using a variety of hydrazide-based reagents or, in some cases, using antibodies directed to nine-carbon acyl derivatives such as 4-hydroxy 2,3 trans nonenal (4).
 
Protein carbonylation is studied most commonly in those systems where increased oxidative stress meets biological membranes or lipid droplets. As such, adipose tissue is a major site for protein carbonylation, and the loss of intrinsic antioxidant enzymes that occurs during the course of an obese inflammatory challenge produces a state of increased lipid aldehyde synthesis. Because lipid aldehydes are capable of diffusing across membranes, mass-spectrometry-based identification of carbonylated proteins reveals that they are widespread in the cell, including the nuclear, mitochondrial and cytoplasmic compartments. However, a major difficulty in carbonylation analysis is that modified peptides do not separate well in the mass spectrometer, and, as a consequence, the site and stoichiometry of modification often are not well defined. However, in some cases, such as the adipocyte fatty-acid binding protein, carbonylation modifies about 10 percent of the polypeptide and results in loss of lipid binding activity (5).
 
In the case of mitochondrial targets of carbonylation, such as enzymes of complex I of the electron transport chain (NDUFA2, NDUFA3), it is not clear if protein carbonylation is causative in the loss of NADH oxidation capacity associated with inflammation or simply correlative (6). However, it is tempting to speculate that protein carbonylation contributes to the mitochondrial dysfunction associated with obesity and insulin resistance. Intriguingly, protein carbonylation recently has been linked to epigenetic processes via carbonylation of lysine groups on histones and via carbonylation of class I and II histone deacetylases (7). Both types of modifications may affect gene expression and, as such, may provide a redox-based connectivity of lipid metabolism to epigenetics.
 
Interestingly, in adipocytes the loss of the major phase II enzyme controlling lipid aldehyde levels, glutathione S-transferase A4, is associated not only with increased protein carbonylation but also with increased superoxide anion production, suggesting protein carbonylation is a key determinant in reactive oxygen species synthesis (8). Superoxide anion synthesis leads to increased hydroxyl radical formation and, in turn, increased protein carbonylation, catalyzing a feed-forward process whereby increased protein carbonylation and reactive oxygen species formation go hand in hand. As reactive oxygen species can oxidize directly the side chains of many amino acids, such as cysteine and methionine, protein carbonylation may initiate an oxidative stress cascade and a change in the cellular redoxome, resulting in pleotropic effects on cellular structure and function. Within the context of diabetes and obesity, oxidative stress often leads to endoplasmic reticulum stress and the unfolded-protein response. As such, protein carbonylation by lipid aldehydes may not be simply another -ation to catalog but rather an important initiating event in a biological cascade affecting major components of cellular homeostatic control and gene expression.

REFERENCES
  1. 1.  Zhang, Z. et al. Nat. Chem. Biol. 7, 58 – 63 (2011).
  2. 2.  Peng, C. et al. Mol. Cell. Proteomics. doi: 10.1074/mcp.M111.012658.
  3. 3.  Esterbauer, H. et al. Free Rad. Biol. Med. 11, 81 – 128, (1991).
  4. 4.  Curtis, J. M., et al. Trends in Endocrinology and Metabolism 23, 399 – 406 (2012).
  5. 5.  Grimsrud, P. A. et al. Mol. Cell. Proteomics. 6, 624 – 637 (2007).
  6. 6.  Curtis, J. M. et al. J. Biol. Chem. 39, 32967 – 32980 (2012).
  7. 7.  Doyle, K., and Fitzpatrick, F. A. J. Biol. Chem. 285, 17417 – 17424 (2010).
  8. 8.  Curtis J. M. et al. Diabetes 59, 1132 – 1142 (2010).

David BernlohrDavid Bernlohr (bernl001@umn.edu) is a Distinguished McKnight University Professor at the University of Minnesota-Twin Cities in the Department of Biochemistry, Molecular Biology and Biophysics.

 

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