Oxidant-Mediated Protein Amino Acid Conversion

Abstract

Biological oxidation plays important roles in the pathogenesis of various diseases and aging. Carbonylation is one mode of protein oxidation. It has been reported that amino acids that are susceptible to carbonylation are arginine (Arg), proline (Pro), lysine, and threonine residues. The carbonylation product of both Arg and Pro residues is glutamyl semialdehyde. While chemically the oxidation reactions of neither Pro to glutamyl semialdehyde nor Arg to glutamyl semialdehyde are reversible, experimental results from our laboratory suggest that the biological system may drive the reduction of glutamyl semialdehyde to Pro in the protein structure. Further, glutamyl semialdehyde can be oxidized to become glutamic acid (Glu). Therefore, I hypothesize that biological oxidation post-translationally converts Arg to Pro, Arg to Glu, and Pro to Glu within the protein structure. Our mass spectrometry experiments provided evidence that, in human cells, 5⁻10% of peroxiredoxin 6 protein molecules have Pro-45 replaced by Glu. This concept of protein amino acid conversion challenges the dogma that amino acid sequences are strictly defined by nucleic acid sequences. I propose that, in the biological system, amino acid replacements can occur post-translationally through redox regulation, and protein molecules with non-DNA coding sequences confer functions.

Keywords: amino acid; carbonylation; oxidation; protein; reactive oxygen species; redox.

Figure 1

Scheme depicting the oxidation of Arg and Pro residues in the protein structure. Iron-catalyzed oxidation of both Arg and Pro residues results in the formation of glutamyl semialdehyde with a carbonyl group (red circle). Oxidation of Arg results in the loss of a bulky amino group-containing structure during the formation of glutamyl semialdehyde. Oxidation of Pro first forms 5-hydroxyproline, which is further oxidized into glutamyl semialdehyde. Studies of purified proteins indicate that the oxidation of Pro to 5-hydroxyproline is irreversible, yet our experiments with live cells suggest that this reaction may be reversible (as indicated with?). Glutamyl semialdehyde can further be oxidized into Glu.

Figure 2

Effects of siRNA knockdown of glutaredoxin 1 (Grx1) on peroxiredoxin 6 (Prx6) decarbonylation. (A) Cultured human smooth muscle cells were transfected with Grx1 siRNA, followed by stimulation with platelet-derived growth factor (PDGF). Cell lysates were prepared, derivatized with 2,4-dinitrophenylhydrazine (DNPH), immunoprecipitated with the DNPH antibody, and immunoblotted with the Prx6 antibody [9]. (B) Cultured human smooth muscle cells were transfected with Grx1 siRNA, and cell lysates were prepared. Cell lysates were then treated with or without 2% (w/v) BME, derivatized with DNPH, immunoprecipitated with the DNPH antibody, and immunoblotted with the Prx6 antibody [9]. Bar graphs represent means ± SEM of the ratio of carbonylated Prx6 to Prx6 protein expression. The symbol * denotes values significantly different from each other at p < 0.05.

Figure 3

Amino acid sequence of human Prx6 protein molecule. Proline 45 (Pro45) and catalytic cysteine 47 (Cys47) are indicated by arrows.

Figure 4

Proposed mechanisms for defining protein amino acid sequences. Protein amino acid sequences are largely defined by the sequence of DNA that is transcribed to mRNA then translated to the protein sequence. In some cases, RNA editing changes the sequence of mRNA thereby altering the protein amino acid sequence not to encode the DNA sequence completely. I proposed that the amino acid sequences can also be altered post-translationally through the oxidant-mediated protein amino acid conversions.

Figure 5

Scheme depicting the fate of amino acid-converted proteins in response to biological oxidation. Reactive oxygen species (ROS) are produced during oxidative stress or oxidant signaling. ROS promote amino acid conversions as described in this article. Some of these amino acid-converted proteins are subjected to protein degradation as a mechanism of oxidative stress. I propose that amino acid-converted proteins also possess biologic functions as nature intended.