ALL Metrics
-
Views
-
Downloads
Get PDF
Get XML
Cite
Export
Track
Research Note

Evidence for the oxidant-mediated amino acid conversion, a naturally occurring protein engineering process, in human cells

[version 1; peer review: 1 approved, 2 not approved]
PUBLISHED 28 Apr 2017
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

Abstract

Reactive oxygen species (ROS) play an important role in the development of various pathological conditions as well as aging. ROS oxidize DNA, proteins, lipids, and small molecules. Carbonylation is one mode of protein oxidation that occurs in response to the iron-catalyzed, hydrogen peroxide-dependent oxidation of amino acid side chains. Although carbonylated proteins are generally believed to be eliminated through proteasome-dependent degradation, we previously discovered the protein de-carbonylation mechanism, in which the formed carbonyl groups are chemically eliminated without proteins being degraded. Major amino acid residues that are susceptible to carbonylation include proline and arginine, both of which are oxidized to become glutamyl semialdehyde, which contains a carbonyl group. The further oxidation of glutamyl semialdehyde produces glutamic acid. Thus, we hypothesize that through the ROS-mediated formation of glutamyl semialdehyde, the proline, arginine, and glutamic acid residues within the protein structure are interchangeable. In support of this hypothesis, mass spectrometry demonstrated that proline 45 (a well-conserved residue within the catalytic sequence) of the peroxiredoxin 6 molecule can be converted into glutamic acid in cultured human cells, establishing a revolutionizing concept that biological oxidation elicits the naturally occurring protein engineering process.

Keywords

Amino acid, Glutamyl semialdehyde, Oxidative stress, Protein carbonylation, Protein engineering, Protein oxidation, Reactive oxygen species

Introduction

Reactive oxygen species (ROS) are produced through the electron reduction of molecular oxygen and include superoxide anion radicals, hydrogen peroxide (H2O2), and hydroxyl radicals (Freeman & Crapo, 1982; Halliwell & Gutteridge, 2007). ROS have been implicated in the pathogenesis of various diseases (Freeman & Crapo, 1982; Halliwell & Gutteridge, 2007), as well as in the aging process (Harman, 1956). One electron reduction of molecular oxygen produces superoxide, which in turn reacts with each other to produce H2O2 and reduces cellular iron ions. Reduced iron donates an electron to H2O2 and produces highly reactive hydroxyl radicals. Hydroxyl radicals in turn react with virtually all biological molecules, including DNA, proteins, lipids and small molecules, damaging the biological system (Freeman & Crapo, 1982; Halliwell & Gutteridge, 2007).

One important event that occurs in response to the metal (iron)-catalyzed oxidation process is the formation of carbonyls in the protein structure. Protein carbonylation has been shown to be increased in various diseases and in aging (Berlett & Stadtman, 1997; Levine & Stadtman, 2001; Levine, 2002; Stadtman et al., 1988). Protein carbonylation occurs in response to the iron-catalyzed, H2O2-dependent oxidation of amino acid side chains (Stadtman, 1990; Suzuki et al., 2010). Protein carbonylation inactivates protein functions and marks damaged proteins for proteasome-dependent degradation (Grune et al., 1997; Levine, 1989). While carbonylated proteins are believed not to undergo electron reduction, we previously discovered the protein de-carbonylation mechanism, in which carbonyl groups can be eliminated without proteins being degraded (Wong et al., 2008). Major amino acid residues that are susceptible to iron-catalyzed oxidation include proline and arginine, both of which are oxidized to become glutamyl semialdehyde, which contains a carbonyl group (Amici et al., 1989). Glutamyl semialdehyde is further oxidized into glutamic acid (Figure 1).

62ae515f-da89-40b3-8a2a-921d896d0336_figure1.gif

Figure 1. Iron-catalyzed oxidations of arginine and proline residues that result in the formation of glutamyl semialdehyde with a carbonyl group (Amici et al., 1989).

Glutamyl semialdehyde is further oxidized into glutamic acid.

We previously demonstrated the role of protein carbonylation in ligand/receptor-mediated cell signaling (Wong et al., 2008). We further noted that the kinetics of ligand-mediated protein carbonylation is transient. Typically, in cultured cells, ligands activate the carbonylation of various proteins within 10 min and the activated protein carbonylation reverts to baseline by 30 min. These results suggest that there is a mechanism for the elimination of the formed carbonyls. We named this process “de-carbonylation” (Wong et al., 2008). To understand the mechanism of de-carbonylation, we tested the hypothesis that protein carbonyls may be reduced. We found that the addition of reductants to rat heart homogenates resulted in a decrease in the protein carbonyl content (Wong et al., 2013). By contrast, reductants had no effect on the carbonyl content in purified proteins, suggesting that protein carbonyls are not reduced in the absence of other cellular components. From these results, we hypothesized that cells contain catalysts for the reduction of protein carbonyls. This hypothesis is supported by our results demonstrating that the heating of heart homogenates to inactivate cellular enzymes inhibits the decrease in protein carbonyls in vitro, and that knocking down glutaredoxin 1 in the cells inhibits protein de-carbonylation (Wong et al., 2013). We used two-dimensional gel electrophoresis and mass spectrometry to identify proteins that can be de-carbonylated and found that peroxiredoxin 6 (Prx6) is one such protein (Wong et al., 2013).

Since both arginine and proline residues can be oxidized to form glutamyl semialdehyde that can further be oxidized to form glutamic acid, we speculated that arginine, proline, and glutamic acid residues may be interchangeable in the biological system, in a process that resembles site-directed mutagenesis. This article reports that the proline residue 45 of the human Prx6 protein molecule can be converted into glutamic acid in cells, indeed demonstrating the existence of a naturally occurring site-directed mutagenesis/protein engineering-like process that may be regulated by ROS.

Methods

Cell culture and immunoprecipitation

Human pulmonary artery smooth muscle cells (ScienCell Research Laboratories, Carlsbad, CA, USA) grown in 10 cm dishes were serum-starved overnight with 10 ml of 0.01% fetal bovine serum-containing Dulbecco’s Modified Eagle’s medium (Mediatech, Inc., Manassas, VA, USA) for cell signaling studies. To prepare lysates, the cells were washed with phosphate buffered saline and solubilized with 1 ml of 50 mM Hepes solution (pH 7.4) containing 1% (v/v) Triton X-100, 4 mM EDTA, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, 2 mM PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin. Cell lysates (1 ml) were immunoprecipitated with the rabbit polyclonal anti-Prx6 antibody (Sigma-Aldrich, St. Louis, MO, USA; Catalogue # P0058; 5 µg) and SureBeads Protein G Magnetic Beads (Bio-Rad Bio-Rad Laboratories, Hercules, CA, USA; 1 mg) for 1 h at room temperature.

Peptide sample preparation

Immunoprecipitation samples were processed with trypsin digestion (12.5 ng/µl) followed by a C18 Zip-tip clean-up (EMD Millipore, Billerica, MA, USA). Tryptic peptide samples were reconstituted in 20 µl of 0.1% formic acid before nanospray liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis was performed.

Nanospray LC/MS/MS analysis

The tryptic peptides mixture from each sample was analyzed using a Thermo Scientific Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Electron, Bremen, Germany) equipped with a Thermo Dionex UltiMate 3000 RSLCnano System (Thermo Dionex, Sunnyvale, CA, USA). Tryptic peptide samples were loaded onto a peptide trap cartridge at a flow rate of 5 μl/min. The trapped peptides were eluted onto a reversed-phase 20-cm C18 PicoFrit column (New Objective, Woburn, MA, USA) using a linear gradient of acetonitrile (3–36%) in 0.1% formic acid. The elution duration was 60 min at a flow rate of 0.3 μl/min. Eluted peptides from the PicoFrit column were ionized and sprayed into the mass spectrometer using a Nanospray Flex Ion Source ES071 (Thermo Scientific, Waltham, MA, USA) under the following settings: spray voltage 1.6 kV and capillary temperature 250°C. The Q Exactive instrument was operated in the data-dependent mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (m/z 300−2,000) were acquired in the Orbitrap with 70,000 resolution (m/z 200) after the accumulation of ions to a 3 × 106 target value based on predictive AGC from the previous full scan. Dynamic exclusion was set to 20 s. The 15 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the Axial Higher Energy Collision-induced Dissociation (HCD) cell using normalized HCD collision energy at 25% with an AGC target of 1e5 and a maximum injection time of 100 ms at 17,500 resolution. Two independent MS analyses in triplicate (a total of six cell samples) were performed.

LC/MS/MS data analysis

The raw MS files were analyzed using the Thermo Proteome Discoverer 1.4.1 platform (Thermo Scientific, Bremen, Germany) for peptide identification and protein assembly. The raw data files were searched against the human protein sequence database obtained from the NCBI website (https://www.ncbi.nlm.nih.gov) using the Proteome Discoverer software based on the SEQUEST algorithm. The carbamidomethylation of cysteines was set as a fixed modification, and Oxidation and Deamidation Q/N-deamidated (+0.98402 Da), and Pro>Glu (+31.990 Da) were set as dynamic modifications. The minimum peptide length was specified to be five amino acids. The precursor mass tolerance was set to 15 ppm, whereas fragment mass tolerance was set to 0.05 Da. The maximum false peptide discovery rate was specified as 0.01.

Results

Identification of the conversion of proline residues into glutamic acid in Prx6

To identify protein carbonylation sites, we enriched Prx6 by immunoprecipitation from cultured human cells. The Prx6 immunoprecipitation samples were processed for digestion by trypsin and the tryptic peptides were analyzed by nanoLC-MS/MS analysis and protein sequence alignment to identify proline sites conversion into glutamic acid in Prx6. The conversion was identified based on a mass shift of + 31.990 Da at the proline residue (Figures 2A and B). The experiments led to the identification of one specific site at Pro 45 in human Prx6 protein (Figure 2C).

62ae515f-da89-40b3-8a2a-921d896d0336_figure2.gif

Figure 2. Identification of the conversion of the proline (P) residue at amino acid 45 into glutamic acid (E) in human peroxiredoxin 6 (Prx6).

(A) Extracted ion chromatograms of Prx6 peptide (DFTP+31.990VCTTELGR, +2 charge, m/z=714.33) (top) and its non-conversion counterpart (DFTPVCTTELGR, +2 charge, m/z=698.33) (bottom). Both peptides were eluted at the same retention time and are from affinity-enriched cultured human cell extract using the anti-Prx6 antibody. (B) High resolution MS spectra of the co-elution of peptides (DFTP+31.990VCTTELGR, +2 charge, m/z=714.33) (right) and its non-conversion counterpart (DFTPVCTTELGR, +2 charge, m/z=698.33) (left). (C) Illustration of the identified proline 45 conversion into glutamic acid in cultured human cells (shown in bold red). Sequence areas containing amino acid residues shown in green are detected by LC-MS/MS analysis after trypsin digestion.

Confirmation of Prx6 peptides containing proline to glutamic acid conversion by MS/MS

We are reasonably confident that the identified mass shift of + 31.990 Da is caused by the conversion of proline into glutamic acid, since the Prx6 was affinity-purified before MS/MS analysis. Since the conversion of proline into glutamic acid in Prx6 is a novel post-translational modification identified so far, it is desirable to confirm the structure of the identified peptides to ensure that the derived mass shifts of +31.99 Da are caused by the conversion into glutamic acid. MS/MS and HPLC co-elution are gold standards for verifying peptide identification. As demonstrated in Figure 3, both peptides, DFTP+31.990VCTTELGR, +2 charge, m/z=714.33, and its non-conversion counterpart DFTPVCTTELGR, +2 charge, m/z=698.33 were co-eluted with a peak shift of less than 0.2 min. Our result showed that the high resolution MS/MS fragmentation patterns of DFTP+31.990VCTTELGR and its non-conversion counterpart DFTPVCTTELGR peptide were almost identical except the addition of +31.990 Da of fragments that contain the proline 45 residue (Figures 3A and B).

62ae515f-da89-40b3-8a2a-921d896d0336_figure3.gif

Figure 3. NanoLC-MS/MS verification of the conversion of proline 45 into glutamic acid at Prx6 (DFTP+31.990VCTTELGR).

(A) High resolution MS/MS spectra of peroxiredoxin 6 (Prx6) proline to glutamic acid conversion peptide (DFTP+31.990VCTTELGR). (B) High resolution MS/MS spectra of Prx6 proline 45 peptide (DFTPVCTTELGR). Spectrum was obtained by LC-MS/MS analysis using the Thermo UltiMate 3000 RSLCnano System and Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer. (C) % of Prx6 molecules with the proline 45 conversion into glutamic acid in cultured human cells. Two independent MS analyses in triplicate (a total of six cell samples) were performed.

Analysis of the ion intensity of the MS spectra of DFTP+31.990VCTTELGR and its non-conversion counterpart DFTPVCTTELGR peptide (Figure 3C) determined that the proline 45 to glutamic acid conversion occurs in 5–10% of the Prx6 molecule in our samples with a mean of 7.43 ± 1.78% (N=6).

Discussion

The present study introduces a revolutionizing concept that a protein engineering-like process could occur naturally in the biological system. Specifically, we identified that proline 45 of the Prx6 protein can be converted into glutamic acid. Proline 45 is in the peroxidase catalytic domain (Fisher, 2011; Fisher, 2017), thus this conversion should have functional significance. Future work should identify if this conversion increases, decreases or modifies the catalytic activity of Prx6. Such studies would open up the possibility that proteins with altered amino acid sequences have functional roles in the biological system.

The results from the present study also open up a new mechanism of ROS, indicating that the amino acid conversion, specifically the proline–glutamic acid conversion, is a consequence of oxidative stress mediated by the formation of glutamyl semialdehyde in the process of protein carbonylation. Through glutamyl semialdehyde, other conversions among arginine, proline, and glutamic acid are possible. Since the caged and site-directed production of hydroxyl radicals and carbonyl formation can occur via metal binding to specific sites of the protein structure (Stadtman & Berlett, 1991; Wong et al., 2010), ROS-mediated amino acid conversion may be a tightly regulated process.

Data availability

The raw MS files from the output of the LC/MS/MS are available: doi, 10.17605/OSF.IO/5FN2E and 10.17605/OSF.IO/RP9J8 (Suzuki, 2017a; Suzuki, 2017b).

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 28 Apr 2017
Comment
Author details Author details
Competing interests
Grant information
Copyright
Download
 
Export To
metrics
Views Downloads
F1000Research - -
PubMed Central
Data from PMC are received and updated monthly.
- -
Citations
CITE
how to cite this article
Suzuki YJ and Hao JJ. Evidence for the oxidant-mediated amino acid conversion, a naturally occurring protein engineering process, in human cells [version 1; peer review: 1 approved, 2 not approved]. F1000Research 2017, 6:594 (https://doi.org/10.12688/f1000research.11376.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
track
receive updates on this article
Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 28 Apr 2017
Views
28
Cite
Reviewer Report 28 Jun 2017
Adelina Rogowska-Wrzesinska, Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark 
Michael J. Davies , Sydney Medical School, University of Sydney, Sydney, NSW, Australia 
Not Approved
VIEWS 28
General overview:

This manuscript presents an interesting aspect of the effect of ROS on proteins – the possibility of converting one type of amino acid into another one. It briefly describes the idea and presents results of ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Rogowska-Wrzesinska A and Davies  MJ. Reviewer Report For: Evidence for the oxidant-mediated amino acid conversion, a naturally occurring protein engineering process, in human cells [version 1; peer review: 1 approved, 2 not approved]. F1000Research 2017, 6:594 (https://doi.org/10.5256/f1000research.12281.r23077)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 1
     
    The paper submitted by Suzuki and Hao highlights the importance of modifications occurring in proteins as a consequence of oxidative stress. In this particular case, the authors ... Continue reading
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 3
     
    General overview:
     
    This manuscript presents an interesting aspect of the effect of ROS on proteins – the possibility of converting one type of amino acid into ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 1
     
    The paper submitted by Suzuki and Hao highlights the importance of modifications occurring in proteins as a consequence of oxidative stress. In this particular case, the authors ... Continue reading
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 3
     
    General overview:
     
    This manuscript presents an interesting aspect of the effect of ROS on proteins – the possibility of converting one type of amino acid into ... Continue reading
Views
34
Cite
Reviewer Report 20 Jun 2017
Dolores Pérez-Sala, Biological Research Center ( CIB), Spanish National Research Council (CSIC), Madrid, Spain 
Not Approved
VIEWS 34
In their manuscript, Suzuki and Hao report the finding of a peptide in Peroxiredoxin 6 that shows a mass increment of 32 in mass spectrometry analysis. NanoLC-MSMS analysis maps this increment at the site of a proline residue (P45 in ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Pérez-Sala D. Reviewer Report For: Evidence for the oxidant-mediated amino acid conversion, a naturally occurring protein engineering process, in human cells [version 1; peer review: 1 approved, 2 not approved]. F1000Research 2017, 6:594 (https://doi.org/10.5256/f1000research.12281.r23628)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 2
     
    In their manuscript, Suzuki and Hao report the finding of a peptide in Peroxiredoxin 6 that shows a mass increment of 32 in mass spectrometry analysis. NanoLC-MSMS ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 2
     
    In their manuscript, Suzuki and Hao report the finding of a peptide in Peroxiredoxin 6 that shows a mass increment of 32 in mass spectrometry analysis. NanoLC-MSMS ... Continue reading
Views
19
Cite
Reviewer Report 14 Jun 2017
Joaquim Ros, Department of Basic Medical Sciences, IRB-Lleida (Biomedical Research Institute of Lleida), University of Lleida, Lleida, Spain 
Approved
VIEWS 19
      The paper submitted by Suzuki and Hao highlights the importance of modifications occurring in proteins as a consequence of oxidative stress. In this particular case, the authors provide data showing that proline residue at position 45 in peroxiredoxin 6 ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Ros J. Reviewer Report For: Evidence for the oxidant-mediated amino acid conversion, a naturally occurring protein engineering process, in human cells [version 1; peer review: 1 approved, 2 not approved]. F1000Research 2017, 6:594 (https://doi.org/10.5256/f1000research.12281.r23475)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 1
     
    The paper submitted by Suzuki and Hao highlights the importance of modifications occurring in proteins as a consequence of oxidative stress. In this particular case, the authors ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 28 Sep 2018
    Yuichiro Suzuki, Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, 20057, USA
    28 Sep 2018
    Author Response
    Reviewer 1
     
    The paper submitted by Suzuki and Hao highlights the importance of modifications occurring in proteins as a consequence of oxidative stress. In this particular case, the authors ... Continue reading

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 28 Apr 2017
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
Sign In
If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password.

The email address should be the one you originally registered with F1000.

Email address not valid, please try again

You registered with F1000 via Google, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Google account password, please click here.

You registered with F1000 via Facebook, so we cannot reset your password.

To sign in, please click here.

If you still need help with your Facebook account password, please click here.

Code not correct, please try again
Email us for further assistance.
Server error, please try again.