Related Experiment Videos
Factors determining the selectivity of protein tyrosine nitration
J M Souza1, E Daikhin, M Yudkoff
1Stokes Research Institute and Department of Pediatrics, Division of Neonatology, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Abstract:
Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.
Insights
Protein tyrosine nitration, a key posttranslational modification, shows biological selectivity. Factors like protein structure and tyrosine accessibility influence which sites are modified by nitrating agents.
Area of Science:
- Biochemistry
- Molecular Biology
- Posttranslational Modifications
Background:
- Tyrosine nitration is a protein modification involving reaction with nitrating agents.
- In vivo protein nitration is selective, not affecting all tyrosine residues or proteins.
Purpose of the Study:
- Investigate factors determining the biological selectivity of protein tyrosine nitration.
- Develop an in vitro model using three proteins of similar size but varying structures and tyrosine content.
Main Methods:
- Exposure of ribonuclease A, lysozyme, and phospholipase A(2) to various nitrating agents (peroxynitrite, nitric oxide, SIN-1, myeloperoxidase system).
- Analysis of preferential tyrosine residue modification in each protein.
- Assessment of protein modification in a mixture of the three proteins under different nitrating conditions.
Main Results:
- Specific tyrosine residues were preferentially nitrated in each protein (e.g., Y(115) in ribonuclease A).
- The nature of the nitrating agent influenced which protein was predominantly modified.
- Factors influencing selectivity include tyrosine residue exposure, loop structure location, and neighboring charge.
Conclusions:
- Protein structure and the specific nitrating agent are critical determinants of tyrosine nitration selectivity.
- Protein surface accessibility, tyrosine location on loops, and local charge influence nitration site selection.
- Understanding these factors is key to elucidating the biological roles of protein tyrosine nitration.