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Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.

Uda M, Kawasaki H, Shigenaga A, Baba T, Yamakura F - Biosci. Rep. (2012)

Bottom Line: We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05).The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus.This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sportology Center, Juntendo University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

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Related in: MedlinePlus

Three-dimensional models of active site region of human PDCE2The results for three-dimensioanl modelling were downloaded from the Protein Data Bank. Theaccession number of the protein is 3B8K. Both Trp616 and Trp483 (shown inyellow), which were nitrated in the present study are located within the CoA-binding region and nearthe catalytic amino acid residues (shown in blue).
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Figure 7: Three-dimensional models of active site region of human PDCE2The results for three-dimensioanl modelling were downloaded from the Protein Data Bank. Theaccession number of the protein is 3B8K. Both Trp616 and Trp483 (shown inyellow), which were nitrated in the present study are located within the CoA-binding region and nearthe catalytic amino acid residues (shown in blue).

Mentions: We have shown that the nitration of single tryptophan at position 32 in human recombinantCu, Zn-SOD (copper/zinc superoxide dismutase) decreases the enzymatic activity [13,25] and the nitration ofthree tryptophan residues in egg-white lysozyme resulted in total loss of the activity in ourprevious study [26]. These observations indicate that thenitration of tryptophan residues in enzymes could affect the activity of enzymes. We haveinvestigated the position of the modified tryptophan residues in the three-dimensional structure ofthe identified proteins in the present study. We found that five of the modified tryptophan residuesare located near substrates or coenzyme-binding sites of the enzymes, namely, Trp616 andTrp483 in PDCE2 [dihydrolipoly(lysine)-residue acetyltransferase component of PDH(pyruvate dehydrogenase) complex] in mitochondria, Trp314 in fructose-bisphosphatealdolase C (Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320521add.htm), Trp85 andTrp311 in GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Supplementary Figure S2 athttp://www.bioscirep.org/bsr/032/bsr0320521add.htm) and Trp383 inphosphoglycerate kinase 1 (Supplementary Figure S3 at http://www.bioscirep.org/bsr/032/bsr0320521add.htm). We show the three-dimensionalstructure of human PDCE2 in Figure 7 as a typicalexample. The amino acid sequences of PDCE2 are 93% identical between human and rat. The positions ofthe nitrated tryptophan residues are shown in black in Figure7. Trp616 and Trp483 are located within the CoA-binding region. Theaddition of a nitro group to these tryptophan residues may cause steric hindrance and a differentstate on the indole π electron, consequently these changes may affect CoA binding andmodulate the activity of the enzyme. However, further studies using isolated proteins are requiredto clarify the effects of the nitration of the tryptophan residues for each of the enzymes.


Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.

Uda M, Kawasaki H, Shigenaga A, Baba T, Yamakura F - Biosci. Rep. (2012)

Three-dimensional models of active site region of human PDCE2The results for three-dimensioanl modelling were downloaded from the Protein Data Bank. Theaccession number of the protein is 3B8K. Both Trp616 and Trp483 (shown inyellow), which were nitrated in the present study are located within the CoA-binding region and nearthe catalytic amino acid residues (shown in blue).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475453&req=5

Figure 7: Three-dimensional models of active site region of human PDCE2The results for three-dimensioanl modelling were downloaded from the Protein Data Bank. Theaccession number of the protein is 3B8K. Both Trp616 and Trp483 (shown inyellow), which were nitrated in the present study are located within the CoA-binding region and nearthe catalytic amino acid residues (shown in blue).
Mentions: We have shown that the nitration of single tryptophan at position 32 in human recombinantCu, Zn-SOD (copper/zinc superoxide dismutase) decreases the enzymatic activity [13,25] and the nitration ofthree tryptophan residues in egg-white lysozyme resulted in total loss of the activity in ourprevious study [26]. These observations indicate that thenitration of tryptophan residues in enzymes could affect the activity of enzymes. We haveinvestigated the position of the modified tryptophan residues in the three-dimensional structure ofthe identified proteins in the present study. We found that five of the modified tryptophan residuesare located near substrates or coenzyme-binding sites of the enzymes, namely, Trp616 andTrp483 in PDCE2 [dihydrolipoly(lysine)-residue acetyltransferase component of PDH(pyruvate dehydrogenase) complex] in mitochondria, Trp314 in fructose-bisphosphatealdolase C (Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320521add.htm), Trp85 andTrp311 in GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Supplementary Figure S2 athttp://www.bioscirep.org/bsr/032/bsr0320521add.htm) and Trp383 inphosphoglycerate kinase 1 (Supplementary Figure S3 at http://www.bioscirep.org/bsr/032/bsr0320521add.htm). We show the three-dimensionalstructure of human PDCE2 in Figure 7 as a typicalexample. The amino acid sequences of PDCE2 are 93% identical between human and rat. The positions ofthe nitrated tryptophan residues are shown in black in Figure7. Trp616 and Trp483 are located within the CoA-binding region. Theaddition of a nitro group to these tryptophan residues may cause steric hindrance and a differentstate on the indole π electron, consequently these changes may affect CoA binding andmodulate the activity of the enzyme. However, further studies using isolated proteins are requiredto clarify the effects of the nitration of the tryptophan residues for each of the enzymes.

Bottom Line: We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05).The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus.This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sportology Center, Juntendo University Graduate School of Medicine, Chiba, Japan.

ABSTRACT
Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

Show MeSH
Related in: MedlinePlus