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Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

de la Mora-de la Mora I, Torres-Larios A, Enríquez-Flores S, Méndez ST, Castillo-Villanueva A, Gómez-Manzo S, López-Velázquez G, Marcial-Quino J, Torres-Arroyo A, García-Torres I, Reyes-Vivas H, Oria-Hernández J - PLoS ONE (2015)

Bottom Line: Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins.The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme.In contrast, the N15D mutant displays all the detrimental effects related to deamidation.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, México, D.F., México.

ABSTRACT
Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

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The two sites of deamidation of HsTIM are found close to each other.(A) Overall view of the WT HsTIM dimer (PDB code 2JK2) showing the position of N15 and N71; each subunit is depicted in blue and orange. (B) Close-up view of the two deamidating residues. N15 is found in loop 1 and N71 in loop 3.
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pone.0123379.g001: The two sites of deamidation of HsTIM are found close to each other.(A) Overall view of the WT HsTIM dimer (PDB code 2JK2) showing the position of N15 and N71; each subunit is depicted in blue and orange. (B) Close-up view of the two deamidating residues. N15 is found in loop 1 and N71 in loop 3.

Mentions: It was also suggested that deamidation of HsTIM is sequential beginning at N71 and followed by N15. Even more, it was proposed that deamidation of N71 is a prerequisite for the deamidation of N15 [20]. Based on the crystal structure of the protein, which showed that N15 of one subunit is closely positioned to N71 of the adjacent subunit (Fig 1), it was suggested that the introduction of negative charges into the dimer interface could affect the stability of the enzyme by a mechanism of charge repulsion. In fact, it was shown that deamidated forms of HsTIM were more susceptible to dissociation [20].


Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

de la Mora-de la Mora I, Torres-Larios A, Enríquez-Flores S, Méndez ST, Castillo-Villanueva A, Gómez-Manzo S, López-Velázquez G, Marcial-Quino J, Torres-Arroyo A, García-Torres I, Reyes-Vivas H, Oria-Hernández J - PLoS ONE (2015)

The two sites of deamidation of HsTIM are found close to each other.(A) Overall view of the WT HsTIM dimer (PDB code 2JK2) showing the position of N15 and N71; each subunit is depicted in blue and orange. (B) Close-up view of the two deamidating residues. N15 is found in loop 1 and N71 in loop 3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401446&req=5

pone.0123379.g001: The two sites of deamidation of HsTIM are found close to each other.(A) Overall view of the WT HsTIM dimer (PDB code 2JK2) showing the position of N15 and N71; each subunit is depicted in blue and orange. (B) Close-up view of the two deamidating residues. N15 is found in loop 1 and N71 in loop 3.
Mentions: It was also suggested that deamidation of HsTIM is sequential beginning at N71 and followed by N15. Even more, it was proposed that deamidation of N71 is a prerequisite for the deamidation of N15 [20]. Based on the crystal structure of the protein, which showed that N15 of one subunit is closely positioned to N71 of the adjacent subunit (Fig 1), it was suggested that the introduction of negative charges into the dimer interface could affect the stability of the enzyme by a mechanism of charge repulsion. In fact, it was shown that deamidated forms of HsTIM were more susceptible to dissociation [20].

Bottom Line: Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins.The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme.In contrast, the N15D mutant displays all the detrimental effects related to deamidation.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Bioquímica-Genética, Instituto Nacional de Pediatría, Secretaría de Salud, México, D.F., México.

ABSTRACT
Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

Show MeSH
Related in: MedlinePlus