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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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Related in: MedlinePlus

The N15D mutation, but not the N71D mutation, decreases the thermal stability of HsTIM.The thermal unfolding of 0.1 mg/mL of WT and deamidated TIMs was monitored by recording the change in the CD signal at 222 nm in response to heating. The temperature was increased from 20 to 90°C at 1°C/2.5 min. The fraction of unfolded protein and the Tm values (inset) were calculated as previously described (24). Experiments were performed in duplicate; standard errors were less than 5%.
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pone.0123379.g003: The N15D mutation, but not the N71D mutation, decreases the thermal stability of HsTIM.The thermal unfolding of 0.1 mg/mL of WT and deamidated TIMs was monitored by recording the change in the CD signal at 222 nm in response to heating. The temperature was increased from 20 to 90°C at 1°C/2.5 min. The fraction of unfolded protein and the Tm values (inset) were calculated as previously described (24). Experiments were performed in duplicate; standard errors were less than 5%.

Mentions: The destabilization of HsTIM is a major characteristic of terminal marking by deamidation; therefore, protein stability was thoroughly studied in WT HsTIM and the mutated proteins. We initially performed thermal induced denaturation assays, recording the change in the circular dichroism signal at 222 nm as function of temperature (Fig 3). The results showed that the Tm for the N71D mutant closely resembles the Tm of the WT enzyme (61.2 vs 60.3°C for the WT and the N71D HsTIM, respectively); in contrast, the Tm decreased at least 7°C with respect to the WT enzyme for the N15D or the N15D/N71D mutants (53.5 and 53.9°C for the N15D and N15D/N71D mutants, respectively). As can be observed, the single deamidation of N15 is sufficient to disrupt the thermal stability of HsTIM.


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 N15D mutation, but not the N71D mutation, decreases the thermal stability of HsTIM.The thermal unfolding of 0.1 mg/mL of WT and deamidated TIMs was monitored by recording the change in the CD signal at 222 nm in response to heating. The temperature was increased from 20 to 90°C at 1°C/2.5 min. The fraction of unfolded protein and the Tm values (inset) were calculated as previously described (24). Experiments were performed in duplicate; standard errors were less than 5%.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123379.g003: The N15D mutation, but not the N71D mutation, decreases the thermal stability of HsTIM.The thermal unfolding of 0.1 mg/mL of WT and deamidated TIMs was monitored by recording the change in the CD signal at 222 nm in response to heating. The temperature was increased from 20 to 90°C at 1°C/2.5 min. The fraction of unfolded protein and the Tm values (inset) were calculated as previously described (24). Experiments were performed in duplicate; standard errors were less than 5%.
Mentions: The destabilization of HsTIM is a major characteristic of terminal marking by deamidation; therefore, protein stability was thoroughly studied in WT HsTIM and the mutated proteins. We initially performed thermal induced denaturation assays, recording the change in the circular dichroism signal at 222 nm as function of temperature (Fig 3). The results showed that the Tm for the N71D mutant closely resembles the Tm of the WT enzyme (61.2 vs 60.3°C for the WT and the N71D HsTIM, respectively); in contrast, the Tm decreased at least 7°C with respect to the WT enzyme for the N15D or the N15D/N71D mutants (53.5 and 53.9°C for the N15D and N15D/N71D mutants, respectively). As can be observed, the single deamidation of N15 is sufficient to disrupt the thermal stability of HsTIM.

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