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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 N15D mutation increases the susceptibility of HsTIM to proteolysis.The proteolysis of WT (A), N71D (B), N15D (C) and N71D/N15D (D) with Proteinase K was followed by SDS-PAGE (upper insets, open symbols) or residual activity (closed symbols). WT or mutant TIMs (1mg/mL) were incubated with Proteinase K at 30°C at a molar ratio of 265:1 (TIM:Proteinase K). At the times indicated in the ordinate axis, the reaction was arrested by the addition of PMSF 5 mM and aliquots withdrawn for analysis. SDS-PAGE was digitalized and analyzed densitometrically; TIM activity was measured under standard conditions. The experiment is representative of a triplicate; standard errors were less than 10%.
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pone.0123379.g004: The N15D mutation increases the susceptibility of HsTIM to proteolysis.The proteolysis of WT (A), N71D (B), N15D (C) and N71D/N15D (D) with Proteinase K was followed by SDS-PAGE (upper insets, open symbols) or residual activity (closed symbols). WT or mutant TIMs (1mg/mL) were incubated with Proteinase K at 30°C at a molar ratio of 265:1 (TIM:Proteinase K). At the times indicated in the ordinate axis, the reaction was arrested by the addition of PMSF 5 mM and aliquots withdrawn for analysis. SDS-PAGE was digitalized and analyzed densitometrically; TIM activity was measured under standard conditions. The experiment is representative of a triplicate; standard errors were less than 10%.

Mentions: Increased susceptibility to proteolysis induced by deamidation was described as a prominent feature of the terminal marking mechanism in HsTIM [22]; we therefore studied the proteolytic digestion with proteinase K of the WT and the deamidated HsTIM mutants (Fig 4). Proteinase K is a broad-spectrum serine protease previously used to characterize stability perturbations of HsTIM [32]. The results showed an increased susceptibility of the N15D and N15D/N71D mutants to proteolysis. Under very soft proteolysis conditions, where the WT and N71D proteins are completely insensitive to proteinase K (Fig 4, panels A-B), the N15D and N15D/N71D mutants were completely hydrolyzed (Fig 4, panels C-D). The results indicate that deamidation of N71 does not contribute to the increased susceptibility of HsTIM to proteolysis.


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 increases the susceptibility of HsTIM to proteolysis.The proteolysis of WT (A), N71D (B), N15D (C) and N71D/N15D (D) with Proteinase K was followed by SDS-PAGE (upper insets, open symbols) or residual activity (closed symbols). WT or mutant TIMs (1mg/mL) were incubated with Proteinase K at 30°C at a molar ratio of 265:1 (TIM:Proteinase K). At the times indicated in the ordinate axis, the reaction was arrested by the addition of PMSF 5 mM and aliquots withdrawn for analysis. SDS-PAGE was digitalized and analyzed densitometrically; TIM activity was measured under standard conditions. The experiment is representative of a triplicate; standard errors were less than 10%.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123379.g004: The N15D mutation increases the susceptibility of HsTIM to proteolysis.The proteolysis of WT (A), N71D (B), N15D (C) and N71D/N15D (D) with Proteinase K was followed by SDS-PAGE (upper insets, open symbols) or residual activity (closed symbols). WT or mutant TIMs (1mg/mL) were incubated with Proteinase K at 30°C at a molar ratio of 265:1 (TIM:Proteinase K). At the times indicated in the ordinate axis, the reaction was arrested by the addition of PMSF 5 mM and aliquots withdrawn for analysis. SDS-PAGE was digitalized and analyzed densitometrically; TIM activity was measured under standard conditions. The experiment is representative of a triplicate; standard errors were less than 10%.
Mentions: Increased susceptibility to proteolysis induced by deamidation was described as a prominent feature of the terminal marking mechanism in HsTIM [22]; we therefore studied the proteolytic digestion with proteinase K of the WT and the deamidated HsTIM mutants (Fig 4). Proteinase K is a broad-spectrum serine protease previously used to characterize stability perturbations of HsTIM [32]. The results showed an increased susceptibility of the N15D and N15D/N71D mutants to proteolysis. Under very soft proteolysis conditions, where the WT and N71D proteins are completely insensitive to proteinase K (Fig 4, panels A-B), the N15D and N15D/N71D mutants were completely hydrolyzed (Fig 4, panels C-D). The results indicate that deamidation of N71 does not contribute to the increased susceptibility of HsTIM to proteolysis.

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