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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 deamidation of N15 does not depend on deamidation of N71.Native gel electrophoresis of the WT (A) or the N71D mutant (B) after incubation at 1mg/mL in TE buffer at 37°C with 3.5 mM GAP in a temporal course of 5 hours. The appearance of additional bands with higher electrophoretic mobility indicates the emergence of negatively charged additional species. The deamidation rate in the WT and the N71D mutant (C) was determined as the percentage decrease of the upper band (native enzyme). The experiments are representative of independent, duplicate assays with differences of less than 10%.
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pone.0123379.g007: The deamidation of N15 does not depend on deamidation of N71.Native gel electrophoresis of the WT (A) or the N71D mutant (B) after incubation at 1mg/mL in TE buffer at 37°C with 3.5 mM GAP in a temporal course of 5 hours. The appearance of additional bands with higher electrophoretic mobility indicates the emergence of negatively charged additional species. The deamidation rate in the WT and the N71D mutant (C) was determined as the percentage decrease of the upper band (native enzyme). The experiments are representative of independent, duplicate assays with differences of less than 10%.

Mentions: The prevailing terminal marking mechanism proposed for HsTIM establishes that deamidation of N71 is a prerequisite for the deamidation of N15 [20–22]. In order to test the influence of N71 deamidation on the deamidation of N15, the deamidation rate, as measured by the decrease of native enzyme with the concomitant appearance of negatively charged bands in a temporal course followed by native gel electrophoresis, was tested in the WT and in the N71D mutant (Fig 7). It was supposed that if deamidation of N71 favors the deamidation of N15, the N71D mutant (where deamidation already had occurred), must be deamidated faster than the WT enzyme. As can be observed (Fig 7), this is not the case, since both proteins are deamidated at very similar rates. This result indicates that deamidation of N71 does not influence the deamidation rate of N15.


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 deamidation of N15 does not depend on deamidation of N71.Native gel electrophoresis of the WT (A) or the N71D mutant (B) after incubation at 1mg/mL in TE buffer at 37°C with 3.5 mM GAP in a temporal course of 5 hours. The appearance of additional bands with higher electrophoretic mobility indicates the emergence of negatively charged additional species. The deamidation rate in the WT and the N71D mutant (C) was determined as the percentage decrease of the upper band (native enzyme). The experiments are representative of independent, duplicate assays with differences of less than 10%.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123379.g007: The deamidation of N15 does not depend on deamidation of N71.Native gel electrophoresis of the WT (A) or the N71D mutant (B) after incubation at 1mg/mL in TE buffer at 37°C with 3.5 mM GAP in a temporal course of 5 hours. The appearance of additional bands with higher electrophoretic mobility indicates the emergence of negatively charged additional species. The deamidation rate in the WT and the N71D mutant (C) was determined as the percentage decrease of the upper band (native enzyme). The experiments are representative of independent, duplicate assays with differences of less than 10%.
Mentions: The prevailing terminal marking mechanism proposed for HsTIM establishes that deamidation of N71 is a prerequisite for the deamidation of N15 [20–22]. In order to test the influence of N71 deamidation on the deamidation of N15, the deamidation rate, as measured by the decrease of native enzyme with the concomitant appearance of negatively charged bands in a temporal course followed by native gel electrophoresis, was tested in the WT and in the N71D mutant (Fig 7). It was supposed that if deamidation of N71 favors the deamidation of N15, the N71D mutant (where deamidation already had occurred), must be deamidated faster than the WT enzyme. As can be observed (Fig 7), this is not the case, since both proteins are deamidated at very similar rates. This result indicates that deamidation of N71 does not influence the deamidation rate of N15.

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