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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 decreases the structural compactness of HsTIM.The derivatization of cysteine residues in TIM (300 μg/mL) by DTNB (1 mM) was followed spectrophotometrically at 412 nm. The experiment is representative of duplicate experiments qualitatively identical.
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pone.0123379.g005: The N15D mutation decreases the structural compactness of HsTIM.The derivatization of cysteine residues in TIM (300 μg/mL) by DTNB (1 mM) was followed spectrophotometrically at 412 nm. The experiment is representative of duplicate experiments qualitatively identical.

Mentions: The susceptibility of the N15D and N15D/N71D mutants to proteolysis suggests that these proteins have a more relaxed 3D structure [32]; therefore, the compactness of WT HsTIM and the mutants was tested by the accessibility of their cysteine residues to Ellman´s reagent (DTNB) (Fig 5). Each WT HsTIM monomer has five cysteine residues, which are totally buried (less than 1% of accessible surface area for each of the ten cysteines in the dimer of the 2JK2 structure; data not shown), distributed throughout the structure of the protein. Therefore, the velocity of chemical modification (derivatization) of the cysteine residues by DTNB can be considered as an index of the structural compactness in the protein [32]. The results showed that the WT or the N71D HsTIM are derivatized slowly in comparison with the N15D and the N15D/N71D mutants (Fig 5). Under identical experimental conditions, DTNB derivatizes only 0.7 cysteines per monomer in the WT or the N71D enzymes after one hour; in contrast, in the N15D or N15D/N71D mutants 4.6 cysteines per monomer are derivatized in the first 30 minutes of the experiment (Fig 5). The result supports the idea of a loosening of the three-dimensional structure of the N15D and N15D/N71D mutants.


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 decreases the structural compactness of HsTIM.The derivatization of cysteine residues in TIM (300 μg/mL) by DTNB (1 mM) was followed spectrophotometrically at 412 nm. The experiment is representative of duplicate experiments qualitatively identical.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123379.g005: The N15D mutation decreases the structural compactness of HsTIM.The derivatization of cysteine residues in TIM (300 μg/mL) by DTNB (1 mM) was followed spectrophotometrically at 412 nm. The experiment is representative of duplicate experiments qualitatively identical.
Mentions: The susceptibility of the N15D and N15D/N71D mutants to proteolysis suggests that these proteins have a more relaxed 3D structure [32]; therefore, the compactness of WT HsTIM and the mutants was tested by the accessibility of their cysteine residues to Ellman´s reagent (DTNB) (Fig 5). Each WT HsTIM monomer has five cysteine residues, which are totally buried (less than 1% of accessible surface area for each of the ten cysteines in the dimer of the 2JK2 structure; data not shown), distributed throughout the structure of the protein. Therefore, the velocity of chemical modification (derivatization) of the cysteine residues by DTNB can be considered as an index of the structural compactness in the protein [32]. The results showed that the WT or the N71D HsTIM are derivatized slowly in comparison with the N15D and the N15D/N71D mutants (Fig 5). Under identical experimental conditions, DTNB derivatizes only 0.7 cysteines per monomer in the WT or the N71D enzymes after one hour; in contrast, in the N15D or N15D/N71D mutants 4.6 cysteines per monomer are derivatized in the first 30 minutes of the experiment (Fig 5). The result supports the idea of a loosening of the three-dimensional structure of the N15D and N15D/N71D mutants.

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