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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 favors the dissociation of HsTIM.The stability of HsTIM dimers was tested by incubating enzymes (500 μg/mL) at 30°C for 3 hours and then loading them onto a size-exclusion chromatography column (Superdex 75) equilibrated and developed in 50 mM Tris pH 8.0 plus 150 mM NaCl and 5% (v/v) glycerol. For simplicity, only the data for the WT, the N15D and the N15D/N71D proteins are shown. The profile for the N71D mutant overlaps with that of the WT enzyme.
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pone.0123379.g006: The N15D mutation favors the dissociation of HsTIM.The stability of HsTIM dimers was tested by incubating enzymes (500 μg/mL) at 30°C for 3 hours and then loading them onto a size-exclusion chromatography column (Superdex 75) equilibrated and developed in 50 mM Tris pH 8.0 plus 150 mM NaCl and 5% (v/v) glycerol. For simplicity, only the data for the WT, the N15D and the N15D/N71D proteins are shown. The profile for the N71D mutant overlaps with that of the WT enzyme.

Mentions: The interfacial charge-repulsion assumption explaining HsTIM destabilization arose from the close position of N15 and N71 in adjacent subunits [20] (Fig 1). To test the contribution of deamidation to dimer stability, we performed size-exclusion chromatography of WT HsTIM and the mutants after incubation at 30°C for 3 hours (Fig 6). Whereas the WT and the N71D mutants elute as single peaks with retention volumes corresponding to native dimers (55 mL, 53 kDa), the N15D and N15D/N71D mutants show two well defined peaks. The first peak corresponds to native dimers, whereas the second closely corresponds to monomers (62 mL and 30 kDa for the N15D mutant and 63 mL and 31 kDa for the double mutant). The results indicate that the N15D and the N15D/N71D mutants are prone to monomerization.


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 favors the dissociation of HsTIM.The stability of HsTIM dimers was tested by incubating enzymes (500 μg/mL) at 30°C for 3 hours and then loading them onto a size-exclusion chromatography column (Superdex 75) equilibrated and developed in 50 mM Tris pH 8.0 plus 150 mM NaCl and 5% (v/v) glycerol. For simplicity, only the data for the WT, the N15D and the N15D/N71D proteins are shown. The profile for the N71D mutant overlaps with that of the WT enzyme.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123379.g006: The N15D mutation favors the dissociation of HsTIM.The stability of HsTIM dimers was tested by incubating enzymes (500 μg/mL) at 30°C for 3 hours and then loading them onto a size-exclusion chromatography column (Superdex 75) equilibrated and developed in 50 mM Tris pH 8.0 plus 150 mM NaCl and 5% (v/v) glycerol. For simplicity, only the data for the WT, the N15D and the N15D/N71D proteins are shown. The profile for the N71D mutant overlaps with that of the WT enzyme.
Mentions: The interfacial charge-repulsion assumption explaining HsTIM destabilization arose from the close position of N15 and N71 in adjacent subunits [20] (Fig 1). To test the contribution of deamidation to dimer stability, we performed size-exclusion chromatography of WT HsTIM and the mutants after incubation at 30°C for 3 hours (Fig 6). Whereas the WT and the N71D mutants elute as single peaks with retention volumes corresponding to native dimers (55 mL, 53 kDa), the N15D and N15D/N71D mutants show two well defined peaks. The first peak corresponds to native dimers, whereas the second closely corresponds to monomers (62 mL and 30 kDa for the N15D mutant and 63 mL and 31 kDa for the double mutant). The results indicate that the N15D and the N15D/N71D mutants are prone to monomerization.

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