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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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Phylogenetic analysis of TIM; relationship between R17, S79 and NG pairs in Eukarya TIMs.The Eukarya domain was extracted from the complete phylogenetic tree and analyzed. NG pairs present in Eukarya sequences are shown in red; residues occurring at positions 17 and 79 are shown in the right columns. Numbering according to the HsTIM sequence is shown at the top of the figure. Color bars indicate major evolutionary grades with a color code as follows: mammals, blue; sauropsidae (reptiles and birds), cyan; anamniotae (fishes and amphibians), purple; arthropodae, brown; nematodae, yellow; green algae, olive; plants, green; kinetoplastidae, orange; fungi, gray; chromalveolata, red.
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pone.0123379.g011: Phylogenetic analysis of TIM; relationship between R17, S79 and NG pairs in Eukarya TIMs.The Eukarya domain was extracted from the complete phylogenetic tree and analyzed. NG pairs present in Eukarya sequences are shown in red; residues occurring at positions 17 and 79 are shown in the right columns. Numbering according to the HsTIM sequence is shown at the top of the figure. Color bars indicate major evolutionary grades with a color code as follows: mammals, blue; sauropsidae (reptiles and birds), cyan; anamniotae (fishes and amphibians), purple; arthropodae, brown; nematodae, yellow; green algae, olive; plants, green; kinetoplastidae, orange; fungi, gray; chromalveolata, red.

Mentions: Finally, considering that the terminal marking mechanism by deamidation seems to depend on the presence of R17 and S79, and that this mechanism is not general for TIM (recall that deamidation is relevant for the human and rabbit enzymes, but not for the yeast or chicken proteins), it was interesting to ask if a phylogenetic relationship between the deamidating pairs, R17 and S79, exists (Fig 11). The results show that the NG pair 15–16 is highly conserved (in 66 of 70 Eukarya sequences) in comparison with the pair 71–72 (which is only found in 25 of 70 sequences, mainly in mammals); in contrast, the remaining NG pairs are poorly conserved (Fig 11). In addition, it can be observed that S79 is highly conserved (it occurs in 65 of the 70 Eukarya sequences) whereas, in contrast, R17 is only found in mammal sequences and is completely absent in the rest of Eukarya TIMs. In fact, positive amino acids are not found in position 17, excepting T. vaginalis where lysine is present (Fig 11). The results indicate a close relationship between R17 and the NG pair 15–16 in the mammalian enzymes, but not for other phylogenetic groups. In addition, it is interesting to note that the NG pair 71–72 is also consistently present in mammalian enzymes, but in contrast to R17, it is also found in lower branches as those of apicomplexans, fungi, green algae, G. lamblia, and T. vaginalis (Fig 11).


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)

Phylogenetic analysis of TIM; relationship between R17, S79 and NG pairs in Eukarya TIMs.The Eukarya domain was extracted from the complete phylogenetic tree and analyzed. NG pairs present in Eukarya sequences are shown in red; residues occurring at positions 17 and 79 are shown in the right columns. Numbering according to the HsTIM sequence is shown at the top of the figure. Color bars indicate major evolutionary grades with a color code as follows: mammals, blue; sauropsidae (reptiles and birds), cyan; anamniotae (fishes and amphibians), purple; arthropodae, brown; nematodae, yellow; green algae, olive; plants, green; kinetoplastidae, orange; fungi, gray; chromalveolata, red.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123379.g011: Phylogenetic analysis of TIM; relationship between R17, S79 and NG pairs in Eukarya TIMs.The Eukarya domain was extracted from the complete phylogenetic tree and analyzed. NG pairs present in Eukarya sequences are shown in red; residues occurring at positions 17 and 79 are shown in the right columns. Numbering according to the HsTIM sequence is shown at the top of the figure. Color bars indicate major evolutionary grades with a color code as follows: mammals, blue; sauropsidae (reptiles and birds), cyan; anamniotae (fishes and amphibians), purple; arthropodae, brown; nematodae, yellow; green algae, olive; plants, green; kinetoplastidae, orange; fungi, gray; chromalveolata, red.
Mentions: Finally, considering that the terminal marking mechanism by deamidation seems to depend on the presence of R17 and S79, and that this mechanism is not general for TIM (recall that deamidation is relevant for the human and rabbit enzymes, but not for the yeast or chicken proteins), it was interesting to ask if a phylogenetic relationship between the deamidating pairs, R17 and S79, exists (Fig 11). The results show that the NG pair 15–16 is highly conserved (in 66 of 70 Eukarya sequences) in comparison with the pair 71–72 (which is only found in 25 of 70 sequences, mainly in mammals); in contrast, the remaining NG pairs are poorly conserved (Fig 11). In addition, it can be observed that S79 is highly conserved (it occurs in 65 of the 70 Eukarya sequences) whereas, in contrast, R17 is only found in mammal sequences and is completely absent in the rest of Eukarya TIMs. In fact, positive amino acids are not found in position 17, excepting T. vaginalis where lysine is present (Fig 11). The results indicate a close relationship between R17 and the NG pair 15–16 in the mammalian enzymes, but not for other phylogenetic groups. In addition, it is interesting to note that the NG pair 71–72 is also consistently present in mammalian enzymes, but in contrast to R17, it is also found in lower branches as those of apicomplexans, fungi, green algae, G. lamblia, and T. vaginalis (Fig 11).

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