Structural Effects of Protein Aging: Terminal Marking by Deamidation in Human Triosephosphate Isomerase

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.

Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum

“…The result obtained show that the FoxTPI protein is a well-folded protein with a secondary structure like that of a canonical TPI barrel. It is important to mention that the intensity pattern in the far UV region shown for FoxTPI is similar to to other secondary structures of TPIs previously purified and characterized, as in G. lamblia (GlTPI) [17], human (HuTPI) [50], Trichomonas vaginalis (TvTPI) [55], Saccharomyces cerevisiae (TPI) [44], and Encephalitozoon intestinalis (EiTPI) [48], between which no significant differences were observed.…”

“…The temperature increases induced denaturation of the protein. In the denaturation profile, the protein remained at its baseline (native structure) until approximately 43 • C ( Figure 7B) and, above this temperature, the enzyme was denatured cooperatively, completing the process at approximately 60 • C. The calculated T m was 51.4 • C, which is slightly inferior to the values reported for other TPI enzymes previously characterized from E. intestinalis (EiTPI; 52.8 • C) [48], T. cruzi (TcTPI; 57.3 • C) [52], Entamoeba histolytica (EhTPI; 58.6 • C) [59], G. lamblia (GlTPI; 57.5 • C) [17], and Homo sapiens (HsTPI; 61.2 • C) [50].…”

Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

“…Structure-function studies of TPIs from animals, protozoa, and bacteria illustrate that these enzymes are regulated by diverse mechanisms such as: altering dimer-monomer equilibrium, deamination, phosphorylation, binding to competitive inhibitors or binding to thiol-conjugated reagents (Ralser et al, 2006; Olivares-Illana et al, 2007; Lee et al, 2010; Enríquez-Flores et al, 2011; Grüning et al, 2014; Lara-Gonzalez et al, 2014, 2015; de la Mora-de la Mora et al, 2015). In contrast, no structure-function studies of TPIs from land plants have been carried out to date.…”

Disulfiram as a novel inactivator of Giardia lamblia triosephosphate isomerase with antigiardial potential

“…Recombinant proteins used in this work (GlTIM C202A, GlTIM C202A/C222A and WT HsTIM) have been previously expressed, purified and characterized ( Reyes-Vivas et al, 2007 , Hernández-Alcántara et al, 2013 , de la Mora-de la Mora et al, 2015 ). In brief, individual genes from GlTIM and HsTIM cloned into the pET-HisTEVP vector were expressed in the Escherichia coli BL21(DE3)pLysS or BL21-CodonPlus (DE3)-RIL strains as His-Tagged proteins and purified by immobilized-metal affinity chromatography ( Hernández-Alcántara et al, 2013 , de la Mora-de la Mora et al, 2015 ). The histidine tag was removed with tobacco etch virus protease as reported previously ( Hernández-Alcántara et al, 2013 , de la Mora-de la Mora et al, 2015 ).…”

“…In brief, individual genes from GlTIM and HsTIM cloned into the pET-HisTEVP vector were expressed in the Escherichia coli BL21(DE3)pLysS or BL21-CodonPlus (DE3)-RIL strains as His-Tagged proteins and purified by immobilized-metal affinity chromatography ( Hernández-Alcántara et al, 2013 , de la Mora-de la Mora et al, 2015 ). The histidine tag was removed with tobacco etch virus protease as reported previously ( Hernández-Alcántara et al, 2013 , de la Mora-de la Mora et al, 2015 ).…”