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

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.

脱酰胺作用（Deamidation）是指天冬酰胺与谷氨酰胺失去铵根基团，进而生成天冬氨酸与谷氨酸的过程，是蛋白质中最常见的翻译后修饰类型之一。
由于脱酰胺速率由蛋白质结构所编码，已有研究提出其可作为生物过程时序的分子时钟，例如蛋白质周转、发育与衰老进程。
尽管该过程具有重要研究价值，但目前仍缺乏详细的结构信息来阐释脱酰胺对蛋白质结构的影响。
本研究针对人源磷酸丙糖异构酶（HsTIM）展开探究，该酶的N15与N71位点发生脱酰胺作用的现象，早已被认定为该蛋白质末端标记的信号。
研究通过定点诱变技术模拟脱酰胺作用，对三种HsTIM突变体（N15D、N71D及N15D/N71D）进行了表征。
结果显示，N71D突变体在结构与功能层面均与野生型酶高度相似。
与之相反，N15D突变体则表现出所有与脱酰胺相关的有害效应。
相较于N15D单突变体，N15D/N71D双突变体仅呈现出轻微的附加效应，这表明N71位点的脱酰胺作用诱导的效应可忽略不计。
晶体结构分析表明，与仅存在微小结构改变的N71D突变体不同，N15D突变体形成了新的相互作用，扰乱了环1与环3的结构——这两个区域均为催化位点与人源磷酸丙糖异构酶界面的关键组成部分。
基于对TIM序列的系统发育分析，本研究提出该调控机制在哺乳动物TIMs中具有保守性。