Aging in individuals and aspartate modification in proteins

蛋白质异天冬氨酸甲基转移酶（PIMT）被认为在与异天冬氨酸残基自发结合的老化蛋白质的修复中起作用。因此，这种修复系统的缺乏可能会在敲除动物的大脑中显示异天冬氨酸的异常积累，这与衰老过程中蛋白质异构化的病理意义有关。最近，我们报道了敲除小鼠缺乏PIMT酶活性。pmt缺陷小鼠意外地在相当年轻的时候表现出致命的癫痫发作，并且未能存活超过12周，这使得研究衰老大脑中异构化蛋白质的病理意义变得不可能。因此，我们接下来研究了pmt缺陷小鼠发育过程中异天冬氨酸在大脑中积累的动态。生化分析显示，与对照小鼠相比，pmt缺陷小鼠的新生儿大脑中已经积累了异构化蛋白。异构化蛋白的积累在2周龄后显著增加，并且在我们观察到的时间内持续增加，直到9周龄。这些数据表明，蛋白质异构化不仅局限于老年大脑的长寿蛋白质，而且早在pmt缺陷小鼠的胚胎阶段就观察到。为了鉴定异构化蛋白，我们利用含有isoAsp的三肽作为免疫原，制备了抗isoAsp抗体。利用该抗体，选择性地检测了pmt缺陷小鼠脑提取物中43 kDa蛋白（p43）的表观分子量。出生后4周检测到P43蛋白，并集中于突触体部分。提示p43蛋白修饰可能对pmt缺陷小鼠致死性癫痫发作具有致病作用。p43蛋白的鉴定应该澄清pmt缺陷小鼠的这一问题。

Protein isoaspartyl methyltransferase (PIMT) has been suggested to play a role in the repair of aged proteins spontaneously incorporated with isoaspartyl residues. Thus the lack of this repair system was expected to show an abnormal accumulation of isoaspartate in the brain of knockout animals with reference to the pathological implications of protein isomerization during aging. Recently, we reported knockout mice lacking the enzymatic activity for PIMT.The PIMT-deficient mice unexpectedly showed a fatal epileptic seizure at a rather young age and failed to survive beyond 12 weeks, making it impossible to investigate the pathological implications of isomerized proteins in aged brains. Therefore, we next investigated the dynamics of accumulated isoaspartate in the brain during the development of PIMT-deficient mice. Biochemical analysis revealed that isomerized proteins have already accumulated in neonatal brains of PIMT-deficient mice compared to control mice. The accumulation of isomerized proteins then markedly progressed after 2 weeks of age and continued to increase as long as we could observe it until 9 weeks of age. These data indicated that protein isomerization is not restricted to long-lived proteins of aged brains, but is observed in as early as embryonic stage of PIMT-deficient mice. In order to identify the isomerized proteins, we developed the anti-isoAsp antibody using isoAsp containing tripeptide as an immunogen. Using this antibody, apparent molecular weight 43 kDa proteins (p43) were selectively detected in brain extract of PIMT-deficient mice. p43 proteins were detected from 4 weeks after birth and concentrated in synaptosomal fraction. The result suggests that modification of p43 protein may have a pathogenic role for the fatal epileptic seizure of PIMT-deficient mice. The identification of p43 protein should clarify this issue of PIMT-deficient mice.