Protein Methylation in Brain

描述(由申请人提供)：本研究的总体目标是 探索蛋白质甲基化反应的生物学功能 特别关注它们在神经功能中可能扮演的角色。我们的研究 将专注于两种不同的甲基转移酶。一、蛋白质 L异天冬氨酸甲基转移酶(PIMT)。催化非典型的甲基化 在某些蛋白质中以一种形式出现的异天冬氨酸 自发性损伤。IsoAsp可以使蛋白质功能失调，和/或高度 在产生它们的同一种动物中具有免疫原性。此外，PIMT 基因敲除小鼠在细胞内积累高水平的isAsp位点并发展为 4-6周出现致命性癫痫发作。要了解更多关于的功能 PIMT和isAsp累积的后果，我们将：(1)确定是否 PIMT在体内的作用是挽救受损的蛋白质或促进其 通过比较组蛋白H_2B(体内主要成分)的周转率来进行降解 在正常细胞和PIMT缺陷细胞中)，并通过比较 在H_2B中消旋isAsp倾向于Asp-25残基；(2)确定isAsp如何 蓄积影响突触素-1和其他突触体的功能 蛋白质；(3)确定isAsp位点是否大大增强了 在同一物种中，isAsp H2B诱导小鼠 自身免疫病理，以及自身免疫患者的血清 系统性红斑狼疮等疾病的抗体或T细胞 选择性地识别H_2B的isAsp形式。待研究的第二种酶 是共激活物相关的精氨酸甲基转移酶1(CARM 1)，是一种 与特定转录因子形成复合体，介导 糖皮质激素调节的基因表达。要了解有关函数的更多信息，请执行以下操作 对于CARM 1，我们将(1)确定纯化的HUD(一种mRNA结合蛋白 与神经元发育有关)是CARM 1的体内底物，并且 用先前的方法在大鼠PC12细胞中寻找额外的Carm1底物 开发了基于甲基转移酶抑制剂的策略，以及(2)确定是否 哺乳动物的大脑(或其他组织)含有一种蛋白质--精氨酸去甲基化 可逆转CARM 1和/或催化甲基化反应的酶 相关蛋白精氨酸甲基转移酶。我们提出的关于PIMT的研究 应提供有关等位天冬氨酸形成的可能贡献的新见解 和PIMT缺陷在困扰大脑和可能的免疫系统的疾病中 系统。同样，对CARM1的研究应提供关于以下方面的重要新信息 糖皮质激素如何调节大脑中的基因表达。

DESCRIPTION (provided by applicant): The overall goal of this research is to explore the biological function of protein methylation reactions, with particular attention to their possible roles in neuronal function. Our studies will focus on two distinct methyltransferase enzymes. One, protein L-isoaspartyl methyltransferase (PIMT). catalyzes methylation of atypical isoaspartyl sites (isoAsp) that arise in certain proteins as a form of spontaneous damage. IsoAsp can render proteins dysfunctional, and/or highly immunogenic in the same animal from which they are derived. Moreover, PIMT knock-out mice accumulate high levels of intracellular isoAsp sites and develop fatal epileptic seizures at 4-6 weeks. To understand more about the function of PIMT and the consequences of isoAsp accumulation, we will: (1) determine if PIMT functions in vivo to rescue damaged proteins or to facilitate their degradation by comparing the turnover rate of histone H2B (a major in vivo substrate for PIMT) in normal vs. PIMT-deficient cells, and by comparing the racemization of the isoAsp prone Asp-25 residue in H2B; (2) identify how isoAsp accumulation affects the function of synapsin-1 and other synaptosomal proteins; (3) determine if isoAsp sites greatly enhance the immunogenicity of mouse H2B in that same species, the extent to which isoAsp H2B induces an autoimmune pathology, and if the sera of patients afflicted with autoimmune diseases such as systemic lupus erythematosus have antibodies or T cells that selectively recognize the isoAsp form of H2B. The second enzyme to be studied is coactivator-associated arginine methyltransferase 1 (CARM 1), an enzyme that forms complexes with specific transcription factors that mediate glucocorticoid-regulated gene expression. To understand more about the function of CARM 1 we will (1) determine if purified HuD (an mRNA-binding protein implicated in neuronal development) is an in vivo substrate for CARM 1 and search for additional CARM 1 substrates in rat PC12 cells using a previously developed methyltransferase inhibitor-based strategy, and (2) determine if mammalian brain (or other tissues) contain a protein-arginine demethylating enzyme that may reverse the methylation reactions catalyzed by CARM 1 and/or related protein arginine methyltransferases. Our proposed studies on PIMT should provide new insights as to the possible contribution of isoAsp formation and PIMT deficiency in diseases afflicting the brain and possibly the immune system. Similarly, studies on CARM1 should provide important new information on how glucocorticoids regulate gene expression in the brain.