Excess O-GlcNAc modification of proteins and myocardial fibrosis

在该提议中要检验的主要假设是，“减少β-N-的过量添加， 乙酰葡萄糖胺（O-GlcNAc）对心脏成纤维细胞中存在的调节因子改善糖尿病 诱发心肌纤维化”。鉴于退伍军人的平均年龄较大，约25%的人患有 2型糖尿病（DM 2）。这些患者中的大量将发展DM 2诱导的心脏纤维化， 其不利地影响舒张功能并经常导致心力衰竭的发展。过量O- 已知蛋白质的GlcNAc修饰随着衰老而发生，并且最显著地发生在DM 2的情况下。我们有 证明了心脏成纤维细胞（CF）蛋白的过量O-GlcNAc酰化与增强的 生产胶原蛋白。由于组织纤维化（如肾小球硬化）是DM 2的一个突出特征，我们的研究 这些发现也可能具有更广泛的意义，作为一种改善胶原蛋白过量产生的策略， 纤维母细胞存在于其他器官中。丝氨酸和苏氨酸残基的翻译后修饰 通过O-连接的单糖β-N-乙酰葡糖胺连接细胞核和细胞质蛋白， 一种高度动态和普遍存在的蛋白质修饰，其继发于β-N- 乙酰葡糖胺转移酶（OGT）。相反，O-GlcNAc的去除是由N- 乙酰氨基葡糖苷酶（O-GlcNAcase）。蛋白质O-GlcNAc化正迅速成为一种关键的调节因子， 关键的生物学过程，包括核转运、翻译和转录、信号转导， 细胞骨架重组、蛋白酶体降解和凋亡。我们证明了在高葡萄糖下 CF处理后，核转录因子Sp1和β-淀粉酶表现出过量的O-GlcNAc酰化。这两 蛋白质与刺激胶原蛋白的产生错综复杂地相关。在CF中的表达式 编码O-GlcNAcase的腺病毒，减少Sp1和精氨酸酶O-GlcNAcylation并恢复高糖- 将过量的胶原蛋白生产恢复到正常水平。然而，没有研究确定哪些特定的 氨基酸残基可以通过O-GlcNAc酰化修饰，以及它们如何改变Sp1和β-淀粉酶功能。 此外，这些观察结果尚未在体内环境中得到证实，并且与以下变化有关： 心脏结构和功能。鉴于这些事实和我们所产生的初步数据，我们建议 研究以下具体目标：目标1。为了确定心肌纤维化的发展，舒张压 功能障碍，Sp1和β-淀粉酶I残基修饰的老年模型DM 2。目标2.表征HG 诱导的CF氨基酸残基O-GlcNAc修饰的Sp1和丝氨酸蛋白酶I及其功能意义。 目标3。表征改变的O-GlcNAcase活性修饰DM 2或HG诱导的改变的能力 在Sp1和β-淀粉酶I残基修饰、成纤维细胞/肌成纤维细胞表型、心脏结构和功能中。

The major hypothesis to be tested in this proposal is that, “Reducing the excess addition of β-N- acetylglucosamine (O-GlcNAc) to regulatory factors present in cardiac fibroblasts ameliorates diabetes mellitus induced myocardial fibrosis”. Given the older average age of the Veteran population, about 25% suffer from type 2 diabetes mellitus (DM2). A large number of these patients will develop DM2 induced cardiac fibrosis, which adversely impacts diastolic function and frequently leads to the development of heart failure. Excess O- GlcNAc modification of proteins is known to occur with aging and most notably in the setting of DM2. We have demonstrated that excess O-GlcNAcylation of cardiac fibroblast (CF) proteins is associated with the enhanced production of collagens. As tissue fibrosis (e.g. glomerulosclerosis) is a prominent feature of DM2 our research findings may also have broader implications as a strategy to ameliorate excess collagen production by fibroblasts present in other organs. The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide β-N-acetylglucosamine is a highly dynamic and ubiquitous protein modification that is secondary to the action of β-N- acetylglucosaminyltransferase (OGT). Conversely, the removal of O-GlcNAc is mediated by N- acetylglucosaminidase (O-GlcNAcase). Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. We demonstrated that in high glucose treated CF, the nuclear transcription factor Sp1 and arginase evidence excess O-GlcNAcylation. Both of these proteins are intricately associated with stimulating the production of collagens. Expression in CF of an adenovirus coding for O-GlcNAcase, decreased Sp1 and arginase O-GlcNAcylation and restores high glucose- induced excess collagen production back to normal levels. However, no studies have identified which specific amino acid residues can be modified by O-GlcNAcylation and how they alter Sp1 and arginase function. Furthermore, these observations have not been evidenced in the in vivo setting and linked with changes in cardiac structure and function. Given these facts and the preliminary data we have generated, we propose to examine the following specific aims: Aim 1. To identify the development of myocardial fibrosis, diastolic dysfunction, Sp1 and arginase I residue modification in an aged model of DM2. Aim 2. To characterize HG induced CF amino acid residue O-GlcNAc modification of Sp1 and arginase I and its functional implications. Aim 3. To characterize the capacity of altered O-GlcNAcase activity to modify DM2 or HG induced alterations in Sp1 and arginase I residue modification, fibroblast/myofibroblast phenotype, cardiac structure and function.