Aberrant post-translational modifications characterize the hepatic proteome of methylmalonic acidemia

项目摘要 有机酸血症(OAS)，如甲基丙二酸血症(MMA)，是一组先天代谢疾病 这通常是由于氨基酸和脂肪酸分解代谢的缺陷引起的。OAS很难治疗，而且有 多系统表现，导致发病率和死亡率增加。酰基辅酶A物种的积累是 推测会引起细胞内的毒性。在这里，我们探讨了另一种病理生理学后果 酰辅酶A代谢受损：异常翻译后修饰(PTM)的积累 关键的细胞内途径中的酶，特别是在压力增加的时期。使用鼠标模型 这概括了MMA(Mmut-/-；TgINS-MCK-Mut)和MMA患者肝脏的线粒体病变 组织中，我用丙酰和丙二酰赖氨酸抗体检测了肝脏提取物中的PTM。我发现 MMA小鼠和MMA患者组织样本中广泛存在的超酰化反应与各自 对照样本，但不是Acsf3缺乏的小鼠，Acsf3缺乏是一种酰基-辅酶A合成障碍。接下来，我准备了反- PTM抗体柱，MMA和对照小鼠的纯化肝提取液，并进行质谱分析 对PTM蛋白质组进行鉴定。谷胱甘肽、尿素、精氨酸、赖氨酸等酶的过度酰化， 在MMA小鼠体内检测到色氨酸、缬氨酸、异亮氨酸、蛋氨酸、苏氨酸和脂肪酸代谢。 但不是对照，并通过免疫沉淀分析进一步证实了质谱学Cps1的超酰化 和Western blotting。同时，我们通过非酶促酰化反应生成了PTM修饰的BSA靶标 用于体外分析。我们纯化了SIRT1-7脱酰基酶活性，然后用BSA-PTM标准进行鉴定 最有效去除MMA相关PTM的SIRT(S)。由于PTM通常会抑制酶功能，我们的 观察表明，在MMA中，已知的通路中关键酶的高酰化可能是不受调控的 有助于改变新陈代谢，并确定一套新的治疗干预目标。

Project Summary Organic acidemias (OAs), such as methylmalonic acidemia (MMA), are a group of inborn errors of metabolism that typically arise from defects in the catabolism of amino- and fatty acids. OAs are difficult to treat and have multisystemic manifestations, leading to increased morbidity and mortality. Accretion of acyl-CoA species is postulated to cause intracellular toxicity. Here, we explore an alternative pathophysiological consequence of impaired acyl-CoA metabolism: the accumulation of aberrant posttranslational modifications (PTMs) that modify enzymes in critical intracellular pathways, especially during periods of increased stress. Using a mouse model that recapitulates the hepatic mitochondriopathy of MMA (Mmut-/-;TgINS-MCK-Mut) as well as MMA patient liver tissues, I surveyed PTMs in hepatic extracts with propionyl- and malonyl-lysine antibodies. I discovered widespread hyper-acylation in the MMA mice and MMA patient tissue samples compared to their respective control samples, but not in mice with Acsf3 deficiency, a disorder of acyl-CoA synthesis. Next, I prepared anti- PTM antibody columns, purified hepatic extracts from MMA and control mice, and performed mass spectrometry to characterize the PTM proteome. Excessive acylation of enzymes involved in glutathione, urea, arginine, lysine, tryptophan, valine, isoleucine, methionine, threonine, and fatty acid metabolism were detected in the MMA mice but not controls, and further validated mass spectrometry Cps1 hyperacylation via immunoprecipitation analysis and Western blotting. In parallel, we generated, via nonenzymatic acylation reactions, PTM-modified BSA targets for in vitro analyses. We purified, then assayed, SIRT1-7 deacylase activity using BSA-PTM standards to identify the SIRT(s) that most efficiently remove MMA related PTMs. Because PTMs usually inhibit enzyme function, our observations suggest that hyperacylation of key enzymes in pathways known to be dysregulated in MMA likely contributes to altered metabolism and identifies a new set of targets for therapeutic intervention.