Label-Free Quantitative Proteomics in a Methylmalonyl-CoA Mutase-Silenced Neuroblastoma Cell Line.
Label-Free Quantitative Proteomics in a Methylmalonyl-CoA Mutase-Silenced Neuroblastoma Cell Line.
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DOI：
10.3390/ijms19113580
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发表时间：
2018-11-13
影响因子：
5.6
通讯作者：
Ruoppolo M
中科院分区：
文献类型：
--
作者：
Costanzo M;Cevenini A;Marchese E;Imperlini E;Raia M;Del Vecchio L;Caterino M;Ruoppolo M
甲基丙二酸血症(MMAS)是由于甲基丙二酰辅酶A变位酶(MUT)活性低下所致的先天性代谢疾病。MUT催化甲基丙二酰辅酶A生成琥珀酰辅酶A，甲基丙二酰辅酶A是丙酰-辅酶A分解产物，来源于奇链脂肪酸β氧化、胆固醇和支链氨基酸降解。甲基丙二酰辅酶A水平的增加使疾病的症状前诊断成为可能，即使没有批准的治疗方法。MMA患者表现为高氨血症、酮症酸中毒、嗜睡、呼吸窘迫、认知障碍和肝肿大。长期的后果是神经损伤和终末性肾功能衰竭，存活的机会很小。在MUT基因敲除的细胞模型上，用定量蛋白质组学方法研究了MUT缺乏对细胞通路的影响。目前，在神经母细胞瘤细胞系(SH-SY5Y)中，通过针对MUT转录本(MUT SiRNA)的小干扰RNA(SiRNA)获得了持续的MUT蛋白表达下调。MUT缺失不影响SH-SY5Y细胞的存活率和凋亡过程。在这项研究中，我们使用两种不同的定量策略，通过基于质谱学的无标记定量分析来评估和量化沉默导致的蛋白质表达谱的变化。这两种定量方法都使我们能够观察到参与线粒体氧化还原动态平衡的蛋白质的表达受到MUT缺乏的影响。在添加丙酸的培养液中，观察到siRNA_mut细胞功能线粒体活性的改变。最后，发现代谢途径中的蛋白质水平发生了变化，如碳水化合物代谢和脂肪代谢。
Methylmalonic acidemias (MMAs) are inborn errors of metabolism due to the deficient activity of methylmalonyl-CoA mutase (MUT). MUT catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, produced from propionyl-CoA catabolism and derived from odd chain fatty acids β-oxidation, cholesterol, and branched-chain amino acids degradation. Increased methylmalonyl-CoA levels allow for the presymptomatic diagnosis of the disease, even though no approved therapies exist. MMA patients show hyperammonemia, ketoacidosis, lethargy, respiratory distress, cognitive impairment, and hepatomegaly. The long-term consequences concern neurologic damage and terminal kidney failure, with little chance of survival. The cellular pathways affected by MUT deficiency were investigated using a quantitative proteomics approach on a cellular model of MUT knockdown. Currently, a consistent reduction of the MUT protein expression was obtained in the neuroblastoma cell line (SH-SY5Y) by using small-interfering RNA (siRNA) directed against an MUT transcript (MUT siRNA). The MUT absence did not affect the cell viability and apoptotic process in SH-SY5Y. In the present study, we evaluate and quantify the alterations in the protein expression profile as a consequence of MUT-silencing by a mass spectrometry-based label-free quantitative analysis, using two different quantitative strategies. Both quantitative methods allowed us to observe that the expression of the proteins involved in mitochondrial oxido-reductive homeostasis balance was affected by MUT deficiency. The alterated functional mitochondrial activity was observed in siRNA_MUT cells cultured with a propionate-supplemented medium. Finally, alterations in the levels of proteins involved in the metabolic pathways, like carbohydrate metabolism and lipid metabolism, were found.
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影响因子：
3
作者：
Melo, Daniela R.;Kowaltowski, Alicia J.;Castilho, Roger F.
通讯作者：
Castilho, Roger F.
DOI：
10.1007/978-1-62703-450-0_14
发表时间：
2013-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Croft, David
通讯作者：
Croft, David
影响因子：
4.2
作者：
de Baulny, HO;Benoist, JF;Saudubray, JM
通讯作者：
Saudubray, JM
影响因子：
--
作者：
Imperlini E;Santorelli L;Orrù S;Scolamiero E;Ruoppolo M;Caterino M
通讯作者：
Caterino M
影响因子：
4.4
作者：
Alberio, Tiziana;Pieroni, Luisa;Fasano, Mauro
通讯作者：
Fasano, Mauro