Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme
Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme
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DOI：
10.1074/jbc.m605421200
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发表时间：
2006-10-27
影响因子：
4.8
通讯作者：
Carter, Wayne G.
中科院分区：
文献类型：
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作者：
Vigneswara, Vasanthy;Lowenson, Jonathan D.;Carter, Wayne G.
我们报告使用蛋白质组学策略来鉴定哺乳动物蛋白l -异天冬氨酸o -甲基转移酶迄今未知的底物。这种甲基转移酶启动体内老化或应激损伤蛋白中异天冬氨酸残基的修复。缺乏甲基转移酶（Pcmt1(-/-)）的小鼠的组织比野生型小鼠积累了更多的异天冬氨酸残留物，其中最“受损”的残留物出现在大脑中。为了鉴定含有这些残基的蛋白质，用外源性修复酶和放射性标记的甲基供体s -腺苷-[甲基- h -3]蛋氨酸对Pcmt1（-/-）小鼠的脑匀浆进行甲基化。通过一维和二维电泳分离匀浆中的甲基化蛋白，与Pcmt1（-/-）幼崽相比，从Pcmt1（-/-）幼崽中分离的甲基转移酶底物通过增加的放射性标记来鉴定。这些分离的脑蛋白的质谱分析首次揭示了微管相关蛋白-2、钙网蛋白、网格蛋白轻链a和b、泛素羧基末端水解酶L1、磷脂酰乙醇胺结合蛋白、stathmin、β -突触核蛋白和α -突触核蛋白都是体内l-异天冬氨酸甲基转移酶的底物。我们的甲基转移酶底物鉴定方法进一步得到了补充，证明了这些甲基转移酶靶标之一微管相关蛋白-2可以在Pcmt1（-/-）脑提取物中使用放射性甲基供体和外源性甲基转移酶进行放射性标记，然后用微管相关蛋白-2抗体特异性免疫沉淀，以恢复具有放射性的共定位蛋白。在甲基转移酶敲除小鼠相关的组织学和表型变化的背景下，我们评论了甲基转移酶靶点内相对高水平异天冬氨酸积累的功能意义。
We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein L-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1(-/-) mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-H-3] methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1(-/-) animals compared with Pcmt1(-/-) littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, beta-synuclein, and alpha-synuclein, are all substrates for the L-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1(-/-) brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.