Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning
Substrates of the Arabidopsis thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning
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DOI：
10.1074/jbc.m110.157008
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发表时间：
2010-11-26
影响因子：
4.8
通讯作者：
Downie, A. Bruce
中科院分区：
文献类型：
--
作者：
Chen, Tingsu;Nayak, Nihar;Downie, A. Bruce
蛋白质异戊酰甲基转移酶（PIMT）在修复宿主生物体中各种各样的受损蛋白质中的作用已经从酶对过多氨基酸背景中的异戊酰残基的亲和力推断出来。植物种子中PIMT靶蛋白的鉴定受到大量含异戊二酸盐的贮藏蛋白的阻碍，在植物种子中PIMT靶蛋白是高活性的并且蛋白质组寿命长。成熟的种子噬菌体展示文库规避了这个问题。在淘选过程中包含PIMT共底物S-腺苷甲硫氨酸（SNAMet）允许PIMT保留比缺乏共底物的对照或当PIMT蛋白结合被S-腺苷高半胱氨酸毒害时更多数量的老化噬菌体。四轮后，噬菌体滴度在含TMPET的盘中达到平台，而在两种对照中滴度下降。该策略鉴定了17个框内PIMT靶蛋白，包括与先前使用印迹甲基化鉴定的那些相似的cupin家族蛋白。所有回收的噬菌体都具有至少一个敏感的Asp或Asn残基。五个目标被独立回收。在大肠杆菌中产生两个框内靶标作为重组蛋白，并通过印迹甲基化显示获得isoAsp，成为PIMT靶标。两者在热损伤后在溶液中迅速获得isoAsp。三个框内PIMT靶中的任何一个缺陷的植物的突变体分析导致可证明的表型。过度代表性的克隆编码蛋白质参与蛋白质生产表明，翻译装置包括一个亚组，PIMT介导的修复是至关重要的正统种子寿命。受损的PIMT活性将阻碍这些靶标中的蛋白质功能，可能导致种子性能差。
The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.