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Mass spectrometric identification of novel posttranslational modification sites in H untingtin
Author(s) -
Dong Gaofeng,
Callegari Eduardo,
Gloeckner Christian J.,
Ueffing Marius,
Wang Hongmin
Publication year - 2012
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100380
Subject(s) - phosphorylation , microbiology and biotechnology , chemistry , tandem mass spectrometry , huntingtin , serine , cytotoxicity , hek 293 cells , western blot , polyglutamine tract , mutant , biochemistry , biology , mass spectrometry , gene , in vitro , chromatography
Huntington's disease ( HD ) is caused by a CAG triplet repeat expansion in exon 1 of the H untingtin ( H tt) gene, encoding an abnormal expanded polyglutamine (poly Q ) tract that confers toxicity to the mutant H tt (m H tt) protein. Recent data suggest that posttranslational modifications of m H tt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of m H tt, we have generated HEK 293 cell models stably expressing S trep‐ and FLAG ‐tagged H tt containing either 19 Q (wild‐type H tt), 55 Q (m H tt), or 94 Q (m H tt) repeats. Following tandem affinity purification, the tagged H tt and associated proteins were subjected to tandem mass spectrometry or 2 D nano‐ LC tandem mass spectrometry and several novel modification sites of m H tt containing 55 Q or 94 Q were identified. These were phosphorylation sites located at S er431 and S er432, and ubiquitination site located at L ys444. The two phosphorylation sites were confirmed by W estern blot analysis using phosphorylation site‐specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered m H tt toxicity and accumulation. These modifications of m H tt may provide novel therapeutic targets for effective treatment of the disorder.