Mass spectrometric identification of novel posttranslational modification sites in H untingtin

G Dong, E Callegari, CJ Gloeckner, M Ueffing… - …, 2012 - Wiley Online Library
G Dong, E Callegari, CJ Gloeckner, M Ueffing, H Wang
Proteomics, 2012Wiley Online Library
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) …
Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep‐ and FLAG‐tagged Htt containing either 19Q (wild‐type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano‐LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site‐specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.
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