Abstract W P206: Analysis of SUMO3-Modified Proteome in Post-Ischemic Mouse Brain
Background and Purpose: Small ubiquitin-like modifier (SUMO) conjugation modulates many key cellular processes. Transient cerebral ischemia dramatically activates SUMO2/3 conjugation, and this is believed to be a protective stress response. It is, therefore, of tremendous clinical interest to characterize the SUMO-modified proteome regulated by transient ischemia. We generated a novel SUMO transgenic mouse and performed the first SUMO proteomics study using post-ischemic brain samples.
Methods: CAG-loxP-STOP-loxP-SUMO (CAG-SUMO) mice were generated in which His-SUMO1, HA-SUMO2, and FLAG-SUMO3 were expressed from a single multicistronic transgene in a Cre-dependent manner. CAG-SUMO mice were mated with Emx1Cre/Cre mice to generate double transgenic CAG-SUMO/Emx1-Cre mice as experimental mice and Emx1Cre/+ mice as control mice. Double transgenic mice were subjected to 10 min global cerebral ischemia followed by 1 h reperfusion or sham operation. FLAG-SUMO3-conjugated proteins were enriched from cortical tissues and analyzed.
Results: Characterization of double transgenic mice demonstrated that exogenous expressed tagged SUMO paralogues were functionally intact and did not perturb the endogenous SUMOylation machinery in the brain. FLAG pulldown of cortical samples from sham and ischemia mice followed by GeLC-MS/MS analysis identified 91 candidates whose SUMOylation states were up-regulated in ischemic samples. Data analysis revealed several potentially important processes in which SUMO3 conjugation may play a key role during ischemia/reperfusion, including the cross-talk between SUMOylation and ubiquitination, glucocorticoid receptor signaling, and modulation of posttranscriptional mRNA processing.
Conclusions: SUMO proteomic analysis identified important processes and pathways modulated by SUMOylation in the post-ischemic brain that warrant future investigations, since they could be the key to understand the overall impact of SUMOylation on the fate and functions of post-ischemic neurons. The conditional SUMO transgenic mouse will be an invaluable tool for in-depth in vivo analysis of the SUMO-modified proteome in various pathological states.
Author Disclosures: W. Yang: None. H. Sheng: None. W. Thompson: None. S. Zhao: None. L. Wang: None. X. Liu: None. W. Paschen: None.
This research has received full or partial funding support from the American Heart Association, National Center.
- © 2014 by American Heart Association, Inc.