Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics.

Transient cerebral ischemia dramatically activates small ubiquitin-like modifier (SUMO2/3) conjugation. In cells exposed to 6 h of transient oxygen/glucose deprivation (OGD), a model of ischemia, SUMOylation increases profoundly between 0 and 30 min following re-oxygenation. To elucidate the effect of transient OGD on SUMO conjugation of target proteins, we exposed neuroblastoma B35 cells expressing HA-SUMO3 to transient OGD and used stable isotope labeling with amino acids in cell culture (SILAC) to quantify OGD-induced changes in levels of specific SUMOylated proteins. Lysates from control and OGD-treated cells were mixed equally, and HA-tagged proteins were immunoprecipitated and analyzed by 1D-SDS-PAGE-LC-MS/MS. We identified 188 putative SUMO3-conjugated proteins, including numerous transcription factors and coregulators, and PIAS2 and PIAS4 SUMO ligases, of which 22 were increased or decreased more than ±2-fold. In addition to SUMO3, the levels of protein-conjugated SUMO1 and SUMO2, as well as ubiquitin, were all increased. Importantly, protein ubiquitination induced by OGD was completely blocked by gene silencing of SUMO2/3. Collectively, these results suggest several mechanisms for OGD-modulated SUMOylation, point to a number of signaling pathways that may be targets of SUMO-based signaling and recovery from ischemic stress, and demonstrate a tightly controlled crosstalk between the SUMO and ubiquitin conjugation pathways.

J. Proteome Res.2012 Feb 3,11(2).

Protein Name:SUMO3
Gene Name:SUMO3 SMT3B SMT3H1

Recombinant Human Small Ubiquitin-Related Modifier 3/SUMO3 produced by our E. coli expression system. The target protein is expressed with sequence (Met1-Phe103) of Human SUMO3 fused with a 6His tag at the N-terminus.

SUMO3 belongs to the SUMO protein family and operates like ubiquitin. Ubiquitin-like protein which can be covalently attached to target lysines either as a monomer or as a lysine-linked polyer. Nevertheless unlike ubiquitin that targets proteins for degration, SUMO3 takes part in several cellular processess, such as nuclear transport, transcription regulation, apoptosis and protein stability. SUMO3 participates in amyloid beta generation and has a key role in the oneset or progression of Alzheimer's disease.