title: Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics.
abstract: 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.
authors.affiliation: Department of Anesthesiology, Multidisciplinary Neuroprotection Laboratories, Duke University Medical Center , Durham, North Carolina 28810, United States. wulf.paschen@duke.edu
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: J Will
authors.initials: JW
authors.lastname: Thompson
authors.forename: Zhengfeng
authors.initials: Z
authors.lastname: Wang
authors.forename: Liangli
authors.initials: L
authors.lastname: Wang
authors.forename: Huaxin
authors.initials: H
authors.lastname: Sheng
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Foster
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.forename: Wulf
authors.initials: W
authors.lastname: Paschen
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D005947:Glucose / Q000172:deficiency / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D009447:Neuroblastoma; D010100:Oxygen / Q000378:metabolism*; D060066:Protein Interaction Maps; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000378:metabolism*; D013312:Stress, Physiological / Q000502:physiology; D054875:Ubiquitination
chemical_list: D011506:Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D005947:Glucose; D010100:Oxygen
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