A stress-dependent SUMO4 sumoylation of its substrate proteins Wenzhong Wei a , Ping Yang a , Junfeng Pang a , Shu Zhang a , Ying Wang a , Mong-Heng Wang b , Zheng Dong c , Jin-Xiong She a , Cong-Yi Wang a, * a Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, GA 30912, USA b Department of Physiology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA c Department of Cellular Biology and Anatomy, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA article info Article history: Received 4 August 2008 Available online 15 August 2008 Keywords: SUMO4 Sumoylation Hydrolase Maturation Intracellular stress Serum starvation . abstract Here we performed studies to demonstrate SUMO4 maturation process. Unlike other SUMO proteins, cells under physiological condition mediate a rapid degradation for SUMO4. However, when cells under stressed condition, SUMO4 can be matured by the stress-induced endogenous hydrolase and be able to covalently conjugate to its substrate proteins. Furthermore, we failed to obtain evidence supporting a role for proline-90 unique to SUMO4 in its activation and functionality. Both wild-type SUMO4 and SUMO4- P90Q can be hydrolyzed by the stressed RAW264.7 cell lysates, and no significant functional difference between SUMO4, SUMO4-P90Q, and SUMO4-GG (matured form) was observed as determined by lucifer- ase assay. However, the C-terminal di-glycine motif, a prerequisite for sumoylation, is necessary for SUMO4 to exert its functional activity. These data not only confirmed our previous published data, but also provided additional evidence suggesting a role for SUMO4 sumoylation in the regulation of intracel- lular stress. Ó 2008 Elsevier Inc. All rights reserved. Sumoylation carried out by the small ubiquitin-like modifiers (SUMO) is a newly identified posttranslational modification mech- anism, contributing to dynamic regulation of gene transcription, cell apoptosis, intracellular stress response and cell cycle progres- sion [1–3]. SUMO constitutes a highly conserved protein family found in all eukaryotes and is required for viability of most eukary- otic cells, including yeasts, nematodes, fruit flies, and vertebrate cells in culture. In 2004, we reported the cloning of a novel human SUMO gene, SUMO4, in the IDDM5 interval on chromosome 6q25, and presented strong genetic and functional evidence suggesting that SUMO4 is a susceptibility gene for type 1 diabetes (T1D) [4]. We discovered the M55V substitution associated with reduced sumoylation function for SUMO4 and, as a result, it confers in- creased risk for T1D [5–10]. Similar effect for M55V on SUMO4 functionality was also reported by another independent study [11]. Despite the consistent association between SUMO4 and T1D observed in the Asian populations, genetic heterogeneity was also detected in certain European Caucasians [4,12,13]. We next per- formed a proteome-wide analysis of the substrate proteins for SUMO4, and identified a total of 90 SUMO4 substrates implicated in the regulation of DNA repair and synthesis, RNA processing, pro- tein degradation and glucose metabolism [14]. Our results sug- gested a possible role for SUMO4 in the regulation of intracellular stress. To be functionally active, SUMO proteins need to be hydrolyzed to expose their C-terminal di-glycine motif. This di-glycine motif is prerequisite for SUMO proteins to covalently conjugate to their substrate proteins. While we were publishing the SUMO4 sub- strate data, Owerbach and colleagues published their biochemical data on SUMO4 [15]. They have artificially added additional 15 amino acids (SKGISRGTAAAGIQT) from the vector sequence in the SUMO4 C-terminus which was then used for the hydrolyzing studies. They demonstrated that SUMO4 does not have the capabil- ity for covalent conjugation to its substrate proteins and concluded that amino acid proline-90 unique to SUMO4 prevents SUMO4 to be hydrolyzed into the active form (with exposed di-glycine motif) [15]. These new data, somehow, rendered all of their previously published data biologically meaningless, as their previous studies were carried out by ectopic expressing the matured form of SUMO4 [11]. On the other hand, their results were contradictory to what we published, since we did use the full-length SUMO4 con- struct for all of our studies [4,14]. To address this discrepancy, we performed biochemical studies using the wild-type SUMO4 pro- tein. Consistent with our previous data, SUMO4 showed the capa- bility to covalently conjugate to its substrate proteins. Unlike other SUMO proteins, cells under physiological condition mediate a rapid degradation for SUMO4. However, when cells under stressed con- dition SUMO4 can be matured by the stress-induced endogenous hydrolase, and therefore, be able to conjugate to its substrate pro- teins. As a result, SUMO4 sumoylation acts as a stress-dependent manner. These results further confirmed our previous published 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.08.028 * Corresponding author. Fax: +1 706 721 3482. E-mail address: cwang@mcg.edu (C.-Y. Wang). Biochemical and Biophysical Research Communications 375 (2008) 454–459 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc