Small Ubiquitin-Like Modifier 3–Modified Proteome Regulated by Brain Ischemia in Novel Small Ubiquitin-Like Modifier Transgenic Mice
Putative Protective Proteins/Pathways
Background and Purpose—Small ubiquitin-like modifier (SUMO) conjugation is a post-translational modification associated with many human diseases. Characterization of the SUMO-modified proteome is pivotal to define the mechanistic link between SUMO conjugation and such diseases. This is particularly evident for SUMO2/3 conjugation, which is massively activated after brain ischemia/stroke, and is believed to be a protective response. The purpose of this study was to perform a comprehensive analysis of the SUMO3-modified proteome regulated by brain ischemia using a novel SUMO transgenic mouse.
Methods—To enable SUMO proteomics analysis in vivo, we generated transgenic mice conditionally expressing tagged SUMO1-3 paralogues. Transgenic mice were subjected to 10 minutes forebrain ischemia and 1 hour of reperfusion. SUMO3-conjugated proteins were enriched by anti-FLAG affinity purification and analyzed by liquid chromatography–tandem mass spectrometry.
Results—Characterization of SUMO transgenic mice demonstrated that all 3 tagged SUMO paralogues were functionally active, and expression of exogenous SUMOs did not modify the endogenous SUMOylation machinery. Proteomics analysis identified 112 putative SUMO3 substrates of which 91 candidates were more abundant in the ischemia group than the sham group. Data analysis revealed processes/pathways with putative neuroprotective functions, including glucocorticoid receptor signaling, RNA processing, and SUMOylation-dependent ubiquitin conjugation.
Conclusions—The identified proteins/pathways modulated by SUMOylation could be the key to understand the mechanisms linking SUMOylation to neuroprotection, and thus provide new promising targets for therapeutic interventions. The new transgenic mouse will be an invaluable platform for analyzing the SUMO-modified proteome in models of human disorders and thereby help to mechanistically link SUMOylation to the pathological processes.
Small ubiquitin-like modifiers (SUMOs) are covalently conjugated to lysine residues of target proteins, and thereby modulate their function, stability, and localization.1,2 SUMO1, SUMO2, and SUMO3 are widely expressed in mammalian tissues. SUMO2 and SUMO3 are almost identical and are often referred to as SUMO2/3. They are distinct from SUMO1, however, with about 50% homology. The SUMO conjugation (SUMOylation) is an energy-dependent process catalyzed by activating enzyme (SAE1/SAE2), conjugating enzyme (Ubc9), and ligating enzyme.1,2 SUMOylation is a dynamic and reversible reaction, as SUMOylated proteins can be readily deconjugated by SUMO proteases.3
SUMOylation modulates many cellular functions including DNA repair, genome maintenance, gene transcription, and protein degradation control4–6 and plays key roles in many human diseases such as cerebral ischemia/stroke, cancer, and heart failure.1,7–9 It is, therefore, of tremendous clinical interest to characterize the SUMO-modified proteome in these disorders. This is particularly evident for cerebral ischemia/stroke because transient cerebral ischemia dramatically activates SUMOylation, and this is believed to be a neuroprotective stress response. It is, therefore, of key interest to identify the proteins SUMOylated after brain ischemia to uncover the mechanisms linking SUMOylation to neuroprotection. However, SUMO proteomics analysis is hampered by the low levels of SUMOylated proteins. A common strategy is, therefore, to perform proteomics analysis on purified SUMO-conjugated proteins from cells stably expressing tagged SUMOs. Recently, a His6-HA-SUMO1 knockin mouse was generated, which allowed researchers, for the first time, to characterize the SUMO1-modified proteome in brains.10 In the present study, we generated a transgenic mouse (CAG-loxP-STOP-loxP-SUMO, hereafter referred to as CAG-SUMO) in which His-SUMO1, HA-SUMO2, and FLAG-SUMO3 are expressed in a Cre-dependent manner. Using this new mouse line, we report here the first profile of the SUMO3-modified proteome in mouse brain after ischemia, a pathological state associated with a dramatic activation of SUMO2/3 conjugation.8,9
Full details of the methods are provided in the online-only Data Supplement.
Animal experiments were approved by the Duke University Animal Care and Use Committee. CAG-SUMO transgenic mice were generated by pronuclear injection of the transgene vector illustrated in Figure 1A.
Transient forebrain ischemia was performed as described previously with minor modifications.8
Sample Preparation for Proteomic Analysis
Each sample for proteomic analysis was generated by FLAG pull-down of nuclear fraction from cortical tissues pooled from 4 mice. Three biological replicates were used for each group.
Liquid Chromatography–Tandem Mass Spectrometric Analysis and Data Analysis
All 9 samples derived from FLAG pull-down were separated on SDS-PAGE gel, and each lane was dissected into 14 slices. After in-gel digestion, peptides from each slice were analyzed by liquid chromatography–tandem mass spectrometry. Data were analyzed by various programs.
Generation and Characterization of CAG-SUMO Mice
We designed a transgene vector based on Cre/lox recombination and 2A-mediated cotranslational cleavage to express His-SUMO1, HA-SUMO2, and FLAG-SUMO3 from a single multicistronic transgene in a conditional manner (Figure 1A). We used the cDNAs encoding precursor SUMO1-3 that allows the endogenous SUMO proteases to remove the extra 2A peptides11 and expose the C-terminal di-glycine motif of SUMOs. Green fluorescent protein (GFP) and mCherry were used as indicators of transgene expression before and after Cre recombination. We obtained 8 founder lines with varying levels and patterns of GFP expression. Because of ubiquitous expression of GFP, line 10 was chosen for the present study. To examine the global pattern of Cre-mediated transgene expression, mice were cross-bred with hemizygous β-actin-Cre mice to generate double transgenic CAG-SUMO/β-actin-Cre mice. CAG-SUMO line 10 exhibited strong GFP fluorescence in all organs examined including brain, heart, lung, kidney, and liver; those organs showed mCherry expression in CAG-SUMO/β-actin-Cre mice. Expression of FLAG-SUMO3 was also confirmed in those organs (data not shown).
In the brain of CAG-SUMO line 10, GFP was ubiquitously expressed with strong signals in hippocampus and cerebellum (Figure 1B). Because we were particularly interested in the SUMO-modified proteome regulated by forebrain ischemia, we mated hemizygous CAG-SUMO mice with homozygous Emx1Cre/Cre mice to generate double transgenic CAG-SUMO/Emx1-Cre as tagged SUMO-expressing mice and littermates Emx1Cre/+ as control mice. In line with a previous report,12 CAG-SUMO/Emx1-Cre mice showed strong mCherry expression in the cerebral cortex and hippocampus (Figure 1C). For both CAG-SUMO and CAG-SUMO/Emx1-Cre mice, we did not find any obvious physical or behavioral abnormalities.
Postischemic SUMOylation in the Brain of Transgenic Mice
To extend our previous findings8 and also demonstrate the suitability of CAG-SUMO/Emx1-Cre mice for in vivo SUMO studies, we investigated the temporal and spatial profiles of postischemic SUMOylation by endogenous and exogenous SUMOs in brains of Emx1Cre/+ control and CAG-SUMO/Emx1-Cre mice. First, the temporal profile studies indicated that SUMO1-3 conjugation was decreased dramatically during and rapidly activated after ischemia (Figure 2A). Similar pattern was found for FLAG-SUMO3 (data not shown). Then, we carefully compared SUMOylation response to brain ischemia in Emx1Cre/+ and CAG-SUMO/Emx1-Cre mice to check for possible off-target effects that might be caused by overexpressing SUMOs. In both sham and ischemia groups, levels of SUMO1 and SUMO2/3 conjugates in the high-molecular-weight regions were comparable in controls and double transgenic mice, although there were substantially higher levels of unconjugated SUMOs in CAG-SUMO/Emx1-Cre mice because of expression of tagged SUMOs (Figure 2B–2D, data not shown). Finally, transient forebrain ischemia induced nuclear accumulation of SUMO2/3-conjugated proteins, and the same pattern was also observed for HA-SUMO2 and FLAG-SUMO3 (Figure 3 and Figure IA in the online-only Data Supplement).
SUMO3-Modified Proteome Regulated by Transient Forebrain Ischemia
To compare results to our previous SUMO3 proteomics analysis using an in vitro ischemia model,13 we focused on the SUMO3-modified proteome in this study. We chose 1 hour of reperfusion when SUMO2/3 conjugation was maximally activated (Figure 2A). Furthermore, we used cortical tissues because we were interested in the neuroprotective role of SUMOylation, and the cortex is spared from damage in this ischemia model.14 For future studies, we also performed a small-scale HA pull-down to confirm enrichment of HA-SUMO2–conjugated proteins (Figure IB in the online-only Data Supplement).
First, we optimized the FLAG pull-down procedure by using nuclear fractions as input for FLAG pull-down. This greatly enhanced specificity because nuclear fractions were devoid of unconjugated FLAG-SUMO3, had markedly less unspecific bands on Western blots (Figure IA in the online-only Data Supplement), and exhibited dramatically lower total protein levels (Figure IC in the online-only Data Supplement). Indeed, FLAG-SUMO3–conjugated proteins were immunoprecipitated effectively from nuclear fractions (Figure ID in the online-only Data Supplement). Interestingly, we did not notice a marked decrease in SUMO2/3 and HA signals in flow-through samples (Figure ID in the online-only Data Supplement). This suggested that FLAG-SUMO3 represented only a small fraction of the total SUMO2/3 pool. We also found HA-SUMO2 in FLAG-SUMO3 pull-down eluates, and, notably, there was a shift toward higher molecular weights in the ischemic sample, implying increased length of SUMO2/3 chains (Figure ID in the online-only Data Supplement).
For the large-scale SUMO3 proteomics study, 3 groups of mice were used: Emx1Cre/+ without surgery (control, to account for background binding to anti-FLAG beads) and CAG-SUMO/Emx1-Cre double transgenic mice with sham (transgenic sham) or ischemia surgery (transgenic ischemia; Figure 4A). All 9 FLAG pull-down samples (n=3/group) were confirmed by Western blotting (Figure IIA in the online-only Data Supplement) and then separated on an SDS-PAGE gel (Figure IIB in the online-only Data Supplement). Fourteen gel slices per lane were cut for liquid chromatography–tandem mass spectrometric analysis (Figure IIB in the online-only Data Supplement).
Proteomics data showed that SUMO2/3 and ubiquitin shared a similar distribution of spectral counts (Figure 4B), suggesting a marked postischemic activation of the cross talk between these 2 post-translational modifications. Indeed, we found ubiquitin conjugation to be activated after ischemia, particularly pronounced in nuclei (Figure IIC in the online-only Data Supplement). Based on selection criteria described in online-only Data Supplement Methods, 112 proteins were considered as putative SUMO3 substrates (Table I in the online-only Data Supplement), and 91 proteins (Table I in the online-only Data Supplement, asterisks) were considered as ischemia-upregulated candidates of which 46 candidates were found only in ischemia samples (Table I in the online-only Data Supplement, triangles), including the general transcription factor IIi (TFII-I/GTF2I), tripartite motif containing 33 (TRIM33), glucocorticoid receptor (GR/GCR), and B-cell lymphoma/leukemia 11B (CTIP2/BCL11B).
Gene ontology annotation analysis by the PANTHER program indicated that 38.5% and 47.3% of the 91 candidates were predicted to have nuclear and cytoplasmic localization, respectively (Figure 4C). We did not expect that most of the identified proteins would have predicted cytoplasmic localization, considering that nuclear fractions were used for proteomics analyses. A plausible explanation is that cytoplasmic SUMO3 target proteins were translocated to the nucleus after ischemia (eg, see below for GR). PANTHER analysis also revealed that most of the 91 proteins belonged to a group of binding proteins of which nucleic acid binding accounted for 55.1% (Figure 4D). Ingenuity Pathway Analysis core analysis of the 91 candidates revealed significant enrichment in the categories of neurological disease (46 targets) and cell death and survival (47 targets; Figure IIIA and IIIB in the online-only Data Supplement). Strikingly, 27 proteins were grouped with functions in RNA processing with high confidence (Ingenuity Pathway Analysis score=72; Figure IIIC in the online-only Data Supplement).
To verify proteomics analysis findings independently, a separate large-scale FLAG pull-down was performed using 3 CAG-SUMO/Emx1-Cre mice per group (sham and ischemia). First, we treated FLAG pull-down samples with SUMO/sentrin-specific protease 2 catalytic fragment (SENP2c) for de-SUMOylation. Western blot analysis confirmed that the strong high-molecular-weight smear of bands indeed represented SUMO-conjugated proteins (Figure 5A). The presence of SUMO1 is consistent with the report that SUMO1-3 can form mixed chains.15 Interestingly, ubiquitinated proteins slightly shifted to lower molecular weight after SENP2c treatment (Figure 5A). These data suggest that ubiquitin and SUMO conjugated to different lysine residues for a subset of postischemic SUMO3 conjugates. Similar findings were also reported in cells exposed to ischemia-like conditions or proteasome inhibitor MG132.13,16
Then, we selected 5 candidate SUMO substrates for further verification, GR, TFII-I, TRIM33, transcriptional intermediary factor 1β (TIF1β), and CTIP2. The unmodified forms of all 5 proteins were detected in sham and ischemia input samples (Figure 5B). In contrast, only slower migrating bands were present in FLAG pull-down samples, with much stronger signals in the ischemia sample. After SENP2c treatment, almost all slower migrating bands disappeared, and bands representing unmodified forms appeared (Figure 5B). These experiments convincingly confirmed that these 5 proteins are authentic SUMO3 substrates and that SUMOylation of these proteins was markedly increased after ischemia (Figure 5B).
We further determined the quantitative trend across molecular weight for 4 of these proteins (GR, TFII-I, TRIM33, and TIF1β) by performing a targeted data extraction from the liquid chromatography–tandem mass spectrometric analysis of each gel band (Figure 5C). TFII-I and TRIM33 were virtually undetectable in sham samples, whereas GR and TIF1β were detectable in sham but highly upregulated in ischemia samples. All 4 proteins showed the highest quantity in molecular weight regions that are significantly higher than the unmodified protein molecular weight (Figure 5C), consistent with multiple SUMO modifications.
Finally, we further characterized GR SUMOylation. SUMO3-conjugated GR was already detected in postischemic nuclear fractions without immunoprecipitation (Figure 5B, input), a remarkable observation considering that only a small fraction of a given protein is usually SUMOylated. This suggests a dramatic postischemic activation of GR SUMOylation. Indeed, we found that ischemia/reperfusion triggered massive SUMO conjugation and nuclear accumulation of GR (Figure 5D).
Here, we presented a novel SUMO transgenic mouse model (CAG-SUMO) and the first study on tissue samples to uncover the SUMO-modified proteome in a pathological state. CAG-SUMO/Emx1-Cre mice did not show any obvious phenotype. This was expected because our data demonstrated that exogenous tagged SUMOs did not disturb global SUMOylation in the brain. Indeed, the tagged SUMOs were functionally intact and processed by the endogenous SUMOylation machinery in the same way as endogenous SUMOs (Figures 2 and 3 and Figure I in the online-only Data Supplement). Moreover, expression of exogenous tagged SUMO1-3 did not induce an increase in levels of SUMO-conjugated proteins, although it resulted in a rise in levels of free SUMOs (Figure 2). These data suggest that in the brain SUMO conjugation is controlled by the activity of SUMOylation machinery and not by levels of free SUMOs. Together, our findings demonstrate that this new transgenic mouse model is well suited for SUMO proteomics studies in vivo.
The new SUMO transgenic mouse model has several advantages compared with previously published approaches to investigate the SUMO2/3-modified proteome using tissue samples.17,18 Protein sequences of SUMO2 and SUMO3 are almost identical and cannot be differentiated by available antibodies. Because SUMO2 and SUMO3 are expressed with different tags in this new transgenic mouse, it is possible for future studies to identify individually the SUMO2- and SUMO3-modified proteome. Furthermore, because tagged SUMOs are expressed in a conditional manner, they can be expressed in any cell/organ type for which the respective Cre mouse is available, thus making this new mouse model a universal tool for characterizing the SUMO-modified proteome.
Increased SUMOylation is thought to be a protective response that shields neurons from ischemia-induced damage.19,20 Transient forebrain ischemia triggered a massive increase in levels of SUMO2/3-conjugated proteins in neurons of the resistant cortex and vulnerable hippocampal CA1 subfield (Figures 2 and 3). Whether this postischemic activation of SUMO2/3 conjugation is a stress response protecting neurons in both regions needs to be established in future studies. Recently, we have generated a novel SUMO knockdown transgenic mouse that will be well suited for these studies.21 It also needs to be verified whether transient cerebral ischemia may activate SUMOylation in non-neuronal cells such as astrocytes and endothelial cells. Notably, SUMOylation of the liver X receptor in brain astrocytes blocks inflammatory responses,22 and inflammation is a major contributing factor to ischemia-induced brain damage. Crossing our CAG-SUMO mice with mice expressing Cre in non-neuronal brain cells will help to identify the postischemic SUMO-modified proteome in these cells and thereby clarify this important aspect.
We have identified 91 protein candidates that exhibit a postischemic upregulated SUMO3 conjugation state in cortex, a brain region relatively resistant to a short interruption of blood supply (Table I in the online-only Data Supplement, asterisks). This high stringency list includes many known SUMO substrates, such as TIF1β, heterogeneous nuclear ribonucleoproteins (hnRNPs), TFII-I, and GR.17,23–25 When we compared the in vivo data presented here with a previous in vitro study,13 we found that 34 of the 91 candidates (37%) were identified in both studies. Furthermore, we confidently confirmed 5 in vivo SUMO3 substrates regulated by ischemia (Figure 5). Taken together, these analyses confirm the validity of the approach and new SUMO mouse model and establish the credibility of our SUMO substrate list.
Our data revealed several potentially important processes modulated by SUMO3 conjugation that may play neuroprotective roles during reperfusion. First, we provided evidence that the cross talk between ubiquitylation and SUMOylation was activated after ischemia. Our data suggested that ubiquitin and SUMO3 are conjugated to different lysine residues in a subset of SUMO3 substrates (Figure 5A). This implies postischemic activation of the SUMO-dependent ubiquitin conjugation pathway that plays pivotal roles in DNA damage repair.26 Second, Ingenuity Pathway Analysis analysis revealed that proteins involved in post-transcriptional RNA modification are highly enriched in postischemia FLAG-SUMO3 pull-down samples (Figure IIIC in the online-only Data Supplement). These included various isoforms of hnRNPs, a group of proteins that are involved in mRNAs splicing, stability, and translation.27 Because transcription and translation processes are dramatically impaired during and after ischemia, the post-transcriptional regulation of existing mRNAs is crucial to determine the expression pattern in this pathological state. Therefore, we speculate that SUMOylation of such a large fraction of hnRNPs after ischemia is likely related to the decision of cell survival or death.
Finally, the GR was identified as a SUMO3 target in postischemia samples at a level of SUMO conjugation so pronounced that the SUMOylated GR could be identified in homogenate even without enrichment (Figure 5D). Stress-induced GR activation is associated with increased cell damage triggered by transient cerebral ischemia.28 GR function is modulated by post-translational modifications, and phosphorylation activates GR SUMOylation, resulting in repression of transcriptional activity.24 Taken together, these observations suggest that the massive postischemic activation of GR SUMOylation is a protective stress response.
In summary, we report here the first proteomics analysis of SUMO3-conjugated proteins in tissue samples from a pathological state, brain ischemia. We identified several pathways modulated by SUMOylation in the postischemic brain that warrant future investigations because they could be therapeutic targets for neuroprotection in brain ischemia. Because a large portion of identified SUMO targets were nuclear proteins involved in gene expression, genome stability, and RNA processing, we expect activation of SUMOylation to have long-lasting effects on postischemic neurons. The new conditional SUMO transgenic mouse and the highly stringent purification approach developed in the present study provide an invaluable platform for in-depth analysis of the SUMO-modified proteome in vivo in physiological or pathological states under investigation.
We thank Laura Dubois and Meredith Turner for their excellent technical support, Dr Christoph Harms and Dr Matthew Foster for helpful discussions, and Kathy Gage for excellent editorial contribution.
Sources of Funding
This study was supported by a DREAM Innovation Grant from the Department of Anesthesiology and an American Heart Association Scientist Development Grant 12SDG11950003 to Dr Yang and National Institutes of Health grants HL095552 and NS081299 to Dr Paschen.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.004315/-/DC1.
- Received December 2, 2013.
- Revision received January 15, 2014.
- Accepted January 21, 2014.
- © 2014 American Heart Association, Inc.
- Ayaydin F,
- Dasso M
- Tirard M,
- Hsiao HH,
- Nikolov M,
- Urlaub H,
- Melchior F,
- Brose N
- Gorski JA,
- Talley T,
- Qiu M,
- Puelles L,
- Rubenstein JL,
- Jones KR
- Sheng H,
- Laskowitz DT,
- Mackensen GB,
- Kudo M,
- Pearlstein RD,
- Warner DS
- Matic I,
- van Hagen M,
- Schimmel J,
- Macek B,
- Ogg SC,
- Tatham MH,
- et al
- Schimmel J,
- Larsen KM,
- Matic I,
- van Hagen M,
- Cox J,
- Mann M,
- et al
- Bruderer R,
- Tatham MH,
- Plechanovova A,
- Matic I,
- Garg AK,
- Hay RT
- Wang L,
- Rodriguiz RM,
- Wetsel WC,
- Sheng H,
- Zhao S,
- Liu X,
- et al
- Li T,
- Evdokimov E,
- Shen RF,
- Chao CC,
- Tekle E,
- Wang T,
- et al
- Vassileva MT,
- Matunis MJ
- Prudden J,
- Pebernard S,
- Raffa G,
- Slavin DA,
- Perry JJ,
- Tainer JA,
- et al
- Balkaya M,
- Prinz V,
- Custodis F,
- Gertz K,
- Kronenberg G,
- Kroeber J,
- et al