Browsing by Subject "Ubiquitins"

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    ItemOpen Access
    Small ubiquitin-like modifier 3-modified proteome regulated by brain ischemia in novel small ubiquitin-like modifier transgenic mice: putative protective proteins/pathways.
    (Stroke, 2014-04) Yang, Wei; Sheng, Huaxin; Thompson, J Will; Zhao, Shengli; Wang, Liangli; Miao, Pei; Liu, Xiaozhi; Moseley, M Arthur; Paschen, Wulf

    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.
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    Transient ischemia induces massive nuclear accumulation of SUMO2/3-conjugated proteins in spinal cord neurons.
    (Spinal cord, 2013-02) Wang, Z; Wang, R; Sheng, H; Sheng, SP; Paschen, W; Yang, W

    Objectives

    The objective of this study is to determine whether transient spinal cord ischemia activates small ubiquitin-like modifier (SUMO1-3) conjugation, a post-translational protein modification that protects neurons from ischemia-like conditions.

    Methods

    Mice were subjected to 8-12 min of spinal cord ischemia and 3-24 h of recovery using a newly developed experimental model. To characterize the model, activation of stress response pathways induced after spinal cord ischemia, previously observed in other experimental models, was verified by western blot analysis. Levels and subcellular localization of SUMO-conjugated proteins in spinal cords were evaluated by western blot analysis and immunohistochemistry, respectively.

    Results

    Following transient spinal cord ischemia, stress responses were activated as indicated by increased phosphorylation of eukaryotic initiation factor 2 (eIF2α), extracellular signal-regulated kinases (ERK1/2) and Akt. SUMO1 conjugation was not altered, but a selective rise in levels of SUMO2/3-conjugated proteins occurred, peaking at 6 h reperfusion. The marked activation of SUMO2/3 conjugation was a neuronal response to ischemia, as indicated by co-localization with the neuronal marker NeuN, and was associated with nuclear accumulation of SUMO2/3-conjugated proteins.

    Conclusion

    Our study suggests that spinal cord neurons respond to ischemic stress by activation of SUMO2/3 conjugation. Many of the identified SUMO target proteins are transcription factors and other nuclear proteins involved in gene expression and genome stability. It is therefore concluded that the post-ischemic activation of SUMO2/3 conjugation may define the fate of neurons exposed to a transient interruption of blood supply, and that this pathway could be a therapeutic target to increase the resistance of spinal cord neurons to transient ischemia.
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    Ubc9 overexpression and SUMO1 deficiency blunt inflammation after intestinal ischemia/reperfusion.
    (Laboratory investigation; a journal of technical methods and pathology, 2018-06) Karhausen, Jörn; Bernstock, Joshua D; Johnson, Kory R; Sheng, Huaxin; Ma, Qing; Shen, Yuntian; Yang, Wei; Hallenbeck, John M; Paschen, Wulf
    The intestinal epithelium constitutes a crucial defense to the potentially life-threatening effects of gut microbiota. However, due to a complex underlying vasculature, hypoperfusion and resultant tissue ischemia pose a particular risk to function and integrity of the epithelium. The small ubiquitin-like modifier (SUMO) conjugation pathway critically regulates adaptive responses to metabolic stress and is of particular significance in the gut, as inducible knockout of the SUMO-conjugating enzyme Ubc9 results in rapid intestinal epithelial disintegration. Here we analyzed the pattern of individual SUMO isoforms in intestinal epithelium and investigated their roles in intestinal ischemia/reperfusion (I/R) damage. Immunostaining revealed that epithelial SUMO2/3 expression was almost exclusively limited to crypt epithelial nuclei in unchallenged mice. However, intestinal I/R or overexpression of Ubc9 caused a remarkable enhancement of epithelial SUMO2/3 staining along the crypt-villus axis. Unexpectedly, a similar pattern was found in SUMO1 knockout mice. Ubc9 transgenic mice, but also SUMO1 knockout mice were protected from I/R injury as evidenced by better preserved barrier function and blunted inflammatory responses. PCR array analysis of microdissected villus-tip epithelia revealed a specific epithelial contribution to reduced inflammatory responses in Ubc9 transgenic mice, as key chemotactic signaling molecules such as IL17A were significantly downregulated. Together, our data indicate a critical role particularly of the SUMO2/3 isoforms in modulating responses to I/R and provide the first evidence that SUMO1 deletion activates a compensatory process that protects from ischemic damage.