Yang Wei, Sheng Huaxin, Thompson J Will, Zhao Shengli, Wang Liangli, Miao Pei, Liu Xiaozhi, Moseley M Arthur, Paschen Wulf
From the Department of Anesthesiology (W.Y., H.S., L.W., P.M., X.L., W.P.), Proteomics Core Facility (J.W.T., M.A.M.), and Department of Neurobiology (S.Z.), Duke University Medical Center, Durham, NC; and Department of Neurosurgery, The Fifth Central Hospital of Tianjin, Tianjin, China (X.L.).
Stroke. 2014 Apr;45(4):1115-22. doi: 10.1161/STROKEAHA.113.004315. Epub 2014 Feb 25.
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.
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.
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.
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.
小泛素样修饰物（SUMO）缀合是一种与多种人类疾病相关的翻译后修饰。SUMO修饰蛋白质组的表征对于确定SUMO缀合与这些疾病之间的机制联系至关重要。这在SUMO2/3缀合中尤为明显，其在脑缺血/中风后大量激活，被认为是一种保护反应。本研究的目的是使用新型SUMO转基因小鼠对脑缺血调节的SUMO3修饰蛋白质组进行全面分析。
为了在体内进行SUMO蛋白质组学分析，我们构建了条件性表达带标签的SUMO1-3旁系同源物的转基因小鼠。转基因小鼠经历10分钟的前脑缺血和1小时的再灌注。通过抗FLAG亲和纯化富集SUMO3缀合蛋白，并通过液相色谱-串联质谱进行分析。
SUMO转基因小鼠的表征表明，所有3种带标签的SUMO旁系同源物均具有功能活性，外源性SUMO的表达未改变内源性SUMO化机制。蛋白质组学分析鉴定出112个推定的SUMO3底物，其中91个候选底物在缺血组中比假手术组更丰富。数据分析揭示了具有推定神经保护功能 的过程/途径，包括糖皮质激素受体信号传导、RNA加工和SUMO化依赖性泛素缀合。
鉴定出的受SUMO化调节的蛋白质/途径可能是理解SUMO化与神经保护之间联系机制的关键，从而为治疗干预提供新的有前景的靶点。这种新的转基因小鼠将成为分析人类疾病模型中SUMO修饰蛋白质组的宝贵平台，从而有助于从机制上将SUMO化与病理过程联系起来。
