Global analysis of SUMO substrate selection using protein microarray technology

使用蛋白质微阵列技术对 SUMO 底物选择进行全局分析

• 批准号: 8260778
• 负责人: Ijeoma K Uzoma
• 金额: $ 4.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260778
• 关键词: Affect; Bioinformatics; Cell Cycle Regulation; Chemicals; DNA Damage; Data; Data Set; Diabetes Mellitus; Enzymes; Exhibits; Functional disorder; HSF1; Immunity; In Vitro; Inflammation; Insulin-Dependent Diabetes Mellitus; Link; Lysine; Malignant Neoplasms; Microarray Analysis; Modification; Nerve Degeneration; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Process; Protein Microarray Assay; Protein Microchips; Proteins; Reaction; Regulation; Role; Signal Pathway; Site-Directed Mutagenesis; Specificity; Substrate Specificity; System; Testing; Transcriptional Regulation; Ubiquitin; Ubiquitination; Validation; Viral; data integration; enzyme substrate; experience; high throughput screening; human disease; improved; in vivo; interest; paralogous gene; protein protein interaction; public health relevance; response; therapy design; tumor progression; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Small Ubiquitin-related Modifier (SUMO) is a small protein posttranslational modification that occurs on lysine residues of proteins. It is reversibly conjugated to a wide range of cellular proteins through a multistep enzymatic cascade analogous to that of the ubiquitin conjugation pathway. The functional consequences of sumoylation are substrate specific and include changes in subcellular localization, protein-protein interactions, and transcriptional regulation. SUMO is involved in regulation of the cell cycle and the DNA damage response, thus linking SUMO to cancer progression. It is also involved in a variety of processes related to human disease: viral replication, Type 1 diabetes, inflammation, immunity and neurodegeneration. Understanding how sumoylation is regulated will help to further elucidate the pathophysiology of a wide range of human diseases. Presently, little is known about regulation of sumoylation in the global sense. The ubiquitylation cascade requires E1, E2, and E3 enzymes for substrate modification; moreover, substrate selection is determined by interplay between the E2 and E3 enzymes. Currently, sumoylation of many known substrates is only dependent on the E1 and E2 enzymes in vitro and it is commonly believed that E3 ligases merely enhance the degree of SUMOylation of target proteins rather than serve an essential role in target modification. However, emerging in vivo evidences suggest that the E3 ligases may play an important role in many sumo-dependent signaling pathways. Therefore, we hypothesize that a subset of SUMO-modified proteins may require the action of E3 ligases, thus conferring substrate specificity. We are also interested in interrogating the contribution of post-translational modification (PTM) crosstalk on SUMO substrate specificity. A small number of SUMO substrates have shown phosphorylation dependent sumoylation and a phosphorylation dependent sumoylation motif has been characterized. We hypothesize that both E3 ligases and phosphorylation by specific kinases contribute to SUMO substrate selection and regulation. We have established a global approach through the use of protein microarrays to identify substrates of the known SUMO E3 ligases. To elucidate the determinants of SUMO target selection, we propose to employ protein microarrays to identify E3- dependent SUMO substrates and to investigate crosstalk between sumoylation and phosphorylation, followed by in vivo characterization of selected targets and pathways. PUBLIC HEALTH RELEVANCE: Small Ubiquitin-related Modifier (SUMO) is a chemical alteration that occurs on proteins, affecting their cellular activities. How and why specific proteins experience this alteration is poorly understood. As SUMO has been linked to human diseases such as cancer, diabetes, and neurodegeneration, understanding the context in which this alteration occurs will help us to design therapies to treat such human diseases.

描述（由申请人提供）：小泛素相关修饰剂（SUMO）是一种发生在蛋白质赖氨酸残基上的小蛋白质翻译后修饰。它通过类似于泛素缀合途径的多步酶级联可逆地缀合到多种细胞蛋白质。 sumoylation 的功能后果是底物特异性的，包括亚细胞定位、蛋白质-蛋白质相互作用和转录调控的变化。 SUMO 参与细胞周期和 DNA 损伤反应的调节，从而将 SUMO 与癌症进展联系起来。它还参与与人类疾病相关的多种过程：病毒复制、1型糖尿病、炎症、免疫和神经变性。了解苏酰化是如何调节的将有助于进一步阐明多种人类疾病的病理生理学。目前，人们对全球范围内的苏酰化调控知之甚少。泛素化级联需要 E1、E2 和 E3 酶进行底物修饰；此外，底物选择由 E2 和 E3 酶之间的相互作用决定。目前，许多已知底物的SUMO化在体外仅依赖于E1和E2酶，通常认为E3连接酶仅增强靶蛋白的SUMO化程度，而不是在靶标修饰中发挥重要作用。然而，新出现的体内证据表明，E3 连接酶可能在许多相扑依赖性信号通路中发挥重要作用。因此，我们假设 SUMO 修饰蛋白的子集可能需要 E3 连接酶的作用，从而赋予底物特异性。我们也有兴趣探讨翻译后修饰 (PTM) 串扰对 SUMO 底物特异性的影响。少数 SUMO 底物已显示出磷酸化依赖性苏酰化，并且已表征了磷酸化依赖性苏酰化基序。我们假设 E3 连接酶和特定激酶的磷酸化都有助于 SUMO 底物的选择和调节。我们通过使用蛋白质微阵列来识别已知 SUMO E3 连接酶的底物，建立了一种全局方法。为了阐明 SUMO 靶点选择的决定因素，我们建议采用蛋白质微阵列来识别 E3 依赖性 SUMO 底物并研究 sumoy 化和磷酸化之间的串扰，然后对所选靶点和途径进行体内表征。 公共健康相关性：小泛素相关修饰剂 (SUMO) 是蛋白质上发生的一种化学变化，会影响其细胞活动。人们对特定蛋白质如何以及为何经历这种改变知之甚少。由于 SUMO 与癌症、糖尿病和神经退行性疾病等人类疾病有关，了解这种改变发生的背景将有助于我们设计治疗此类人类疾病的疗法。