Global analysis of SUMO substrate selection using protein microarray technology

描述（由申请人提供）：小泛素相关修饰物（SUMO）是一种发生在蛋白质赖氨酸残基上的小蛋白翻译后修饰。它通过类似于泛素缀合途径的多步酶促级联反应可逆地缀合至广泛的细胞蛋白。类小泛素化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参与调节细胞周期和DNA损伤反应，因此将SUMO与癌症进展联系起来。它还参与与人类疾病相关的各种过程：病毒复制，1型糖尿病，炎症，免疫和神经退行性疾病。了解sumoylation是如何调节将有助于进一步阐明广泛的人类疾病的病理生理学。目前，很少有人知道在全球意义上的sumoylation的调控。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，并且通常认为E3连接酶仅增强靶蛋白的SUMO化程度，而不是在靶修饰中起重要作用。然而，新出现的体内证据表明，E3连接酶可能在许多sumo依赖的信号通路中发挥重要作用。因此，我们假设SUMO修饰蛋白的一个子集可能需要E3连接酶的作用，从而赋予底物特异性。我们也有兴趣询问翻译后修饰（PTM）串扰对SUMO底物特异性的贡献。少数SUMO底物已显示磷酸化依赖性SUMO化，并且已表征磷酸化依赖性SUMO化基序。我们推测E3连接酶和特异性激酶的磷酸化都有助于SUMO底物的选择和调节。我们已经建立了一个全球性的方法，通过使用蛋白质微阵列，以确定基板的已知SUMO E3连接酶。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依赖的SUMO底物，并研究SUMO化和磷酸化之间的串扰，然后在体内表征选定的靶点和途径。 公共卫生相关性：小泛素相关修饰物（SUMO）是发生在蛋白质上的化学改变，影响其细胞活性。具体的蛋白质如何以及为什么经历这种改变还知之甚少。由于相扑与癌症、糖尿病和神经退行性疾病等人类疾病有关，了解这种变化发生的背景将有助于我们设计治疗这些人类疾病的疗法。

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.