Blomster Henri A, Hietakangas Ville, Wu Jianmin, Kouvonen Petri, Hautaniemi Sampsa, Sistonen Lea
Department of Biology, Abo Akademi University and University of Turku, FI-20521 Turku, Finland.
Mol Cell Proteomics. 2009 Jun;8(6):1382-90. doi: 10.1074/mcp.M800551-MCP200. Epub 2009 Feb 24.
Small ubiquitin-like modifier (SUMO) is covalently conjugated to its target proteins thereby altering their activity. The mammalian SUMO protein family includes four members (SUMO-1-4) of which SUMO-2 and SUMO-3 are conjugated in a stress-inducible manner. The vast majority of known SUMO substrates are recognized by the single SUMO E2-conjugating enzyme Ubc9 binding to a consensus tetrapeptide (PsiKXE where Psi stands for a large hydrophobic amino acid) or extended motifs that contain phosphorylated or negatively charged amino acids called PDSM (phosphorylation-dependent sumoylation motif) and NDSM (negatively charged amino acid-dependent sumoylation motif), respectively. We identified 382 SUMO-2 targets using a novel method based on SUMO protease treatment that improves separation of SUMO substrates on SDS-PAGE before LC-ESI-MS/MS. We also implemented a software SUMOFI (SUMO motif finder) to facilitate identification of motifs for SUMO substrates from a user-provided set of proteins and to classify the substrates according to the type of SUMO-targeting consensus site. Surprisingly more than half of the substrates lacked any known consensus site, suggesting that numerous SUMO substrates are recognized by a yet unknown consensus site-independent mechanism. Gene ontology analysis revealed that substrates in distinct functional categories display strikingly different prevalences of NDSM sites. Given that different types of motifs are bound by Ubc9 using alternative mechanisms, our data suggest that the preference of SUMO-2 targeting mechanism depends on the biological function of the substrate.
小泛素样修饰物(SUMO)通过共价连接到其靶蛋白上,从而改变它们的活性。哺乳动物SUMO蛋白家族包括四个成员(SUMO-1 - 4),其中SUMO-2和SUMO-3以应激诱导的方式进行连接。绝大多数已知的SUMO底物是由单一的SUMO E2连接酶Ubc9识别的,Ubc9与一个共有四肽(PsiKXE,其中Psi代表一个大的疏水氨基酸)或扩展基序结合,这些扩展基序分别包含被称为PDSM(磷酸化依赖性SUMO化基序)和NDSM(负电荷氨基酸依赖性SUMO化基序)的磷酸化或带负电荷的氨基酸。我们使用一种基于SUMO蛋白酶处理的新方法鉴定了382个SUMO-2靶标,该方法在进行液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)分析之前,能改善SUMO底物在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)上的分离效果。我们还开发了一个名为SUMOFI(SUMO基序查找器)的软件,以方便从用户提供的一组蛋白质中识别SUMO底物的基序,并根据SUMO靶向共有位点的类型对底物进行分类。令人惊讶的是,超过一半的底物缺乏任何已知的共有位点,这表明许多SUMO底物是通过一种尚未知晓的不依赖共有位点的机制被识别的。基因本体分析表明,不同功能类别的底物中NDSM位点的出现频率显著不同。鉴于不同类型的基序通过不同机制与Ubc9结合,我们的数据表明SUMO-2靶向机制的偏好取决于底物的生物学功能。