SENP1 and SUMO in mouse development

通过逆转录病毒插入突变，我们发现SENP1是一种调节泛素样蛋白SUMO翻译后修饰动态过程的蛋白酶，是发育必需基因。各种蛋白质的SUMO修饰，通过一个类似但不同于泛素化的过程，调节了一些关键的生物过程。SENP1是与酵母Ulp1相关的几个高级真核基因之一，它编码一种半胱氨酸蛋白酶，可以从修饰蛋白中切割SUMO，并进行SUMO前体的成熟。SENP1的插入突变导致许多蛋白质的聚合形式的稳态水平急剧增加。然而，只有SUMO-1偶联的水平增加；相关修饰语SUMO-2和-3的等级不受影响。此外，突变细胞显示出自由SUMO-1的减少和SUMO-1未加工形式的积累。这些结果证明了SENP1在SUMO-1的解偶联和成熟过程中的作用，就像在酵母Ulp1中发现的那样。发育过程中SENP1功能丧失的生理后果在胎盘妊娠中期后首先显现出来。这些缺陷似乎与正常的胎盘功能和胚胎活力不相容。目前的工作集中在突变体中显示水平增加的聚合蛋白上。我们从突变型和野生型中分离了原代小鼠胚胎成纤维细胞（mef），并引入了SUMO-1表位标记形式。初步结果表明，这种形式的SUMO-1可以有效地偶联到蛋白质上，并且这些偶联蛋白可以特异性免疫沉淀。突变体中代谢蛋白稳态水平的显著增加，现在应该允许蛋白质组学方法来确定它们的身份，并允许评估它们在突变表型中的潜在作用。作为鉴定发育胚胎中SENP1去氧基化底物的第二种方法，我们正在培养携带条件活性显性阴性SENP1基因形式的转基因小鼠。组织培养结果表明，无催化活性的酶可以结合底物并形成稳定的复合物，从而使聚合蛋白被免疫共沉淀。

Using retroviral insertional mutagenesis, we identified SENP1, a protease regulating the dynamic process of post-translational modification by the ubiquitin-like protein SUMO, as a developmentally essential gene. SUMO modification of various proteins, by a process similar to but distinct from ubiquitination, regulates a number of critical biological processes. SENP1 is one of several higher eukaryotic genes related to yeast Ulp1, which encodes a cysteine protease that can cleave SUMO from modified proteins as well as carry out the maturation of the SUMO precursor. Insertional mutation of SENP1 causes a dramatic increase in the steady state levels of the sumoylated forms of a number of proteins. However, only the level of SUMO-1 conjugation increases; levels of the related modifiers SUMO-2,-3 are unaffected. In addition, mutant cells show a decrease in free SUMO-1 and an accumulation of the unprocessed form of SUMO-1. These results have proven a role for SENP1 in both deconjugation and maturation of SUMO-1, as found for yeast Ulp1. The physiological consequences of loss of SENP1 function during development are first apparent after midgestation in the placenta. These defects appear incompatible with normal placental function and embryonic viability. Current work is focused on the sumoylated proteins that show increased levels in mutants. We have isolated primary mouse embryonic fibroblasts (MEFs) from mutants and wild types and introduced an epitope tagged form of SUMO-1. Preliminary results show that this form of SUMO-1 can be efficiently conjugated to proteins and that these sumoylated proteins can be specifically immunoprecipitated. The significant increase in steady state levels of sumoylated proteins in the mutant should now permit proteomic approaches to determine their identities and allow an assessment of their potential roles in the mutant phenotype. As a second approach to identify SENP1 desumoylation substrates in the developing embryo, we are generating transgenic mice carrying a conditionally active dominant negative form of the SENP1 gene. Results in tissue culture indicate that the catalytically inactive enzyme can bind substrates and form stable complexes, allowing the sumoylated proteins to be co-immunoprecipitated.