SENP1 and SUMO in mouse development

使用逆转录病毒插入突变，我们确定SENP 1，蛋白酶调节的动态过程中的翻译后修饰的泛素样蛋白SUMO，作为一个发育必需的基因。SUMO修饰的各种蛋白质，通过类似于但不同于泛素化的过程，调节了许多关键的生物学过程。SENP 1是与酵母Ulp 1相关的几个高等真核基因之一，其编码半胱氨酸蛋白酶，该半胱氨酸蛋白酶可以从修饰的蛋白质中切割SUMO，以及进行SUMO前体的成熟。SENP 1的插入突变导致许多蛋白质的类小泛素化形式的稳态水平急剧增加。然而，只有SUMO-1结合水平增加;相关修饰物SUMO-2，-3的水平不受影响。此外，突变体细胞显示游离SUMO-1的减少和未加工形式的SUMO-1的积累。这些结果已经证明SENP 1在SUMO-1的去缀合和成熟中的作用，如在酵母Ulp 1中所发现的。发育过程中SENP 1功能丧失的生理后果在妊娠中期后胎盘中首次显现。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sumoylated蛋白质，显示在突变体中的水平增加。我们已经从突变体和野生型中分离出原代小鼠胚胎成纤维细胞（MEFs），并引入了SUMO-1的表位标记形式。初步结果表明，这种形式的SUMO-1可以有效地与蛋白质偶联，并且这些SUMO化蛋白可以被特异性免疫沉淀。突变体中类小泛素化蛋白的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体表型中的潜在作用。作为鉴定发育中胚胎中SENP 1去小泛素化底物的第二种方法，我们正在产生携带SENP 1基因的条件活性显性阴性形式的转基因小鼠。在组织培养的结果表明，无催化活性的酶可以结合底物和形成稳定的复合物，允许sumoylated蛋白质共免疫沉淀。

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.