Ubiquitin And SUMO Post-Translational Modifications In Development And Disease

为了确定泛素介导的蛋白质降解在正常发育中起关键作用的靶标，以及当失调时可能导致肿瘤发生的靶标，我们对E3泛素连接酶Nedd4的相互作用伙伴进行了酵母双杂交筛选。该筛选确定了N4BP1和N4BP3，这是两种新的发育表达蛋白，由于它们在肿瘤发生中的潜在参与，现在正在进一步表征它们。N4BP1是一个包含NYN基序的蛋白小家族的成员，该结构域被预测具有核糖核酸酶活性。N4BP1经过Nedd4介导的泛素化，也经过与SUMO的偶联，SUMO反过来调控N4BP1的泛素化和稳定性。最近的研究表明，N4BP1也与相关的E3连接酶ITCH相互作用，但不是ITCH介导的泛素化的底物。相反，N4BP1与ITCH结合，负向调节ITCH E3活性，直接针对其底物，包括p53相关的肿瘤抑制蛋白p73和p63，以及c-Jun。这些结果表明，N4BP1可能在调节肿瘤进展和癌细胞对化疗的反应中发挥作用。我们对SUMO通路的研究主要集中在对脱苏酶SENP1的分析，特别是其在调节细胞增殖中的作用。我们之前的研究表明，小鼠Senp1基因的突变导致胎盘缺陷，其原因是滋养层前体细胞持续增殖，而分化为成熟的胎盘迷宫细胞。与野生型相比，源自SENP1突变体的原代细胞系也显示出更高的增殖率。与这一发现一致，我们观察到突变细胞中E2F靶基因如cdc2和cyclinA2的上调以及视网膜母细胞瘤（RB）蛋白磷酸化的增加。有趣的是，RB最近被证明是被聚合的，聚合形式对E2F活性的抑制作用较小。我们自己的分析表明RB是SENP1介导的去氧基化的靶标。因此，我们假设，在SENP1突变细胞中，RB稳态sumomylation的增加可能是细胞增殖增加的基础。我们目前正在评估一种突变形式的RB的能力，它不能被总结，以挽救细胞周期缺陷。除了这些研究外，我们正在进行基于SENP1靶点的蛋白质组学质谱分析。对原代胚胎成纤维细胞的初步研究已经证明，这种方法可以识别出稳定状态下sumo化水平增加的蛋白，这些蛋白可能是SENP1突变表型的基础。我们还在SUMO中引入了特定的序列变异，这些变异不会影响SUMO的偶联活性，但确实有助于用质谱鉴定SUMO在靶蛋白上偶联的实际位点，这是验证和理解SUMO在单个蛋白质功能中的生理作用的重要步骤。作为对SENP1研究的补充，我们正在研究SUMO基因在发育过程中丢失的后果。令人惊讶的是，我们发现SUMO-1基因发生功能丧失突变的小鼠是正常的。进一步分析表明，相关的SUMO-2/3蛋白存在代偿性利用，这显然挽救了SUMO-1的损失。这一发现对开发针对SUMO通路的合理方法的持续努力具有重要意义，说明需要考虑这些相关的SUMO分子可以相互替代。为了确定泛素介导的蛋白质降解在正常发育中起关键作用的靶标，以及当失调时可能导致肿瘤发生的靶标，我们对E3泛素连接酶Nedd4的相互作用伙伴进行了酵母双杂交筛选。该筛选确定了N4BP1和N4BP3，这是两种新的发育表达蛋白，由于它们在肿瘤发生中的潜在参与，现在正在进一步表征它们。N4BP1是一个包含NYN基序的蛋白小家族的成员，该结构域被预测具有核糖核酸酶活性。N4BP1经过Nedd4介导的泛素化，也经过与SUMO的偶联，SUMO反过来调控N4BP1的泛素化和稳定性。最近的研究表明，N4BP1也与相关的E3连接酶ITCH相互作用，但不是ITCH介导的泛素化的底物。相反，N4BP1与ITCH结合，负向调节ITCH E3活性，直接针对其底物，包括p53相关的肿瘤抑制蛋白p73和p63，以及c-Jun。这些结果表明，N4BP1可能在调节肿瘤进展和癌细胞对化疗的反应中发挥作用。我们对SUMO通路的研究主要集中在对脱苏酶SENP1的分析，特别是其在调节细胞增殖中的作用。我们之前的研究表明，小鼠Senp1基因的突变导致胎盘缺陷，其原因是滋养层前体细胞持续增殖，而分化为成熟的胎盘迷宫细胞。与野生型相比，源自SENP1突变体的原代细胞系也显示出更高的增殖率。与这一发现一致，我们观察到突变细胞中E2F靶基因如cdc2和cyclinA2的上调以及视网膜母细胞瘤（RB）蛋白磷酸化的增加。有趣的是，RB最近被证明是被聚合的，聚合形式对E2F活性的抑制作用较小。我们自己的分析表明RB是SENP1介导的去氧基化的靶标。因此，我们假设，在SENP1突变细胞中，RB稳态sumomylation的增加可能是细胞增殖增加的基础。我们目前正在评估一种突变形式的RB的能力，它不能被总结，以挽救细胞周期缺陷。除了这些研究外，我们正在进行基于SENP1靶点的蛋白质组学质谱分析。对原代胚胎成纤维细胞的初步研究已经证明，这种方法可以识别出稳定状态下sumo化水平增加的蛋白，这些蛋白可能是SENP1突变表型的基础。我们还在SUMO中引入了特定的序列变异，这些变异不会影响SUMO的偶联活性，但确实有助于用质谱鉴定SUMO在靶蛋白上偶联的实际位点，这是验证和理解SUMO在单个蛋白质功能中的生理作用的重要步骤。作为对SENP1研究的补充，我们正在研究SUMO基因在发育过程中丢失的后果。令人惊讶的是，我们发现SUMO-1基因发生功能丧失突变的小鼠是正常的。进一步分析表明，相关的SUMO-2/3蛋白存在代偿性利用，这显然挽救了SUMO-1的损失。这一发现对开发针对SUMO通路的合理方法的持续努力具有重要意义，说明需要考虑这些相关的SUMO分子可以相互替代。

To identify targets of ubiquitin mediated protein degradation playing key roles in normal development, and that might potentially contribute to neoplasia when dysregulated, we carried out a yeast two hybrid screen for interaction partners of the E3 ubiquitin ligase Nedd4. This screen identified N4BP1 and N4BP3, two novel developmentally expressed proteins, each of which are now being characterized further because of the potential involvement of each in tumorigenesis. N4BP1 is a member of a small family of proteins containing a NYN motif, a domain predicted to have ribonuclease activity. N4BP1 undergoes polyubiquitination mediated by Nedd4, and also undergoes conjugation with SUMO, which in turn regulates N4BP1 ubiquitination and stability. Recent work has shown that N4BP1 also interacts with the related E3 ligase, ITCH, but is not a substrate for ITCH mediated ubiquitination. Rather, N4BP1 binding to ITCH, negatively regulates ITCH E3 activity directed toward its substrates, including the p53 related tumor suppressor proteins p73 and p63, as well as c-Jun. These results suggest that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. Our studies on the SUMO pathway have as a primary focus the analysis of the desumoylating enzyme SENP1, and specifically its role in regulating cell proliferation. We previously showed that mutation of the Senp1 gene in mice leads to placental defects stemming from continued proliferation of trophoblast precursor cells at the expense of differentiation into mature placental labyrinth cells. Primary cell lines derived from SENP1 mutants also show an increased rate of proliferation as compared to wild type. Consistent with this finding, we observe an up-regulation of E2F target genes such as cdc2 and cyclinA2 in mutant cells and an increase in Retinoblastoma (RB) protein phosphorylation. Interestingly, RB recently was shown to be sumoylated, with the sumoylated form exerting less repressive potential on E2F activity. Our own analysis indicates that RB is a target SENP1 mediated desumoylation. Thus, we hypothesize that increased steady state sumoylation of RB may underlie the increased cellular proliferation seen in SENP1 mutant cells. We are currently assessing the ability of a mutant form of RB, which cannot be sumoylated, to rescue the cell cycle defects. In addition to these studies we are carrying out a proteomic scale mass spectrometry based analysis of SENP1 targets. Preliminary studies in primary embryonic fibroblasts have demonstrated the power of this approach to identify protein(s) whose increased steady state level of sumoylation potentially underlies the SENP1 mutant phenotype. We have also introduced specific sequence variations into SUMO that do not compromise conjugation activity but do facilitate mass spectrometric identification of the actual site of SUMO conjugation on target proteins, an essential step in allowing validation and in understanding the physiological role of sumoylation in individual protein function. Complementing our work on SENP1, we are investigating the consequences of genetic loss of SUMO during development. Surprisingly, we have found that mice with loss-of-function mutations in the SUMO-1 gene are normal. Further analysis has shown that there is compensatory utilization of the related SUMO-2/3 proteins, which apparently rescues the loss of SUMO-1. This finding has important implications for ongoing efforts to develop rational approaches to targeting the SUMO pathway, illustrating the need to take into account that these related SUMO molecules can substitute for each other.To identify targets of ubiquitin mediated protein degradation playing key roles in normal development, and that might potentially contribute to neoplasia when dysregulated, we carried out a yeast two hybrid screen for interaction partners of the E3 ubiquitin ligase Nedd4. This screen identified N4BP1 and N4BP3, two novel developmentally expressed proteins, each of which are now being characterized further because of the potential involvement of each in tumorigenesis. N4BP1 is a member of a small family of proteins containing a NYN motif, a domain predicted to have ribonuclease activity. N4BP1 undergoes polyubiquitination mediated by Nedd4, and also undergoes conjugation with SUMO, which in turn regulates N4BP1 ubiquitination and stability. Recent work has shown that N4BP1 also interacts with the related E3 ligase, ITCH, but is not a substrate for ITCH mediated ubiquitination. Rather, N4BP1 binding to ITCH, negatively regulates ITCH E3 activity directed toward its substrates, including the p53 related tumor suppressor proteins p73 and p63, as well as c-Jun. These results suggest that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. Our studies on the SUMO pathway have as a primary focus the analysis of the desumoylating enzyme SENP1, and specifically its role in regulating cell proliferation. We previously showed that mutation of the Senp1 gene in mice leads to placental defects stemming from continued proliferation of trophoblast precursor cells at the expense of differentiation into mature placental labyrinth cells. Primary cell lines derived from SENP1 mutants also show an increased rate of proliferation as compared to wild type. Consistent with this finding, we observe an up-regulation of E2F target genes such as cdc2 and cyclinA2 in mutant cells and an increase in Retinoblastoma (RB) protein phosphorylation. Interestingly, RB recently was shown to be sumoylated, with the sumoylated form exerting less repressive potential on E2F activity. Our own analysis indicates that RB is a target SENP1 mediated desumoylation. Thus, we hypothesize that increased steady state sumoylation of RB may underlie the increased cellular proliferation seen in SENP1 mutant cells. We are currently assessing the ability of a mutant form of RB, which cannot be sumoylated, to rescue the cell cycle defects. In addition to these studies we are carrying out a proteomic scale mass spectrometry based analysis of SENP1 targets. Preliminary studies in primary embryonic fibroblasts have demonstrated the power of this approach to identify protein(s) whose increased steady state level of sumoylation potentially underlies the SENP1 mutant phenotype. We have also introduced specific sequence variations into SUMO that do not compromise conjugation activity but do facilitate mass spectrometric identification of the actual site of SUMO conjugation on target proteins, an essential step in allowing validation and in understanding the physiological role of sumoylation in individual protein function. Complementing our work on SENP1, we are investigating the consequences of genetic loss of SUMO during development. Surprisingly, we have found that mice with loss-of-function mutations in the SUMO-1 gene are normal. Further analysis has shown that there is compensatory utilization of the related SUMO-2/3 proteins, which apparently rescues the loss of SUMO-1. This finding has important implications for ongoing efforts to develop rational approaches to targeting the SUMO pathway, illustrating the need to take into account that these related SUMO molecules can substitute for each other.