Notch-induced protein degradation in lymphopoiesis

描述（由申请人提供）：Notch信号传导在多个水平上控制真核细胞中的多种分化过程。我们先前发现了在淋巴细胞分化中重要的机制，即Notch连接导致E2A转录因子的降解。进一步的研究表明，这如何有助于B与T淋巴细胞系的命运决定，以及如何E2 A水平是仔细调节分化的胸腺中进行。最近，该研究进入了一个新的令人兴奋的阶段，发现Notch可以控制许多其他蛋白质的稳定性，包括Janus激酶，细胞因子反应的重要介质。此外，已经鉴定了Notch信号传导的下游效应物，并显示其介导Notch靶向的底物的降解。因此，我们很好地解决全球性的问题Notch诱导降解的广泛的底物的cullin环型泛素连接酶。具体目标#1将扩展我们令人兴奋的发现，即Notch信号刺激锚蛋白重复序列和SOCS盒蛋白（Asb）家族的转录，这可以促进Notch靶向蛋白的降解。将在动物中使用AsB功能获得和丧失方法，与Notch功能获得或丧失在肿瘤发生、B与T谱系决定和边缘区（MZ）B细胞形成中引起的作用进行比较，评价AsB表达与Notch功能的生物学相关性。此外，Asb蛋白，特别是Asb 2，促进泛素化反应催化cullin为基础的E3连接酶的生化机制将进行研究。我们将测试的假设，即Asb2桥Cul1和Cul5相关的E3连接酶的异聚体复合物的形成，以提高水平的neddylated形式的cullin含复合物，这是已知的催化活性。这可能会建立一个新的范式，解释E3连接酶如何在高阶复合物中运作，以及Notch信号如何控制不同底物的周转。具体目标#2将描述Notch诱导的E2A和Jak3降解在淋巴发育期间的几个谱系决定中的作用。将研究Notch诱导的Jak3降解的潜在机制，并创建Notch抗性Jak3突变体。然后，这些突变体将与先前建立的Notch抗性E2A蛋白一起在体外和动物中表达。这些蛋白质对骨髓和胸腺中B细胞分化的影响将与mastermid1的显性负性突变体（其抑制Notch功能并因此用作阳性对照）的影响一起进行沿着检查。边缘区B细胞分化也依赖于Notch信号传导，但尚不清楚Notch诱导的E2 A和Jak 3降解是否是原因。在表达Notch抗性E2 A和Jak 3的小鼠中检查MZ B细胞形成将有助于解决这个问题。总之，这些研究将把我们对Notch功能的理解带到一个新的领域，并建立与其他调控途径的串扰。Notch诱导的蛋白质周转的深入研究将产生相当多的基础信息，并可能为免疫缺陷，自身免疫和恶性疾病提出新的治疗方法。公共卫生相关性：该项目将产生关于一个重要蛋白质家族的开创性信息。这些Notch受体对许多器官和组织的正常发育至关重要。然而，Notch的不当调节导致癌症，并且关于Notch功能的许多内容仍然是一个谜。例如，我们的初步数据表明，Notch作为一个环主，控制许多其他蛋白质的水平。虽然我们怀疑它是通过调节这些蛋白质的稳定性来实现这一点的，并且有很有希望的线索，但需要进行广泛的研究来揭示它是如何工作的。我们有充分的理由相信，这些问题的答案将导致几种疾病的新疗法。这些疾病包括白血病、乳腺癌或肠癌、自身免疫性疾病和发育缺陷。

DESCRIPTION (provided by applicant): Notch signaling controls diverse differentiation processes in eukaryotic cells at multiple levels. We previously discovered a mechanism that is important in lymphocyte differentiation, i.e. Notch ligation results in degradation of the E2A transcription factor. Further study showed how this contributes to the B versus T lymphocyte lineage fate decision, and how E2A levels are carefully regulated as differentiation proceeds in the thymus. More recently, the study entered a new and exciting phase with the discovery that Notch can control the stability of many other proteins including the Janus kinases, essential mediators of cytokine responses. Furthermore, a downstream effector of Notch signaling has been identified and shown to mediate the degradation of substrates targeted by Notch. Thus, we are well situated to address global issues concerning Notch-induced degradation of a wide spectrum of substrates of cullin-ring type ubiquitin ligases. Specific aim #1 will extend our exciting finding that Notch signaling stimulates the transcription of a family of ankyrin repeats and SOCS box containing proteins (Asb), which can promote the degradation of proteins targeted by Notch. The biological relevance of Asb expression to Notch function will be evaluated using gain and loss of Asb function approaches in animals, in comparison to the effects caused by gain or loss of Notch function in tumorigenesis, B versus T lineage decision and marginal zone (MZ) B cell formation. Furthermore, the biochemical mechanism by which Asb proteins, particularly Asb2, facilitates ubiquitination reactions catalyzed by cullin-based E3 ligases will be investigated. We will test the hypothesis that Asb2 bridges the formation of heteromeric complexes of Cul1 and Cul5 associated E3 ligases to enhance the levels of neddylated forms of cullin-containing complexes, which are known to be catalytically active. This could potentially establish a new paradigm explaining how E3 ligases operate in higher-order complexes and how Notch signaling controls the turnover of diverse substrates. Specific aim #2 will delineate the role of Notch-induced E2A and Jak3 degradation in several lineage decisions during lymphoid development. Mechanisms underlying Notch-induced Jak3 degradation will be investigated and Notch-resistant Jak3 mutants will be created. These mutants will then be expressed together with previously established Notch-resistant E2A proteins in vitro and in animals. The effects of these proteins on B cell differentiation in the bone marrow and thymus will be examined along with that of the dominant- negative mutant of mastermind1, which inhibits Notch function and thus serves as a positive control. Marginal zone B cell differentiation also depends on Notch signaling but it is not known if Notch-induced E2A and Jak3 degradation is responsible. Examination of MZ B cell formation in mice expressing Notch-resistant E2A and Jak3 will help address this issue. Taken together, these studies will take our understanding of Notch function to a new realm and establish crosstalk with other regulatory pathways. This in-depth investigation of Notch-induced protein turnover will yield considerable basic information and may suggest new therapies for immunodeficiency, autoimmune and malignant diseases. PUBLIC HEALTH RELEVANCE: This project will yield groundbreaking information about an important family of proteins. These Notch receptors are essential for the normal development of many organs and tissues. However, improper regulation of Notch leads to cancer and there is much about Notch functions that remains a mystery. For example, our preliminary data suggest that Notch acts as a ringmaster, controlling the levels of many other proteins. While we suspect that it does this by regulating the stability of these proteins, and have promising leads, extensive study will be needed to reveal exactly how this works. There is good reason to believe the answers to these questions will lead to new treatments for several types of diseases. These include leukemias and cancers of the breast or intestine, autoimmune diseases and developmental defects.