Notch-induced protein degradation in lymphopoiesis

Notch 诱导淋巴细胞生成中的蛋白质降解

• 批准号: 8099313
• 负责人: Xiao-Hong Sun
• 金额: $ 24.84万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-12 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099313
• 关键词: Address; Animals; Ankyrin Repeat; Autoimmune Diseases; Autoimmune Process; B cell differentiation; B-Cell Development; B-Lymphocytes; Biochemical; Biological; Bone Marrow; Boxing; Breeding; Coculture Techniques; Collection; Complex; Cytokine Signaling; Data; Defect; Development; Disease; Dominant-Negative Mutation; Eukaryotic Cell; Event; Family; Gene Expression; Genetic Transcription; Immunologic Deficiency Syndromes; In Vitro; Individual; Intestines; Investigation; Janus kinase; Knock-in Mouse; Lead; Learning; Ligation; Lymphocyte; Lymphoid; Lymphomagenesis; Lymphopoiesis; Malignant - descriptor; Malignant Neoplasms; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinases; Mus; Mutant Strains Mice; Organ; Phase; Process; Production; Protein Family; Proteins; Reaction; Receptor Signaling; Regulation; Regulatory Pathway; Research; Resistance; Role; Signal Transduction; Stromal Cells; Surface; System; T-Lymphocyte; TCF3 gene; Testing; Therapeutic Intervention; Thymus Gland; Tissues; Ubiquitination; Up-Regulation; Work; base; cell type; cytokine; leukemia; leukemogenesis; malignant breast neoplasm; mouse model; mutant; notch protein; overexpression; protein degradation; public health relevance; response; transcription factor; tumorigenesis; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：Notch信号传导控制多个级别的真核细胞中的各种分化过程。我们以前发现了一种在淋巴细胞分化中很重要的机制，即缺结扎导致E2A转录因子降解。进一步的研究表明，这是如何有助于B与T淋巴细胞谱系命运的决策，以及随着胸腺中的分化的进行，如何仔细调节E2A水平。最近，这项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，这是细胞因子反应的基本介体。此外，已经确定了Notch信号的下游效应子，并证明可以介导Notch靶向的底物的降解。因此，我们良好地解决了有关Notch诱导的各种Cullin-Ring型泛素连接酶底物的降解的全球问题。具体的目标＃1将扩展我们令人兴奋的发现，即Notch信号传导刺激了arnkyrin重复序列和含有蛋白质的SOCS盒（ASB）的转录，这可以促进Notch靶向的蛋白质的降解。与动物中ASB功能方法的增益和丧失相比，将评估ASB表达与Notch功能的生物学相关性，这与肿瘤发生中缺口功能引起的效果相比，B与T谱系决策和边缘区域（MZ）B细胞形成。此外，将研究基于Cullin的E3连接酶催化的ASB蛋白（尤其是ASB2）的生化机制。我们将检验以下假设：ASB2桥接CUL1和CUL5相关的E3连接酶的异源复合物的形成，以增强含Cullin的含Cullin含量的复合物的水平，这些复合物已知被催化活性。这可能会建立一个新的范式，以解释E3连接酶在高阶复合物中的运行方式，以及Notch信号如何控制不同底物的周转率。具体目标＃2将描述Notch诱导的E2A和JAK3降解在淋巴发育过程中的几个谱系决策中的作用。将研究由Notch诱导的JAK3降解的机制，并将创建抗缺口的JAK3突变体。然后，这些突变体将在体外和动物中与先前建立的耐凹口E2A蛋白一起表达。这些蛋白质对骨髓和胸腺中B细胞分化的影响将与Mastermind1的显性阴性突变体一起检查，该突变体抑制了Notch功能，因此可以作为阳性对照。边缘区域B细胞分化也取决于Notch信号传导，但尚不清楚Notch诱导的E2A和JAK3降解是否负责。检查表达耐凹口E2A和JAK3的小鼠中MZ B细胞形成的检查将有助于解决此问题。综上所述，这些研究将使我们对Notch功能的理解成为一个新领域，并与其他监管途径建立串扰。对Notch诱导的蛋白质更新的深入研究将产生大量的基本信息，并可能提出有关免疫缺陷，自身免疫性和恶性疾病的新疗法。公共卫生相关性：该项目将产生有关重要蛋白质家族的开创性信息。这些缺口受体对于许多器官和组织的正常发育至关重要。但是，对Notch的调节不当会导致癌症，并且关于Notch功能的许多仍然是一个谜。例如，我们的初步数据表明Notch充当环形大师，控制许多其他蛋白质的水平。尽管我们怀疑它通过调节这些蛋白质的稳定性并具有有希望的潜在客户来实现这一目标，但需要进行广泛的研究以揭示其工作原理。有充分的理由相信这些问题的答案将为几种类型的疾病带来新的治疗方法。其中包括白血病和乳腺癌或肠道的癌症，自身免疫性疾病和发育缺陷。