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(申请人提供)：Noch信号在多个水平上控制真核细胞的不同分化过程。我们以前发现了一个在淋巴细胞分化中很重要的机制，即Notch结扎导致E2A转录因子的降解。进一步的研究表明，这如何有助于B淋巴细胞对T淋巴细胞谱系命运的决定，以及随着胸腺分化的进行，E2A水平是如何受到仔细调节的。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，包括细胞因子反应的基本介体Janus激酶。此外，Notch信号的下游效应器已经被发现，并被证明介导了Notch靶向底物的降解。因此，我们很好地解决了有关Notch诱导的广泛的cullin环型泛素连接酶底物降解的全球性问题。具体目标#1将扩展我们令人兴奋的发现，即Notch信号刺激一组Ankyrin重复序列和SoCS盒包含蛋白(Asb)的转录，这可以促进Notch靶向蛋白的降解。Asb表达与Notch功能的生物学相关性将通过动物Asb功能的获得和丧失来评估，比较Notch功能的获得或丧失在肿瘤发生、B与T谱系决定和边缘带(MZ)B细胞形成中的影响。此外，将研究Asb蛋白，特别是Asb2，促进基于cullin的E3连接酶催化的泛素化反应的生化机制。我们将检验这样的假设，即Asb2桥接了Cul1和cul5相关的E3连接酶的异构体复合体的形成，以提高已知具有催化活性的含有cullin的复合体的水平。这可能会建立一个新的范例，解释E3连接酶如何在高阶复合体中工作，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降解在淋巴发育过程中的几个谱系决定中的作用。将研究Notch诱导JAK3降解的机制，并创建抗Notch的JAK3突变体。然后，这些突变体将与先前建立的抗Notch的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抑制Notch功能的显性-负性突变体Master Mindend1一起进行检测，从而作为阳性对照。边缘区B细胞的分化也依赖于Notch信号，但目前尚不清楚Notch诱导的E2a和JAK3降解是否与此有关。在表达Notch抗性E2a和JAK3的小鼠中检测MZ B细胞的形成将有助于解决这个问题。综上所述，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。这种对Notch诱导的蛋白质周转的深入研究将产生大量的基本信息，并可能为免疫缺陷、自身免疫和恶性疾病提供新的治疗方法。与公共卫生相关：该项目将产生有关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是必不可少的。然而，对Notch的不正确调控会导致癌症，而且关于Notch的许多功能仍然是一个谜。例如，我们的初步数据表明，Notch扮演着环长的角色，控制着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做到这一点的，并且有很有希望的线索，但还需要进行广泛的研究来揭示这一过程的确切机制。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和乳腺癌或肠癌、自身免疫性疾病和发育缺陷。