Targeting Daxx-mediated complex for autoimmune diseases

描述（由申请人提供）：本项目的重点是研究Daxx新功能中涉及的分子机制，可用于开发自身免疫性疾病的策略。多功能蛋白Daxx最初被鉴定为Fas诱导的细胞凋亡的潜在介质。由于淋巴细胞和其他免疫细胞的凋亡受损，Fas基因突变导致自身免疫淋巴细胞增殖综合征（ALPS）。虽然Fas诱导的细胞死亡是在膜上开始的，但Daxx不仅存在于细胞质中，而且存在于细胞核中。许多潜在的Daxx结合蛋白已被鉴定，包括肿瘤抑制因子p53家族成员以及癌蛋白MDM 2。然而，Daxx的体内功能定义不清，因为生殖细胞中Daxx的缺失导致早期胚胎致死。在淋巴细胞发育和功能过程中，细胞增殖、存活和死亡受到各种基因的严格调控。这些过程的失调可能导致自身免疫性疾病。我们和其他人鉴定了Fas相关死亡结构域（FADD）蛋白作为Fas在凋亡信号传导过程中募集和激活起始剂胱天蛋白酶8的关键衔接子。有趣的是，FADD缺陷也具有胚胎致死效应。而淋巴细胞特异性FADD缺陷小鼠中的淋巴细胞对Fas诱导的细胞凋亡具有抗性，这些条件性FADD敲除小鼠不发展自身免疫性疾病，不像Fas突变小鼠。我们最近的研究解释了这种矛盾，该研究揭示了在FADD不存在的情况下释放的有效的RIP 1依赖性坏死途径。我们的初步数据表明，RIP 1家族成员RIP 3的缺失，恢复FADD缺陷小鼠的正常发育。重要的是，所得的RIP 3-/- FADD-/-双敲除（DKO）小鼠发展出进行性自身免疫性疾病，类似于Fas突变小鼠。这些数据与表明Daxx促进Fas途径中FADD非依赖性细胞凋亡的模型形成对比。事实上，我们的初步数据表明，条件Daxx敲除小鼠的Daxx缺陷型T细胞在Fas诱导的细胞凋亡中没有缺陷。出乎意料的是，当通过T细胞抗原受体（TCR）刺激时，Daxx-/- T细胞对活化诱导的细胞死亡（AICD）超敏感。我们假设，在初级淋巴细胞中，有一个额外的死亡途径，这是独立的Fas-FADD-半胱天冬酶8轴，并抑制Daxx。因此，靶向Daxx将促进细胞死亡，并作为自身免疫性疾病治疗的策略。然而，Daxx在免疫系统中的这种新功能的分子机制尚不清楚。因此，我们建议：1）阐明Daxx抑制的原代淋巴细胞中的细胞死亡途径; 2）测试Daxx功能的遗传和药理学消除对抑制自身免疫性疾病的影响。这些具体目标的成功完成将为Daxx的作用机制提供新的见解，并导致更好地设计预防和治疗自身免疫性疾病和相关疾病的措施。

DESCRIPTION (provided by applicant): The focus of this project is to investigate the molecular mechanism involved in a novel function of Daxx, which can be explored in developing strategies for autoimmune diseases. The multifunctional protein Daxx was originally identified as a potential mediator of Fas-induced apoptosis. Mutations in the Fas gene lead to an autoimmune-lymphoproliferative syndrome (ALPS), due to impaired apoptosis in lymphocytes and other immune cells. While Fas-induced cell death is initiated at the membrane, Daxx is present not only in the cytoplasm but also in the nucleus. Many potential Daxx-binding proteins have been identified, including the tumor suppressor p53 family members as well as the oncoprotein MDM2. However, the in vivo function of Daxx is poorly defined, as deletion of Daxx in germ cells leads to early embryonic lethality. During lymphocyte development and function, cell proliferation, survival and death are tightly regulated by various genes. Dysregulation in these processes may lead to autoimmune diseases. We and others identified the Fas- associated death domain (FADD) protein as a critical adaptor for Fas to recruit and activate the initiator caspase 8 during apoptotic signaling. Interestingly, FADD deficiency also has an embryonic lethal effect. Whereas lymphocytes in lymphocyte-specific FADD-deficient mice are resistant to Fas-induced apoptosis, these conditional FADD knockout mice do not develop autoimmune diseases, unlike that in Fas mutant mice. This paradox is explained by our recent study which reveals a potent, RIP1-dependent necrotic pathway unleashed in the absence of FADD. Our preliminary data show that deletion of RIP3, a member of the RIP1 family, restores normal development in FADD-deficient mice. Importantly, the resulting RIP3-/- FADD-/- double knockout (DKO) mice develop progressive autoimmune diseases, similar to that in Fas mutant mice. These data contrast a model suggesting that Daxx promotes FADD-independent apoptosis in the Fas pathway. Indeed, our preliminary data shows that Daxx-deficient T cells from conditional Daxx knockout mice have no defect in Fas-induced apoptosis. Unexpectedly, Daxx-/- T cells are hypersensitive to activation-induced cell death (AICD) when stimulated through the T cell antigen receptor (TCR). We hypothesize that in primary lymphocytes, there is an additional death pathway which is independent of the Fas-FADD-Caspase 8 axis, and is suppressed by Daxx. Therefore, targeting Daxx would promote cell death and serves as a strategy for autoimmune disease therapy. However, the molecular mechanism of this novel function of Daxx in the immune system is unclear. Therefore, we propose: 1) to elucidate the cell death pathway(s) in primary lymphocytes that is inhibited by Daxx; 2) to test for the effect o genetic and pharmacological ablation of Daxx function on suppressing autoimmune diseases. Successful completion of these specific aims will provide new insight into the mechanisms of the action by Daxx and lead to better design of measures for prevention and treatment of autoimmune and related diseases.