Nit1-mediated signaling in T cells

描述(申请人提供)：哺乳动物的Nit1蛋白与细菌和植物腈酶同源。以往的研究表明，Nit1在创伤和除草剂诱导的细胞凋亡中起作用。在苍蝇和蠕虫中，Nit1与FHIT肿瘤抑制因子的5‘端融合。因此，有人认为Nit1可能在功能上与FHIT途径相互作用。FHIT蛋白是一种肿瘤抑制因子。FHIT的躯体缺失与多种癌症有关。FHIT基因缺失导致小鼠发生自发性淋巴系统恶性肿瘤。也有证据表明，Nit1缺乏会导致小鼠诱发肿瘤的发生率增加。FHIT和Nit1抑瘤作用的分子机制尚不清楚。以往的研究表明，FHIT或Nit1的异位表达可诱导肿瘤细胞的凋亡，这需要caspase的激活，但不依赖于Bcl2和Bclxl。此外，显性负性突变体FADD可抑制FHIT诱导的细胞凋亡。我们和其他人之前已经证明，FADD是由死亡受体启动的外在凋亡信号通路的中介。Fas死亡受体诱导的细胞凋亡在免疫系统的动态平衡中起着重要作用。为了帮助了解Nit1在T细胞中的生理功能，我们对Nit1基因敲除小鼠进行了初步分析。这些突变小鼠的胸腺和外周T细胞亚群数量正常。我们的数据显示，Nit1-/-T细胞存在轻微的Fas缺陷，并诱导了细胞凋亡。虽然Nit1可能在DNA损伤诱导的肾脏细胞凋亡中发挥作用，但我们的初步数据表明，包括3-射线或化学处理引起的DNA损伤在内的各种刺激诱导的细胞凋亡对Nit1缺陷的T细胞没有影响。出乎意料的是，我们发现Nit1缺乏导致了通过抗原受体刺激诱导的T细胞的过度增殖反应。在这一应用中，我们提出了一系列的生化、遗传学和免疫学分析，以帮助理解Nit1在负调控T细胞增殖中的新功能。具体地说，我们的目的是(1)分析Nit1在T细胞凋亡中的功能；(2)了解Nit1在T细胞增殖中的非凋亡功能；(3)通过反向遗传学确定Nit1的功能域。公共卫生相关性：在本申请中，我们描述了一项研究Nit1蛋白的科学计划，Nit1蛋白在免疫系统功能和癌症中发挥重要作用。研究结果将有助于制定有效的预防和治疗方法。

DESCRIPTION (provided by applicant): The mammalian Nit1 protein is homologous to bacterial and plant nitrilases. Previous studies have shown that Nit1 plays a role in wound and herbicide induced apoptosis. In flies and worms Nit1 is fused to the 5' end of the Fhit tumor suppressor. Therefore, it has been suggested that Nit1 may interact functionally with the Fhit pathway. The Fhit protein is a tumor suppressor. Somatic loss of Fhit is associated with a wide variety of cancers. Deletion of Fhit results in predisposition to spontaneous lymphoid malignancy in mice. There was also evidence showing that Nit1 deficiency lead to increased incidence of induced tumors in mice. The molecular mechanism involved in the tumor suppressing function of Fhit and Nit1 is not clear. Previous studies have shown that ectopic expression of Fhit or Nit1 can induce apoptosis in tumor cells, which requires caspase activation but is independent of Bcl-2 and Bcl-xL. Furthermore, Fhit- induced apoptosis can be inhibited by dominant negative mutant FADD. We and others have previously demonstrated that FADD is a mediator of the extrinsic apoptotic signaling pathways initiated by death receptors. Apoptosis induced by the Fas death receptor plays an important role in homeostasis in the immune system. To help understand the physiological function of Nit1 in T cells, we have performed preliminary analyses of Nit1 knockout mice. These mutant mice contained normal numbers of thymic and peripheral T cell sub populations. Our data showed that Nit1-/- T cells had a mild defect in Fas and Cainduced apoptosis. Although Nit1 may play a role in DNA-damage induced apoptosis in kidney cells, our preliminary data indicated that that apoptosis induced by a variety of stimuli including DNA damages due to 3- irradiation or chemical treatments was not affected in Nit1-deficient T cells. Unexpectedly, we found that Nit1 deficiency resulted in hyperproliferative responses in T cells induced through the antigen receptor stimulation. In this application, we propose an array of biochemical, genetic and immunological analyses to help understand the novel function of Nit1 in negatively regulating T cell proliferation. Specifically, our aims are (1) to analyze the function of Nit1 in T cell apoptosis; (2) to understand the non-apoptotic function of Nit1 in T cell proliferation; (3) to determine the functional domains of Nit1 by reverse genetics. PUBLIC HEALTH RELEVANCE: In this application, we describe a scientific plan to study the Nit1 protein which plays an essential role in immune system functions, and cancer. The results will facilitate the development of effective preventive and therapeutic approaches.