TRAIL receptor in apoptosis and the immune system

描述：（由申请人提供）：TRAIL（肿瘤坏死因子相关 凋亡诱导配体）是肿瘤坏死因子的成员 超家族它可以杀死多种肿瘤细胞，但没有明显的效果 在大多数正常细胞上。已经在人类中鉴定了几种TRAIL受体。 其中包括DR 4、DR 5、DcR 1和DcR 2。DR 4和DR 5含有一个死亡结构域， 它们的细胞质尾，并且当过表达时可以引发细胞死亡。在 相反，DcRl和DcR 2不编码任何死亡结构域，并且不能启动 凋亡这些所谓的诱饵受体，DcR 1和DcR 2，可以作为一个替代。 DR 4和DR 5的显性阴性突变体，至少在瞬时转染中 实验TRAIL及其受体的生理功能较差， 明白最近，可溶性人DRS蛋白被证明加剧了 胶原诱导的自身免疫和外源性TRAIL显示出抑制T细胞 增殖因此，TRAIL可能作为一种负调控分子， 免疫系统了解TRAIL的生理功能及其在肿瘤发生发展中的作用， 受体，一种小鼠cDNA克隆，编码与 分离人DR 4和DR 5（rnDR 4/5）。其选择性剪接， 发现该基因产生没有死亡结构域的相同蛋白质（mDcR）。 瞬时转染mDR 4 I5而不是mDcR导致细胞凋亡，并且稳定表达。 FADD+细胞中mDR 4/5的表达赋予TRAIL敏感性。阐明本 这种TRAIL受体及其胞质截短形式（mDcR）在 免疫系统，缺乏这两种蛋白质的小鼠和缺乏DcR的小鼠将 生成的.还将使用cre-loxP制备组织特异性空mDR 4/5 技术. DR 4/5和DcR突变对淋巴细胞发育、T淋巴细胞增殖和T淋巴细胞增殖的影响 并检查B细胞耐受性和免疫应答。与FasL类似， TRAIL介导的凋亡通过FADD介导。这就提出了一个问题， 同样的细胞如何对TRAIL有抗性，但对FasL介导的 凋亡受体胞质-尾交换实验， 提出了其根本原因。编码mDR 4/5-Fas的逆转录病毒 (DR4/5细胞外结构域融合至Fas死亡结构域）或相互 Fas-mDR 4/5嵌合蛋白将用于感染野生型或lpr/lpr T 细胞TRAIL或FasL对表达它们的T细胞的凋亡作用是通过诱导T细胞凋亡来实现的。 将评估嵌合蛋白。成功完成这些实验 这将有助于我们更好地理解癌症与正常人的不同之处 细胞以及TRAIL受体如何在免疫系统中发挥作用。

DESCRIPTION: (provided by applicant): TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a member of the tumor necrosis factor superfamily. It can kill a variety of tumor cells but has no appreciable effect on most normal cells. Several TRAIL receptors have been identified in human. These include DR4, DR5, DcR1 and DcR2. DR4 and DR5 contain a death domain in their cytoplasmic tails and can initiate cell death when over-expressed. In contrast, DcRl and DcR2 do not encode any death domain and cannot initiate apoptosis. These so-called decoy receptors, DcR1 and DcR2, can instead act as a dominant negative mutant for DR4 and DR5, at least in transient transfection experiments. The physiological function of TRAIL and its receptors are poorly understood. Recently, soluble human DRS protein was shown to exacerbate collagen-induced autoimmunity and exogenous TRAIL was shown to inhibit T cell proliferation. Thus, TRAIL might act as a negative regulatory molecule for the immune system. To understand the physiological function of TRAIL and its receptor(s), a mouse cDNA clone that encodes a protein with equal homology to human DR4 and DR5 was isolated (rnDR4/5). An alternative splicing of the same gene was found to yield an identical protein without a death domain (mDcR). Transient transfection of mDR4I5 but not mDcR leads to apoptosis, and stable expression of mDR4/5 in FADD+ cells confers TRAIL sensitivity. To elucidate the role of this TRAIL receptor and its cytoplasmic-truncated form (mDcR) in the immune system, mice lacking both proteins and mice lacking DcR will be generated. Tissue-specific null mDR4/5 will also be made using the cre-loxP technology. The effect of DR4/5 and DcR mutation on lymphocyte development, T and B cell tolerance and immune responses will be examined. Similar to FasL, TRAIL-mediated apoptosis is mediated through FADD. This raises the question to how the same cells can be resistant to TRAIL but are sensitive to FasLmediated apoptosis. A receptor cytoplasmic-tail swapping experiment to dissect the underlying reasons for this is proposed. Retroviruses encoding mDR4/5-Fas (DR4/5 extracellular domain fused to the Fas death domain) or the reciprocal Fas-mDR4/5 chimeric proteins will be used to infect wild type or lpr/lpr T cells. The apoptotic effect of TRAIL or FasL on T cells expressing these chimeric proteins will be assessed. Successful completion of these experiments should lead to a significant understanding of how cancer differs from normal cells and how TRAIL receptor functions in the immune system.