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。可杀灭多种肿瘤细胞，但效果不明显 对大多数正常细胞而言。已在人体中鉴定出几种 TRAIL 受体。 其中包括 DR4、DR5、DcR1 和 DcR2。 DR4 和 DR5 包含死亡结构域 它们的细胞质尾部，并且在过度表达时可以引发细胞死亡。在 相反，DcR1和DcR2不编码任何死亡结构域并且不能启动 细胞凋亡。这些所谓的诱饵受体 DcR1 和 DcR2 可以充当 DR4 和 DR5 的显性失活突变体，至少在瞬时转染中 实验。 TRAIL及其受体的生理功能较差 明白了。最近，可溶性人类 DRS 蛋白被证明会加剧 胶原诱导的自身免疫和外源 TRAIL 被证明可以抑制 T 细胞 扩散。因此，TRAIL可能作为负调控分子 免疫系统。了解TRAIL的生理功能及其 受体，一种小鼠 cDNA 克隆，编码与 分离出人 DR4 和 DR5 (rnDR4/5)。相同的替代拼接 发现基因产生相同的蛋白质，但没有死亡结构域（mDcR）。 瞬时转染 mDR4I5 而不是 mDcR 会导致细胞凋亡，并且稳定 FADD 细胞中 mDR4/5 的表达赋予 TRAIL 敏感性。为了阐明 TRAIL 受体及其胞质截短形式 (mDcR) 在 免疫系统，缺乏蛋白质的小鼠和缺乏DcR的小鼠将 生成的。组织特异性无效 mDR4/5 也将使用 cre-loxP 制备 技术。 DR4/5和DcR突变对淋巴细胞发育、T的影响 将检查 B 细胞耐受性和免疫反应。与 FasL 类似， TRAIL 介导的细胞凋亡是通过 FADD 介导的。这就提出了一个问题 相同的细胞如何能够抵抗 TRAIL 但对 FasL 介导敏感 细胞凋亡。受体胞质尾交换实验来剖析 提出了造成这种情况的根本原因。编码 mDR4/5-Fas 的逆转录病毒 （DR4/5 胞外结构域与 Fas 死亡结构域融合）或倒数 Fas-mDR4/5嵌合蛋白将用于感染野生型或lpr/lpr T 细胞。 TRAIL 或 FasL 对表达这些的 T 细胞的凋亡作用 将评估嵌合蛋白。成功完成这些实验 应该会导致人们对癌症与正常人有何不同有一个重要的了解 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.