FAS ASSOCIATED DEATH DOMAIN (FADD)

描述：（改编自研究者摘要）Fas是一种45 kD 属于TNF受体家族的跨膜蛋白。 对 通过其配体（FasL）或通过抗体交联，Fas 传递导致细胞凋亡的死亡信号。 在小鼠中自发丢失 在Fas（lpr）和FasL（gld）中已经描述了功能突变。 在这两种突变品系的小鼠中， 提示Fas和FasL参与了正常的淋巴细胞凋亡。 淋巴细胞死亡 这与最近的观察结果一致， Fas系统参与活化T细胞的清除， 对外来抗原有反应 如果没有一个有效的Fas- FasL系统，如在lpr或gld小鼠中，活化的淋巴细胞积聚 并导致自身免疫性疾病的发展。 朝着正确的 遗传背景（MRL）的lpr和gld小鼠发展成致命的狼疮样 综合征 尽管Fas在程序性细胞死亡中的重要性， 自身免疫，Fas杀伤的分子机制仍然是一个重要的机制。 谜。 Fas和TNF受体家族的其他成员一样， 不具有固有的信号能力（例如，激酶活性）， 这表明信号转导可能是通过结合 分子。 Fas受体胞质结构域的突变分析 描绘了一段大约70个氨基酸的序列， 足以转导死亡信号（称为死亡信号 域）。 为了鉴定相关的信号转导分子，我们 利用酵母双杂交系统克隆编码蛋白的cDNA 结合Fas胞质结构域。 一种这样的相互作用蛋白， 命名为FADD（Fas相关死亡结构域），结合天然Fas，但 而不是功能不活跃的突变体。 FADD本身包含一个死亡领域 与Fas的死亡结构域同源，提示死亡的死亡结构域 域相互作用 FADD的表达诱导细胞凋亡。 这些 研究结果表明，FADD可能发挥重要作用， Fas的近端信号转导。 鉴于此，具体目标是 如下所示： 具体目的1：确认内源性FADD结合Fas，并且FADD的表达与Fas的表达相关。 死亡结构域确实负责受体结合。 此外，使用酵母双杂交和互补生化 方法来表征FADD相互作用蛋白。 具体目标2：使用显性阴性方法，确定FADD是否 是Fas死亡途径的内在和必要组成部分。

DESCRIPTION: (adapted from the investigator's abstract) Fas is a 45kD transmembrane protein that belongs to the TNF receptor family. On activation, either by its ligand (FasL) or by antibody cross linking, Fas delivers a death signal resulting in apoptosis. In mice spontaneous loss of function mutations have been described in Fas (lpr) and FasL (gld). In both these mutant strains of mice, an abnormal accumulation of lymphocytes occurs suggesting that Fas and FasL are involved in normal lymphocyte death. This is consistent with recent observations that the Fas system is involved in the elimination of activated T cells after they have responded to foreign antigens. In the absence of a functioning Fas- FasL system, such as in lpr or gld mice, activated lymphocytes accumulate and contribute to the development of auto-immune disease. In the right genetic background (MRL) the lpr and gld mice develop a fatal lupus-like syndrome. Despite the importance of Fas in programmed cell death and auto-immunity, the molecular mechanism by which Fas kills remains an enigma. Fas, like other members of the TNF receptor family appears to possess no intrinsic signaling capacity (e.g., kinase activity), suggesting that signal transduction is likely mediated by associating molecules. Mutational analysis of the Fas receptor cytoplasmic domain has delineated a stretch of about 70 amino acids that is necessary and sufficient for transduction of the death signal (referred to as a death domain). To identify associated signal transducing molecules, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the Fas cytoplasmic domain. One such interacting protein, designated FADD (for Fas associated death domain), bound native Fas but not functionally inactive mutants. FADD itself contains a death domain homologous to the death domain of Fas suggesting a death domain to death domain interaction. Expression of FADD induces apoptosis. These findings suggest that FADD potentially plays an important role in the proximal signal transduction of Fas. Given this, the Specific Aims are as follows: Specific Aim 1: Confirm that endogenous FADD binds Fas and that the death domain is indeed responsible for receptor association. Additionally, use the yeast two-hybrid and complementary biochemical approaches to characterize FADD interacting proteins. Specific Aim 2: Using a dominant negative approach, determine if FADD is an intrinsic and necessary component of the Fas death pathway.