Nod1: An Apaf-like Activator of Apoptosis and NF-kB

对微生物病原体的免疫反应是由宿主细胞在细胞表面和细胞质中对特定病原体成分的识别发起的。虽然病原体产物在免疫细胞表面引发的反应被很好地表征，但在细胞质中引发的反应却知之甚少。Nod1是一个不断增长的蛋白家族的成员，与凋亡调节因子Apaf-1/Ced-4和植物抗病R基因产物具有结构同源性。Nod1在细胞中过表达时促进细胞凋亡，但与Apaf-1不同，它诱导NF-kappaB活化。Nod1诱导的NF-KappaB活化是由Nod1的CARD与RICK（一种激活NF-KappaB的蛋白激酶）的CARD相关联介导的。对野生型（wt）和突变型Nod1和RICK的分析表明，Nod1和RICK在NF-kappaB激活的相同途径中起作用，其中RICK作为Nod1信号传导的下游介质起作用。Nod1通过其核苷酸结合结构域（NBD）自结合，Nod1寡聚化促进RICK分子的接近和NF-kappaB的激活。与Nod1一样，细胞内植物R蛋白含有与NBD连接的n端效应域和位于NBD c端的多个富亮氨酸重复序列（lrr）。R蛋白的lrr高度多样化，似乎参与识别一系列广泛的病原体成分。值得注意的是，我们发现细菌脂多糖（LPS），而不是其他病原体成分，诱导表达微量Nod1的人胚胎肾293T细胞中TLR4-和MyD88-不依赖的NF-kappaB活化。与植物抗病R蛋白一样，Nod1的lrr也是lps诱导NF-kappaB激活所必需的。此外，LPS结合活性可以与细胞质提取物中的Nod1共同免疫纯化。我们的假设是Nod1是植物R基因产物的哺乳动物对应物，可能作为来自入侵细菌的病原体成分的细胞质受体。我们提出了三个具体目标，以了解细菌LPS和Nod1相互作用的生化机制，并确定Nod1在体内对细菌LPS的反应中的作用。

The immune response to microbial pathogens is initiated by recognition of specific pathogen components by host cells both at the cell surface and in the cytosol. While the response triggered by pathogen products at the surface of immune cells is well characterized, that initiated in the cytosol is poorly understood. Nod1 is a member of a growing family of proteins with structural homology to apoptosis regulators Apaf-1/Ced-4 and plant disease resistant R gene products. Nod1 promotes apoptosis when overexpressed in cells, but unlike Apaf-1, it induces NF-kappaB activation. NF-KappaB activation induced by Nod1 is mediated by the association of the CARD of Nod1 with the corresponding CARD of RICK, a protein kinase that activates NF-kappaB. Analyses with wild-type (wt) and mutant forms of both Nod1 and RICK have suggested that Nod1 and RICK act in the same pathway of NF-kappaB activation, where RICK functions as a downstream mediator of Nod1 signaling. Nod1 self-associates through its nucleotide-binding domain (NBD) and Nod1 oligomerization promotes proximity of RICK molecules and NF-kappaB activation. Like Nod1, intracellular plant R proteins contain N-terminal effector domains linked to a NBD and multiple leucine-rich repeats (LRRs) located C-terminally of the NBD. The LRRs of R proteins are highly diverse and appear to be involved in the recognition of a wide array of pathogen components. Remarkably, we find that bacterial lipopolysacharides (LPS), but not other pathogen components, induced TLR4- and MyD88- independent NF-kappaB activation in human embryonic kidney 293T cells expressing trace amounts of Nod1. Like plant disease resistant R proteins, the LRRs of Nod1 were required for LPS-induced NF-kappaB activation. Furthermore, a LPS binding activity could be co-immunopurified with Nod1 from cytosolic extracts. Our hypothesis is that Nod1 is a mammalian counterparts of plant R gene products that may function as a cytosolic receptor for pathogen components derived from invading bacteria. We propose three Specific Aims to understand the biochemical mechanism by which bacterial LPS and Nod1 interact and to determine the role of Nod1 in the response to bacterial LPS in vivo.