MECHANISMS OF COMPLEMENT ATTACK ON NUCLEATED CELLS

我们研究的长期目标是了解 末端补体复合体(TCC)，包括C5b-7、C5b-8和 C5B-9。C5b-9介导的有核细胞杀伤受多步骤控制 工艺：TCC在膜中的顺序组装，消除 从细胞表面潜在地裂解C5b-9，以及细胞的过程 由C5b-9引起的死亡。一旦TCC组装完成，它将受到快速 从细胞表面清除。消除TCC，对细胞至关重要 存活率，是通过增加蛋白质中的胞内钙离子，[钙]i来提高的 当TCC数量有限时，以PKC依赖的方式表达。 由于补码通道的消除取决于由 已知TCC和TCC是一种有效的细胞激活诱导剂， C5b-7、C5b-8和亚溶血C5b-9通道的作用可能是主要的 细胞在有限补体攻击中存活的生物学重要性 在活体内。除了钙离子内流和[Ca~(2+)]i升高外，几乎没有 了解TCC是否以及如何生成其他信号信使。 有趣的是，C5b-9的一些生物活性可以实现 C5b-7和/或C5b-8很少或没有钙内流，这表明 可能涉及钙离子以外的其他介质。我们已经能够 证明TCC，特别是C5b-7，增加了 人体内有效的第二信使二酰甘油(DAG)和神经酰胺 JY B细胞系和小鼠C2肌肉细胞系。这些发现是 对了解受影响细胞的病理生物学具有潜在的重要意义 在自身免疫性疾病中，具有重要功能的细胞 B细胞和肌肉细胞是自身抗体的靶点。 在这个提案中，我们将研究(1)TCC诱导的信号信使， 特别是DAG和神经酰胺，以及它们的产生机制 分析G蛋白的激活，(2)TCC对细胞周期的调节作用 检测C2肌管中肌肉特异性蛋白的表达 编码这些蛋白的mRNAs转录后的稳定性，以及(3) 探讨C5b-9介导的有核细胞杀伤机制 失控钙内流对心肌细胞功能状态的影响 线粒体。

The long term objective of our research is to understand the biology of the terminal complement complex (TCC) which includes C5b-7, C5b-8, and C5b-9. C5b-9 mediated nucleated cell killing is governed by a multi-step process: sequential assembly of TCC in the membrane, elimination of potentially lytic C5b-9 from the cell surface, and the process of cell death elicited by C5b-9. Once TCC is assembled, it is subjected to rapid elimination from the cell surface. TCC elimination, vital for cell survival, is enhanced by increased cytosolic Ca2+, [Ca2+] i, in a protein kinase C (PKC) -dependent manner when the number of TCC is limiting. Since elimination of complement channels depends on signals generated by TCC and TCC is known to be a potent inducer of cell activation, the effect of C5b-7, C5b-8, and sublytic C5b-9 channels may be of major biological importance in cells surviving from limited complement attack in vivo. Other than Ca2+ influx and increase in [Ca 2+ ]i, little is known about whether and how TCC generates other signal messengers. Interestingly, some of the biological activities of C5b-9 can be achieved by C5b-7 and/or C5b-8 with little or no Ca2+ influx, which suggests that mediators other than Ca2+ may be involved. We have been able to demonstrate that TCC, especially C5b-7, increased the mass levels of diacylglycerol (DAG) and ceramide, potent second messengers, in a human JY B cell line and in a murine C2 muscle cell line. These findings are potentially significant to understand the pathobiology of affected cells in autoimmune diseases in which functionally important cells such as B-cells and muscle cells are targeted by auto-antibodies. In this proposal, we will examine (1) TCC-induced signal messengers, specifically DAG and ceramide, and the mechanisms of their generation by analyzing G protein activation, (2) the regulatory effect of TCC on the expression of muscle-specific proteins in C2 mytotubes by examining post-transcriptional stability of mRNAs encoding these proteins, and (3) the mechanisms of nucleated cell killing mediated by C5b-9 by exploring the effect of uncontrolled calcium influx on the functional status of mitochondria.