MECHANISMS OF COMPLEMENT ATTACK ON NUCLEATED CELLS

我们研究的长期目标是了解 末端补体复合物（TCC），其包括C5 b-7、C5 b-8和C5 b-10; C5b-9 C5 b-9介导的有核细胞杀伤受多步骤调控 过程：TCC在膜中的顺序组装， 从细胞表面潜在地裂解C5 b-9，并且细胞的过程 由C5 b-9引起的死亡。 一旦TCC组装完成， 从细胞表面清除。 TCC消除，对细胞至关重要 蛋白质中胞浆Ca 2 +[Ca 2 +] i的增加可提高存活率 当TCC数量有限时，以激酶C（PKC）依赖的方式。 由于互补通道的消除取决于 已知TCC和TCC是细胞活化的有效诱导剂， C5 b-7、C5 b-8和亚裂解C5 b-9通道的作用可能是主要的 细胞从有限补体攻击中存活的生物学意义 in vivo. 除Ca ~（2+）内流和[Ca ~（2+）]i增加外， 了解TCC是否以及如何产生其他信号信使。 有趣的是，C5 b-9的一些生物学活性可以通过 通过C5 b-7和/或C5 b-8，几乎没有或没有Ca 2+内流，这表明， 除Ca ~（2+）外，还可能有其他介质参与。 我们已经能够 证明TCC，特别是C5 b-7，增加了 二酰基甘油（DAG）和神经酰胺，有效的第二信使，在人类 JY B细胞系和鼠C2肌细胞系中。 这些发现 这对了解受影响细胞的病理生物学具有潜在意义， 在自身免疫性疾病中， B细胞和肌肉细胞是自身抗体的目标。 在这个建议中，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蛋白的活化，（2）TCC对G蛋白活化的调节作用。 通过检测C2肌管中肌肉特异性蛋白的表达， 编码这些蛋白质的mRNA的转录后稳定性，和（3） 探讨C5 b-9介导的有核细胞杀伤机制 不受控制的钙内流对功能状态的影响， 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.