Molecular Responses of Macrophages-- Lipopolysaccharides

细菌脂多糖（LPS）是导致全身炎症反应综合征（SIRS）的毒性表现的主要因素，据报道在美国每年导致100，000例死亡。 最近Toll样受体（TLR）4被鉴定为鼠LPS基因产物;其胞质尾区的错义突变导致C3 H/HeJ小鼠中LSP低应答表型。 TLR 4及其衔接蛋白MyD 88在LPS信号传导中的关键作用开始出现，但所涉及的分子事件仍然知之甚少。 我们推测，TLR 4可能被激活的过程以外的LPS的直接结合; LPS的反应涉及TLR 4的寡聚化和TLR 4与其他蛋白质在除了MyD 88的协会;和热休克蛋白90结合TLR 4激活复合物，并参与其信号转导。 为了检验这些假设，我们产生了一组携带正常、突变或缺失的TLR 4或MyD 88基因的永生化巨噬细胞系。 使用这些细胞系，我们将产生表达完整或改变形式的TLR 4或MyD 88的稳定转染子。 实验方法包括（1）比较这些稳定细胞系之间的LPS或Taxol反应以确定TLR 4和MyD 88的推定结构域的作用，（2）在LPS/Taxol处理后通过差异标记的TLR 4分子的共IP检测TLR 4二聚化，（3）通过酵母双杂交系统和亲和纯化/ MALDI-reTOR质谱鉴定单体或二聚体TLR 4相互作用蛋白，（4）通过co-IP检测Hsp 90与TLR 4复合物之间的相互作用，并检查格尔德霉素对TLR 4、MyD 88和IRAK的半衰期的影响。 这些研究应该丰富和完善我们目前对微生物产品如何启动宿主反应的理解。

Bacterial lipopolysaccharide (LPS) is a major factor responsible for toxic manifestations of systemic inflammatory response syndrome (SIRS), reported to cause 100,000 deaths annually in the US. Recently Toll like receptor (TLR) 4 was identified as the murine lps gene product; a missense mutation in its cytoplasmic tail leads to an LSP- hyporesponsive phenotype in C3H/HeJ mice. Critical roles for TLR4 and its adaptor protein MyD88 in LPS signaling are starting to emerge, yet the molecular events involved remain poorly understood. We hypothesize that TLR4 may be activated by a process other than direct binding of LPS; that the response to LPS involves the oligomerization of TLR4 and the association of TLR4 with other proteins in additional to MyD88; and that Hsp90 binds the TLR4 activation complex and participates in its signaling. To test these hypotheses, we have generated a panel of immortalized macrophage cells lines bearing either normal, mutated or deleted TLR4 or MyD88 genes. Using these cells lines, we will generate stable transfectants expressing either intact or altered versions of TLR4 or MyD88. The experimental approaches include (1) comparison of the LPS or Taxol responses between these stable cell lines to define the role of the putative domains of TLR4 and MyD88, (2) detection of TLR4 dimerization by co-IP of differentially tagged-TLR4 molecules after LPS/Taxol treatment, (3) identification of monomeric or dimeric TLR4 interacting proteins by the yeast two- hybrid system and affinity purification/ MALDI-reTOR mass spectrometry, and (4) detection of interactions between Hsp90 and TLR4 complex by co-IP and examination of the effect of geldanamycin on the half-lives or TLR4, MyD88 and IRAK. These studies should enrich and refine our current understanding of how microbial products initiate host responses.