Molecular Responses of Macrophages-- Lipopolysaccharides

细菌脂多糖(LPS)是导致全身炎症反应综合征(SIRS)中毒表现的主要因素，据报道，在美国每年导致10万人死亡。最近，Toll样受体(TLR)4被鉴定为小鼠LPs基因产物，其胞浆尾部的错义突变导致C3H/HeJ小鼠LSP低反应表型。TLR4及其接头蛋白MyD88在脂多糖信号转导中的关键作用已经开始显现，但涉及的分子事件仍然知之甚少。我们假设TLR4可能是通过与内毒素直接结合以外的其他过程激活的；对内毒素的反应涉及TLR4的寡聚以及TLR4与MyD88以外的其他蛋白质的结合；Hsp90结合TLR4激活复合体并参与其信号转导。为了验证这些假设，我们产生了一组永生巨噬细胞系，它们携带正常、突变或缺失的TLR4或MyD88基因。使用这些细胞系，我们将产生稳定的转染体，表达完整或改变版本的TLR4或MyD88。这些实验方法包括(1)比较这些稳定的细胞系之间的内毒素或紫杉醇反应，以确定TLR4和MyD88结构域的作用，(2)通过差异标记的TLR4分子在脂多糖/紫杉醇处理后的共IP检测TLR4的二聚化，(3)通过酵母双杂交系统和亲和纯化/MALDI-reTOR质谱仪鉴定TLR4相互作用的单体或二聚体，以及(4)通过共IP检测Hsp90与TLR4复合体的相互作用，并检测格尔达霉素对TLR4、MyD88和IRAK半衰期的影响。这些研究应该会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.