Molecular Responses of Macrophages-- Lipopolysaccharides

巨噬细胞的分子反应——脂多糖

• 批准号: 6326408
• 负责人: Aihao Ding
• 金额: $ 36.23万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6326408
• 关键词: biological signal transduction; cell line; crosslink; cytokine receptors; enzyme linked immunosorbent assay; flow cytometry; gel mobility shift assay; gene expression; glycoproteins; heat shock proteins; immunoprecipitation; interleukin 1; leukocyte activation /transformation; lipopolysaccharides; macrophage; mass spectrometry; membrane proteins; mitogen activated protein kinase; nuclear factor kappa beta; paclitaxel; protein protein interaction; protein purification; protein structure function; western blottings; yeast two hybrid system

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.

细菌脂多糖（LPS）是导致系统性炎症反应综合征（SIRS）毒性表现的主要因素，据报道，SIRS在美国每年导致10万人死亡。近年来，Toll样受体（TLR） 4被鉴定为小鼠lps基因产物；在C3H/HeJ小鼠中，其细胞质尾部的错义突变导致LSP-低反应表型。TLR4及其接头蛋白MyD88在LPS信号传导中的关键作用开始显现，但所涉及的分子事件仍然知之甚少。我们假设TLR4可能通过LPS直接结合以外的其他过程被激活；对LPS的反应涉及TLR4的寡聚化以及TLR4与MyD88以外的其他蛋白的结合；Hsp90结合TLR4激活复合物并参与其信号传导。为了验证这些假设，我们生成了一组携带正常、突变或缺失TLR4或MyD88基因的永生化巨噬细胞系。利用这些细胞系，我们将产生稳定的表达完整或改变版本的TLR4或MyD88的转染物。实验方法包括：(1)比较这些稳定细胞系对LPS或Taxol的反应，以确定TLR4和MyD88的假设结构域的作用；(2)通过LPS/Taxol处理后差异标记的TLR4分子的共ip检测TLR4二聚体；(3)通过酵母双杂交系统和亲和纯化/ MALDI-reTOR质谱法鉴定单体或二聚体TLR4相互作用蛋白。(4)通过co-IP检测Hsp90与TLR4复合物的相互作用，检测格尔达霉素对TLR4、MyD88和IRAK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物如何启动宿主反应的理解。