Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself
Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself
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DOI：
10.1186/1476-9255-9-11
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发表时间：
2012-03-26
影响因子：
5.1
通讯作者：
Hall, J. Perry
中科院分区：
文献类型：
--
作者：
Luong, Michael;Zhang, Yanyu;Hall, J. Perry
背景：Toll样受体4（TLR 4）被细菌内毒素激活，是一种病原体相关分子模式（PAMP）。已经表明TLR 4也可以被损伤相关分子模式（DAMP）蛋白如HSP 70激活。它仍然是一个挑战，提供明确的证据表明DAMP蛋白本身发挥作用，在TLR 4激活，作为DAMP蛋白使用的是经常被污染的内毒素和其他TLR配体引入蛋白质表达和/或purification.Results：在这里，我们报告说，TLR 4的激活对原代人巨噬细胞培养物重组HSP 70是不是仅仅由于污染的内毒素。多粘菌素B预处理HSP 70制剂以中和污染的内毒素，导致重组蛋白诱导的TNF-α的量显著减少。然而，消化的HSP 70与蛋白酶K-琼脂糖珠也显着降低了TNF-α的巨噬细胞对HSP 70的反应，而留下的污染内毒素水平基本上不变相对于controls.Conclusions：这些结果表明，重组HSP 70的刺激作用需要的热休克蛋白本身的内毒素和结构完整性的存在。
Background: Toll-like receptor 4 ( TLR4) is activated by bacterial endotoxin, a pathogen-associated molecular pattern ( PAMP). It has been suggested that TLR4 can also be activated by damage-associated molecular pattern ( DAMP) proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification.Results: Here we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-alpha induced by the recombinant protein. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-alpha response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls.Conclusions: These results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.