Proteasome-Independent Activation of Nuclear Factor κB in Cytoplasmic Extracts from Human Endothelial Cells byRickettsia rickettsii
Proteasome-Independent Activation of Nuclear Factor κB in Cytoplasmic Extracts from Human Endothelial Cells byRickettsia rickettsii
复制标题
立克次体人内皮细胞胞浆提取物中核因子 κB 的蛋白酶体依赖性激活
作者：
S. Sahni;D. V. Van Antwerp;M. Eremeeva;D. Silverman;V. Marder;L. Sporn
摘要 已知许多传染原与真核宿主细胞的相互作用会引起普遍存在的转录因子核因子 κB (NF-κB) 的激活（U. Siebenlist、G. Franzoso 和 K. Brown, Annu. Rev. Cell Biol. 10:405–455, 1994）。最近，我们报道了培养的人脐静脉内皮细胞感染立克次体后出现 NF-κB 激活的双相模式，立克次体是一种专性细胞内革兰氏阴性细菌，也是落基山斑疹热的病原体（L. A. Sporn、S. K. Sahni、N. B. Lerner、V. J. Marder、D. J. Silverman、L. C. Turpin，和 A. L. Schwab，《感染》65：2786-2791，1997 年。在本研究中，我们描述了无细胞系统中 NF-κB 的激活，这是通过向内皮细胞胞质提取物中添加部分纯化的立氏立克次氏体来实现的。这种激活很快，在 60 分钟时达到最大水平，并且取决于 R 的数量。添加立克次氏体。使用针对 NF-κB 亚基（p50 和 p65）的单特异性抗血清进行的抗体超位移测定证实了凝胶位移复合物的真实性，并鉴定了 p50-p50 同二聚体和 p50-p65 异二聚体作为激活的 NF-κB 库的组成部分。激活的发生与内皮细胞膜的存在无关，并且不会因内皮细胞蛋白酶体的去除而受到抑制。使用肽醛蛋白酶体抑制剂 MG 132 进行的测定进一步证实了蛋白酶体的参与。激活不依赖于 ATP，因为添加过量的不可水解 ATP 类似物 ATPγS、补充外源 ATP 或用 ATP 酶水解内源 ATP 不会导致激活发生变化。此外，体外激活前后的蛋白质印迹分析未能证明丝氨酸 32 的磷酸化或 IκBα 细胞质池的降解。 IκBα 参与的缺乏得到了以下发现的支持：立氏立克次体可以诱导 T24 膀胱癌细胞和用 IκBα 超阻抑磷酸化突变体稳定转染的人胚胎成纤维细胞制备的细胞质提取物中的 NF-κB 活化，从而使 NF-κB 无法被许多已知信号失活。因此，为潜在的新型 NF-κB 激活途径提供了证据，其中立氏立克次体可能与宿主细胞转录机制相互作用并激活宿主细胞转录机制，而与蛋白酶体或已知信号转导途径的参与无关。
ABSTRACT Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor κB (NF-κB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405–455, 1994). Recently, we reported a biphasic pattern of NF-κB activation in cultured human umbilical vein endothelial cells consequent to infection withRickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786–2791, 1997). In the present study, we describe activation of NF-κB in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number ofR. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-κB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-κB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPγS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IκBα. This lack of IκBα involvement was supported by the finding that R. rickettsii can induce NF-κB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IκBα, rendering NF-κB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-κB activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.
培养的人内皮细胞的立克次体感染诱导组织因子表达。
影响因子：
20.3
作者：
Sporn,LA;Haidaris,PJ;Shi,RJ;Nemerson,Y;Silverman,DJ;Marder,VJ
通讯作者：
Marder,VJ
DOI：
10.1073/pnas.90.16.7436
发表时间：
1993-08-15
影响因子：
11.1
作者：
HEGDE, AN;GOLDBERG, AL;SCHWARTZ, JH
通讯作者：
SCHWARTZ, JH
DOI：
10.1093/infdis/153.4.694
发表时间：
1986-04
期刊：
The Journal of infectious diseases
影响因子：
--
作者：
D. J. Silverman
通讯作者：
D. J. Silverman