Nuclear translocation of IFN-γ is an intrinsic requirement for its biologic activity and can be driven by a heterologous nuclear localization sequence
Nuclear translocation of IFN-γ is an intrinsic requirement for its biologic activity and can be driven by a heterologous nuclear localization sequence
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DOI：
10.1089/107999001753289569
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发表时间：
2001-11-01
影响因子：
2.3
通讯作者：
Johnson, HM
中科院分区：
文献类型：
--
作者：
Subramaniam, PS;Green, MM;Johnson, HM
我们以前已经确定了一个核定位序列（NLS）的干扰素-γ（IFN-γ）。该NLS通过与其转录因子Stat 1 α和Stat 1 α的核输入物（输入素α类似物NPI-1）形成复合物而在细胞内发挥功能。该复合物的稳定性和随后的复合的Stat 1 α的核转位依赖于该NLS的完整性，表明Stat 1 α核输入由IFN-γ NLS介导。在这项研究中，为了直接评估核IFN-γ对其生物活性的内在要求，我们设计了一种嵌合体，其中IFN-γ NLS已被异源NLS取代，即SV 40大T抗原的原型NLS，其将以序列非特异性方式驱动IFN-γ的核转位。嵌合的IFN-gamma -SV在抗病毒和抗增殖试验中与野生型IFN-gamma具有同等活性。有趣的是，IFN-γ-SV也易位到细胞核，并且也作为与Stat 1 α输入者NPI-1的复合物在细胞内回收，就像野生型IFN-γ一样。与NLS缺失突变体的比较表明，IFN-γ的NLS基序内的缺失或变化对IFN-γ受体复合物的高亲和力细胞外结合是无关紧要的，然而NLS的存在对IFN-γ的生物活性及其NPI-1络合能力的表达是至关重要的。我们的数据最终表明，IFN-γ的核转位是IFN-γ的生物活性的充分表达的内在要求，并加强了这样的结论，即核伴侣的Stat 1 α是IFN-γ核转位的主要作用。这种类型的配体印迹通过螯合活化的Stat可能有助于Stat核转录的特异性。
We have previously identified a nuclear localization sequence (NLS) in interferon-gamma (IFN-gamma). This NLS functions intracellularly by forming a complex with its transcription factor Stat1 alpha and the nuclear importer of Stat1 alpha, the importin-alpha analog NPI-1. The stability of this complex and the subsequent nuclear translocation of the complexed Stat1 alpha are dependent on the integrity of this NLS, showing that Stat1 alpha nuclear import is mediated by the IFN-gamma NLS. In this study, to directly evaluate the intrinsic requirement of nuclear IFN-gamma toward its biologic activities, we engineered a chimeric in which the IFN-gamma NLS has been substituted by a heterologous NLS, namely, the prototypical NLS of the SV40 large T antigen, which would drive nuclear translocation of IFN-gamma in a sequence-nonspecific manner. The chimeric, IFN-gamma -SV, was equally active in antiviral and antiproliferative assays as the wild-type IFN-gamma. Interestingly, IFN-gamma -SV was also translocated to the nucleus and was also recovered intracellularly as a complex with the Stat1 alpha importer NPI-1, like wild-type IFN-gamma. Comparison with an NLS deletion mutant showed that deletion or changes within the NLS motif of IFN-gamma were inconsequential to the high-affinity extracellular binding to the IFN-gamma receptor complex, yet the presence of an NLS was critical to the expression of the biologic activities of IFN-gamma and its NPI-1 complexation ability. Our data conclusively demonstrate that nuclear translocation of IFN-gamma is an intrinsic requirement for the full expression of the biologic activities of IFN-gamma and strengthen the conclusion that nuclear chaperoning of Stat1 alpha is the primary role of IFN-gamma nuclear translocation. This type of ligand imprinting by sequestering of activated Stat may contribute to the specificity of Stat nuclear transcription.