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Intracellular Stability of Precursor IL-33 is Maintained through Interaction with Importin-5

机译：通过与Importin-5相互作用维持前体IL-33的细胞内稳定性

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摘要

Interleukin (IL)-33 is a member of the IL-1 family and a potent regulator of immunity. The precursor, or full-length (FLIL33), form behaves differently from the mature (MIL33) cytokine. MIL33 is a potent pro-Th2 cytokine acting through the T1/ST2 receptor, whereas FLIL33 is constitutively expressed and resides predominantly in the nucleus of epithelial and endothelial cells, as well as fibroblasts. Increasing evidence suggests that this intracellular FLIL33 can mediate inflammatory responses and wound healing in a non-Th2 and receptor-independent manner. Additionally, nuclear retention of FLIL33 is essential to maintain homeostasis and prevent unintentional sterile inflammation. FLIL33 has been shown to bind histones and chromatin, but the mechanism of nuclear localization remains elusive. Though FLIL33 contains a conserved bipartite nuclear localization sequence (NLS), this is not required for nuclear localization.;Importins are a family of proteins that act as chaperones for import of proteins through the nuclear pore complex (NPC). Here, importin-5 (IPO5) is identified as a unique intracellular binding partner of IL-33 based on co-immunoprecipitation of HA-tagged IL-33 from normal human lung fibroblasts (NHLF). It was hypothesized that IPO5 binds the N-terminal domain of FLIL33 and that nuclear localization of FLIL33 is dependent on IPO5. To test this hypothesis, HA-tagged FLIL33 and HA-tagged peptide fragments of FLIL33 were expressed in cell culture and immunoprecipitated. We show that FLIL33, but not MIL33, co-immunoprecipitates with IPO5 and that this interaction localizes to the N-terminal domain of FLIL33. Knockdown of IPO5 in NHLFs using RNA interference did not prevent nuclear localization of FLIL33. Instead, attenuation of IPO5 led to a significant decrease in the intracellular protein levels of overexpressed FLIL33, which was reversed by treatment of cells with bortezomib, a proteasome inhibitor. Furthermore, FLIL33 mutant proteins deficient in IPO5-binding remained intra-nuclear. These mutants also had reduced protein levels that were restored by proteasome inhibition with bortezomib. These data indicate that the interaction between FLIL33 and IPO5 localizes to specific segments of the FLIL33 protein, is not required for nuclear localization of FLIL33, and results in its protection from proteasome-dependent degradation.
机译：白介素（IL）-33是IL-1家族的成员，也是一种有效的免疫调节剂。前体或全长（FLIL33）形式的行为与成熟（MIL33）细胞因子不同。 MIL33是通过T1 / ST2受体起作用的强Th2促细胞因子，而FLIL33组成型表达，主要存在于上皮和内皮细胞以及成纤维细胞的核中。越来越多的证据表明，这种细胞内FLIL33可以非Th2和受体独立的方式介导炎症反应和伤口愈合。此外，FLIL33的核保留对于维持体内平衡和防止意外的无菌炎症至关重要。已经显示FLIL33结合组蛋白和染色质，但核定位的机制仍然难以捉摸。尽管FLIL33包含一个保守的二聚核定位序列（NLS），但对于核定位而言并不需要。导入素是一族蛋白，可作为伴侣蛋白通过核孔复合体（NPC）导入蛋白。在这里，important-5（IPO5）被鉴定为基于正常人肺成纤维细胞（NHLF）的HA标记的IL-33的共免疫沉淀，是IL-33的独特细胞内结合伴侣。据推测，IPO5结合FLIL33的N-末端结构域，并且FLIL33的核定位依赖于IPO5。为了检验该假设，在细胞培养物中表达HA-标记的FLIL33和HA-标记的FLIL33的肽片段并进行免疫沉淀。我们显示FLIL33，但不是MIL33，与IPO5共免疫沉淀，并且这种相互作用定位于FLIL33的N末端域。使用RNA干扰在NHLF中敲除IPO5并不能阻止FLIL33的核定位。取而代之的是，IPO5的衰减导致过表达的FLIL33的细胞内蛋白质水平显着下降，这可以通过用蛋白酶体抑制剂硼替佐米处理细胞来逆转。此外，缺乏IPO5结合的FLIL33突变蛋白仍保留在核内。这些突变体还具有降低的蛋白质水平，这些蛋白质水平通过硼替佐米的抑制而得以恢复。这些数据表明，FLIL33和IPO5之间的相互作用定位于FLIL33蛋白的特定片段，对于FLIL33的核定位不是必需的，并导致其免受蛋白酶体依赖性降解的影响。

著录项

  • 作者

    Clerman, Andrew.;

  • 作者单位

    University of Maryland, Baltimore.;

  • 授予单位 University of Maryland, Baltimore.;
  • 学科 Immunology.;Molecular biology.
  • 学位 Ph.D.
  • 年度 2018
  • 页码 151 p.
  • 总页数 151
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 地球物理学;
  • 关键词

  • 入库时间 2022-08-17 11:38:53

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