TRIB1 Is Regulated Post-Transcriptionally by Proteasomal and Non-Proteasomal Pathways

The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCPβ did not play a role in mediating TRIB1 instability although TRCPβ suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level.

TRIB1基因与多种恶性肿瘤、血浆甘油三酯代谢异常及冠状动脉疾病（CAD）密切相关。尽管该假激酶（pseudo-kinase）具有重要临床意义，但目前关于TRIB1的调控机制研究仍较为匮乏。既往研究显示TRIB1 mRNA稳定性较差，提示TRIB1可能受到转录后调控（post-transcriptional regulation）。本研究围绕TRIB1的调控机制展开探索，重点聚焦其转录后调控环节。
在三种不同的模型系统（HEK293T细胞、HeLa细胞及动脉平滑肌细胞）中，通过蛋白质印迹（Western blot）检测未能检出TRIB1蛋白。以重组TRIB1为实验替代物，我们证实TRIB1在蛋白质及RNA水平均呈现高度不稳定性。与之相反，重组TRIB1在细胞裂解液中表现出良好的稳定性。
阻断蛋白酶体（proteasome）功能可使TRIB1蛋白的稳态水平升高，但无法挽救其蛋白不稳定性，这表明TRIB1的蛋白降解与稳态维持是两个相互解偶联的过程。与针对TRIB2的相关研究结果不同，CUL1及TRCPβ并未参与介导TRIB1的蛋白不稳定性，尽管抑制TRCPβ可提升TRIB1的表达水平。最后，我们证实TRIB1的蛋白不稳定性与其亚细胞定位（subcellular localization）无关。在鉴定出TRIB1的核定位信号（nuclear localization signal）后，我们构建了仅定位于胞质的TRIB1突变体。尽管该突变体被限制在胞质中，TRIB1仍表现出不稳定性，这提示其不稳定性发生于核转位及下游核功能发挥之前的阶段。
本研究通过识别两条独立的转录后调控轴，揭示了可用于调控TRIB1功能的潜在干预途径。