TRx/TBP2在晶状体上皮细胞自噬中的作用及调控机制

The redox regulator thioredoxin (TRx) has multiple physiological functions in cells and the TRx activity is negatively controlled by thioredoxin binding protein 2 (TBP2). Previously, we have found that overexpression of TBP2 increased oxidation sensitivity and apoptosis through TRx-ASK1-Bax pathway in human lens epithelial cells (LECs). Interestingly, low intensity hydrogen peroxide (H2O2) did not affect the cell viability, but significantly induced autophagy which regulated by TBP2. To elucidate the role of TRx/TBP2 in regulation of autophagy and its underlying mechanism, this project will 1) optimize the treatment conditions of H2O2 to establish low level oxidative stress induced LECs autophagy model with helps of transmission electron microscopy (TEM), cellular immunofluorescence staining (IF) and Western blot (WB) and the effect of this oxidative stress on TRx/TBP2 protein expression, subcellular localization and TBP2-TRx protein interaction by WB, IF and co-immunoprecipitation (Co-IP) respectively; 2) investigate the role of TRx/TBP2 in low level oxidative stress induced LECs autophagy with TBP2 or TRx over-expression stable LECs and TBP2 or TRx stable knocking-down LECs, and examine the role of TBP2-TRx protein interaction in the autophagy with dominant TBP2 mutant (TRx binding domain) LECs; 3) determine types of the TRx/TBP2 mediated low level oxidative stress induced LECs autophagy (i.e., macroautophagy, microautophagy, chaperone- mediated autophagy) by TEM and the signaling pathways involved (i.e., canonical, non-nanonical, or common pathway) by WB; 4) identify the protein-protein disulfide (PSSP) in TRx regulating autophagy by mass spectrum (MS), and synthesize the TRx regulated PSSP by in vitro biochemical assay and evaluate their effects on autophagy. This study will describe the role of TRx/TBP2 in autophagy, cast light on the molecular mechanisms of LECs' response to low level oxidative stress, and identify PSSP relating autophagy. These expected findings will provide new therapeutic strategies for cataract by targeting oxidative stress, and will be beneficial to the aging population and communities.

本课题组曾首次报道硫氧还蛋白（TRx）对晶状体上皮细胞（LECs）具有抗氧化的保护作用；TRx结合蛋白2（TBP2）负性调节TRx活性并介导高水平氧化应激诱导的细胞凋亡；而低浓度的氧化刺激未能诱导细胞活性明显降低，但自噬水平显著上升，且受TBP2的调节。为深入研究，本项目拟通过建立低浓度氧化刺激诱导的LECs自噬模型，利用Western Blot、过表达质粒载体、干扰RNA、细胞质线粒体分离等技术首次探讨TRx/TBP2在LECs自噬中的调节作用及机制；同时采用RP-HPLC-MS/MS质谱分析及鉴定，寻找受TRx调控并引发自噬的特异性PSSP，为细胞自噬的启动提供解释。本项目预期阐明TRx/TBP2调控LECs自噬的机理，揭示低水平氧化刺激下LECs进行自我防御和保护的分子机制，确定具有自噬诱导特异性的PSSP。有望为白内障的靶向防治研究提供理论依据，抑制和延缓年龄相关性白内障的发病。

本课题组曾首次报道硫氧还蛋白（Trx）对晶状体上皮细胞（LECs）具有抗氧化的保护作用；Trx 结合蛋白2（TBP-2）负性调节Trx 活性并介导高水平氧化应激诱导的细胞凋亡；而低浓度的氧化刺激未能诱导细胞活性明显降低，但自噬水平显著上升，且受TBP-2的调节。. 为深入研究，本项目以人晶状体上皮细胞为研究模型，分析Trx/TBP-2在调节人晶状体上皮细胞（HLEC-B3）响应外界氧化损伤导致自噬中的作用，并进一步分析Trx/TBP-2 调控自噬相关的发生机制。本课题组应用western blot、qRT-PCR、免疫荧光、透射电镜、CCK-8、RP-HPLC-MS/MS 质谱分析等技术和方法深入研究发现：.(1)通过不同浓度的H2O2处理人晶状体上皮细胞24 小时，发现随着浓度增加，细胞活性的丧失增加，选用居中的处理浓度，进行时间梯度的处理后，发现随着时间的增加，细胞活性随之下降，并通过Western blot、GFP-LC3 点聚集实验、透射电镜等验证了在50μM的H2O2处理细胞12h时，细胞会发生自噬。(2)在该自噬条件下，TBP-2、Trx-2表达量明显升高，而Trx-1表达无变化。在该过程中，Trx-1与TBP-2共定位更明显，Trx-2与TBP-2共定位减少。间接说明TBP-2主要与Trx-1结合减低Trx-1的活性来调节自噬，而非Trx-2。(3)通过建立过表达TBP-2稳转细胞株以及设计针对TBP-2 的特异性siRNA瞬时敲减TBP-2，发现TBP-2 能够促进人晶状体上皮细胞在氧化应激下发生自噬，活性丧失更多； (4)探索TBP-2涉及的机制发现TBP-2 对人类晶状体上皮细胞在氧化应激下自噬调控主要发生在自噬的启动阶段，通过抑制Akt 的磷酸化来影响HLEC-B3 在氧化应激下的自噬，并且是通过非mTOR 途径起效应的。(5)通过建立过表达Trx-1稳转细胞株的建立，发现过表达Trx-1能够增加细胞自噬，但是该自噬对细胞具有保护作用。. 本项目阐明Trx/TBP-2在晶状体上皮细胞响应外界氧化损伤、抗氧化和调节细胞自噬功能的作用及其分子机制，有助于深入探究晶状体氧化损伤的防御机制和白内障的发病机制，为白内障防治的研究提供新思路。