溶酶体蛋白TMEPAI在细胞内运输调控机制与功能研究

TMEPAI (Transmembrane prostate androgen-induced protein) has been reported to be elevated in several cancers. TMEPAI expression is induced by TGF-β signaling, and in turn, expression of TMEPAI negatively regulates the TGF-β signaling, but the molecular mechanisms of TMEPAI induced TGF-β signaling inhibition are not well understood. Our preliminary studies indicated that TMEPAI is localized to the lysosome and late endosome; association of TMEPAI with the E3 ubiquitin ligase Nedd4 is required for its transport to the lysosome; TMEPAI interacts with the Dynactin pointed end complex subunit protein Dynactin6. This project will further study the regulatory mechanism of the intracellular transport of TMEPAI and its function in the lysosome, including: 1. Identification of the ubiquitination sites of TMEPAI, and study the role of ubiquitination of TMEPAI in regulating its intracellular transport and function; 2. The role of ubiquitin-binding proteins in regulating the intracellular transport of TMEPAI; 3. Identification of the binding domain between TMEPAI and Dynactin complex, and study the role of the TMEPAI-Dynactin interaction in regulating the intracellular transport of TMEPAI and vesicle trafficking; 4. The role of TMEPAI in regulating lysosomal membrane permeabilization and autophage. These studies will reveal a novel function for TMEPAI in regulating TGF-β signaling and tumorigenesis.

前列腺跨膜蛋白TMEPAI在多种肿瘤细胞中高表达，并与TGF-β信号相关。我们前期研究表明：TMEPAI在细胞内定位于溶酶体和胞内体；TMEPAI与E3泛素连接酶Nedd4相互作用调控其在细胞内的定位；TMEPAI与细胞内微管运输相关的动力蛋白激活蛋白Dynactin6互作。本项目将在分子和细胞水平上深入研究和阐明TMEPAI在细胞内运输的调控机制和定位于溶酶体的功能，研究内容包括：1.鉴定TMEPAI泛素化位点和研究TMEPAI泛素化对其在细胞内运输和功能的作用；2.泛素结合蛋白对TMEPAI在细胞内运输的作用；3.鉴定TMEPAI与Dynactin复合体的结合位点和研究TMEPAI与Dynactin复合体互作对TMEPAI运输和囊泡运输的作用；4.TMEPAI对溶酶体通透性的影响和对细胞自噬的作用。本项目研究将有助于揭示TMEPAI调控TGF-β信号及其在肿瘤发生和发展中的作用机制。

前列腺跨膜蛋白TMEPAI在多种肿瘤细胞中高表达。我们前期研究表明：TMEPAI在细胞内定位于溶酶体和胞内体；TMEPAI与E3泛素连接酶Nedd4相互作用调控其在细胞内的定位；TMEPAI与细胞内微管运输相关的动力蛋白激活蛋白Dynactin6互作。本项目在分子和细胞水平上深入研究和阐明了TMEPAI在细胞内运输的调控机制和定位于溶酶体的功能,取得以下研究结果： .1. TMEPAI蛋白发生泛素化修饰，其C-端4个K277、K279、K281和K283赖氨酸位点是TMEPAI发生泛素化所必需的。在A549细胞中表达泛素化突变体TMEPAI-4KR，结果表明TMEPAI泛素化突变体定位到细胞膜上。研究了ESCRT系统中的泛素结合蛋白是否调节TMEPAI的运输过程，结果表明TMEPAI与Hrs，STAM相互作用，而与GGA3，Tsg101，Eap45没有相互作用。敲低Hrs和STAM表达导致TMEPAI定位到细胞膜上，这表明泛素结合蛋白Hrs，STAM调控泛素化修饰的TMEPAI向溶酶体的运输。.2. 利用酵母双杂交系统鉴定表明TMEPA1与Dynactin复合体中的Dynactin 5，Dynactin 6相互作用, 但不能与Dynactin 4, Arp11相互作用, TMEPAI与Dynactin 5, Dynactin 6的结合区域在aa 132-155。在A549细胞中表达突变体TMEPAI (Δ132-155)主要定位在细胞膜上，表明突变体TMEPAI (Δ132-155)由于不与Dynactin 5，Dynactin 6结合，导致其不能被正常运输到溶酶体。 .3. 敲低A549细胞中TMEPAI表达导致溶酶体稳定性降低，使溶酶体发生通透化，并导致组织蛋白酶释放到细胞质中。敲低TMEPAI表达会降低LC3-II的蛋白水平和抑制自噬体的形成。.在本项目研究的基础上，我们也深入研究了调控TMEPAI表达的分子机制，发现转录因子Sp1促进肿瘤细胞中TMEPAI的表达并促进肿瘤细胞增殖。以此为研究基础，我们筛选到抑制TMEPAI表达和肿瘤细胞增殖的小分子化合物。