生物大分子穿膜常需伴侣蛋白协助,但后者具体作用机制不明。我们前期发现肿瘤细胞线粒体伴侣蛋白GRP75在Cdc42激活内吞中富集并有调节功能。已知Cdc42 激活受Dbl样RhoGEF催化且后者原癌蛋白受Hsc70介导呈现自抑制。我们后续发现GRP75不与HSPG、Cdc42、Dbl相互作用，只与proto-Dbl结合且位于Dbl上游发挥调节。故提出内吞新调节轴:GRP75-Dbl。本项目拟(1)分析二者调节内吞和相互作用的功能结构域,(2)明确GRP75对proto-Dbl泛素化、成熟和 GEF活性影响,(3)探明GRP75与Hsc70间竞争关系,(4)检测二者亚细胞转位、修饰变化和富集来源，最终阐明GRP75富集通过竞争释放Hsc70对proto-Dbl抑制而激活Cdc42促进大分子内吞机制。这可为Cdc42激活内吞途径上游调节提出新认识,为优化HSPG依赖大分子药物内化递送提供实验支撑

Chaperones are universally recognized for roles in facilitating transmembrane endocytosis of macromolecules. However, underlying mechanism for this process, especially for HSPG-dependent macromolecular drug endocytosis, is unclear. We had showed noticeable enrichment of one chaperone protein, GRP75, which often locates inside mitochondrion, in components of endocytic vesicles of tumor cells. We further provided evidences for its regulatory role in Cdc42 activation-dependent endocytosis of macromolecular drugs such as delivered antibody-Fe nanoparticle, Tat/DNA etc., In tumor cells, it is documented activation of Cdc42 GTPase is dominantly catalyzed by upstream Dbl guanine nucleotide exchange factor (GEF), which proto-form-oncoprotein maintaining an autoinhibitory state by Hsc70-mediated intramolecular attenuation in unstimulated cell. Meanwhile, by direct or indirect protein-protein interaction, translocated GRP75 protein has been implied to facilitate receptor internalization, vesicles/membrane trafficking in different cells. Despite existing some blurry colocalization by cofocal imaging, our co-IP data further showed that GRP75 do not bind to HSPG/HS, GTP/GDP-CDC42 and Dbl, but selectively bind to proto-Dbl protein both in vitro and in vivo. In addition, our further data showed GRP75 acts upstream of Dbl on Cdc42 activation and macromolecule drug endocytosis. Based on these lines, an endocytosis-regulatory axis is emerging, and we reason here macromolecular drug endocytosis stimulates enrichment of GRP75, which promotes Cdc42 activation-dependent endocytosis by competitively release of Hsc70 from autoinhibitory proto-Dbl protein. Therefore, we propose to focus research topic on endocytosis regulatory role of GRP75-proto-Dbl interaction. One series experiments are designed to pursuit functional domain(s) determining Cdc42 activation and macromolecular drug endocytosis. another series experiments aim to find key domain(s) mediate GRP75-proto-Dbl functional interaction and downstream changes of Cdc42 activation level correspondingly. Stem experiments are designed to check the competition relationship to Proto-Dbl protein between GRP75 and Hsc70, and to detect ubiquitination and degradation changes of proto-Dbl by overexpression and down-regulation/interference of GRP75. Last but not least, certain experiments are settled to check subcellular location, dynamic translocation and cell surface enrichment of GRP75 and Dbl protein. Combined with these data, it would uncover endocytosis regulatory mechanism of GRP75 and intermolecular functional association of GRP75-Dbl axis. Once approved and done, it would challenge traditional knowledge on chaperone protein, present novel insight into upstream regulation of Cdc42 activation-dependent endocytosis. It will also be great benefited for optimization of HSPG-dependent macromolecular drug endocytic delivery strategy.