乙酰化修饰在大肠癌细胞GRP78蛋白分泌中的调控作用

Glucose regulated protein 78 (GRP78) has a signal peptide sequence which targets it to the endoplasmic reticulum (ER) as a molecular chaperone. GRP78 is an important functional factor regulated by the tumor microenvironment, and contributes to the formation of several cancer hallmarks. Besides being located in the ER, GRP78 is also found to be present in the cell plasma membrane, cytoplasm, mitochondria, nucleus as well as cellular secretions of tumor cells. However, the secretory pathway of GRP78 and the regulatory molecular mechanism in its secretory process are currently unknown. Our present experimental data indicates that colorectal cancer (CRC) cells can secrete GRP78 protein; Sodium butyrate, an HDAC inhibitor, can induce total GRP78 expression but block its extracellular secretion. In addition, the sodium butyrate treatment promotes the acetylated modification of GRP78. Based on these findings, we speculate that acetylated modification is involved in regulating GRP78 secretion in CRC cells. In this project, we are going to determine the secretory forms of GRP78 in the conditioned medium from CRC cells and in CRC patient samples, identifying the secretory pathway of GRP78. Moreover, the roles of acetylated modification in GRP78's cellular localization and secretion will be investigated using the cellular differentiation model by sodium butyrate treatment. This project will provide new clues for GRP78-based tumor diagnosis and treatment, and has important theoretical significances and application value.

葡萄糖调节蛋白78（GRP78）是位于内质网中的重要分子伴侣，它在肿瘤细胞中的定位呈多样化，并促进肿瘤细胞恶性特征的形成。最近发现，GRP78可以被某些肿瘤细胞分泌到细胞外，但其分泌途径和分泌过程的分子调控机制尚不清楚。本课题组前期研究发现，大肠癌细胞也可以分泌GRP78；丁酸钠处理能够阻断GRP78的细胞外分泌过程，并提高细胞内GRP78的乙酰化修饰水平，表明乙酰化修饰可能参与调控GRP78的细胞外分泌过程。本项目拟研究肠癌细胞条件培养液、肠癌病人样本中GRP78是以可溶性蛋白、Exosome还是Microparticle的形式存在，根据其分泌形式确定GRP78的分泌途径。进一步以丁酸钠诱导肠癌细胞分化为细胞模型，研究乙酰化修饰对GRP78细胞定位和分泌的影响，并确定发挥效应的乙酰化修饰位点，为以GRP78为靶点的肿瘤诊断、治疗提供新的思路，具有重要的理论意义和应用价值。

葡萄糖调节蛋白78（GRP78）是位于内质网中的重要分子伴侣，它在肿瘤细胞中的定位呈多样化。GRP78 的表达受肿瘤微环境的调控，作为肿瘤微环境的“感应因子”，参与肿瘤细胞恶性特征的形成，因此GRP78蛋白可以作为肿瘤治疗的分子靶点。最近发现，GRP78还可以被肿瘤细胞分泌到细胞外微环境中，可以作为肿瘤细胞与间质细胞间的通讯因子。肿瘤细胞中GRP78的细胞外分泌途径和具体的分子调控机制还不清楚。我们研究发现，结肠癌细胞能够以外泌体（exosome）的形式分泌GRP78蛋白；组蛋白脱乙酰化酶（HDAC）抑制剂诱导GRP78在内质网中聚集，从而阻断其细胞外的分泌过程。HDAC抑制剂通过抑制HDAC6活性增加GRP78蛋白的乙酰化水平。乙酰化修饰的GRP78蛋白与细胞内III型PI3K激酶VPS34相互作用形成蛋白复合物，从而抑制GRP78进入多泡内体（MVB）。我们进一步将GRP78上HDAC6的去乙酰化位点（K633）突变为谷氨酰胺（K633Q）,发现K633Q突变体能够明显导致GRP78分泌减少，并抑制裸鼠移植肿瘤的生长。综上所述，我们发现GRP78能够以exosome的形式分泌到细胞外环境发挥细胞通讯作用，并且分泌过程受到乙酰化修饰的调控。本研究能够为HDAC抑制剂的临床应用提供重要参考。