RPS27A类泛素化修饰STBD1锚定内质网膜诱导糖自噬介导apelin-13促单核细胞-血管内皮细胞粘附

Applicant reported that apelin-13, the ligand of APJ receptor, increased monocyte (MCs) -vascular endothelial cells (ECs) adhesion mediated by autophagy to promote atherosclerosis (AS) genesis and progression. Our pre-experiments showed that apelin-13 induced glycophagy by increasing the expression of glycophagy marker starch-binding domain-containing protein 1 (STBD1) in vascular endothelial cells. Moreover, apelin-13 promoted expression of ribosomal protein S27A (RPS27A) in vascular endothelial cells. RPS27A acts as a small ubiquitin-like modifier (SUMO) protein to participate in post-transcriptional modification. Thus, we give our hypothesis that STBD1 is SUMOylated by RPS27A and then anchors to endoplasmic reticulum membrane to induce glycophagy which is essential for apelin-13 driven monocyte-vascular endothelial cells adhesion. In this study, we will use RNA interference and other methods to test whether apelin-13 promotes endothelial cells glycophagy activation or not. We will confirm that apelin-13 promotes RPS27A expression in human umbilical vein endothelial cells. And, we will perform studies to explore that RPS27A SUMOlates STBD1 to promote STBD1 anchoring endoplasmic reticulum to induce glycophagy. In a word, we will perform assays to reveal that glycophagy mediates monocyte-vascular endothelial cells adhesion induced by apelin-13. This study will reveal the new mechanism that apelin-13 promotes monocyte-vascular endothelial cell adhesion via glycophagy. Apelin/APJ is expected to be a new target for atherosclerosis therapy.

申请者报道自噬介导APJ受体的配体apelin-13促单核细胞-血管内皮细胞粘附，促动脉粥样硬化（AS）发生发展。预实验发现apelin-13诱导血管内皮细胞糖自噬发生，上调糖自噬标志蛋白STBD1表达；促进核糖体蛋白RPS27A表达，RPS27A具有类泛素化修饰底物蛋白功能，据此提出“RPS27A类泛素化修饰STBD1锚定内质网膜诱导糖自噬介导apelin-13促单核细胞-血管内皮细胞粘附”的新假说。课题拟采用RNA干扰等方法，探讨apelin-13是否诱导血管内皮细胞糖自噬发生；明确apelin-13是否促血管内皮细胞RPS27A表达；分析RPS27A是否类泛素化修饰STBD1锚定内质网膜参与糖自噬；阐明糖自噬是否介导apelin-13促单核细胞-血管内皮细胞粘附。课题将揭示糖自噬介导apelin-13促单核细胞-血管内皮细胞粘附的新机制。Apelin/APJ有望成为抗AS治疗新靶点。

课题从核糖体蛋白S27a诱导糖自噬这一新视角出发，证实了apelin-13/APJ系统促单核细胞-血管内皮细胞粘附过程，为探索动脉粥样硬化发生发展的分子机制提供了理论依据。研究发现：1.Apelin-13通过APJ受体上调STBD1、GABARAPL1、GAA和GDE蛋白水平。2.Apelin-13通过APJ受体促进人脐静脉内皮细胞STBD1与LC3B荧光共定位，诱导内质网发生糖自噬，降低细胞内糖原含量。3.Apelin-13通过APJ受体诱导人脐静脉内皮细胞RPS27a的表达。4.沉默RPS27a抑制Apelin-13诱导的人脐静脉内皮细胞STBD1、GABARAPL1、Beclin-1和LC3B的表达；相反，高表达RPS27a促进以上蛋白表达。5.沉默RPS27a或STBD1抑制Apelin-13诱导的人脐静脉内皮细胞糖自噬，增加细胞糖原含量，而高表达RPS27a或STBD1促人脐静脉内皮细胞糖自噬，降低细胞糖原含量。6.Apelin-13促RPS27a与STBD1的共定位。7.沉默RPS27a后，Apelin-13诱导人脐静脉内皮细胞内质网糖自噬水平下降。8.点突变STBD1 184位赖氨酸抑制了Apelin-13诱导的人脐静脉内皮细胞STBD1、GABARAPL1、Beclin-1和LC3B的表达。9.Apelin-13/APJ系统促人脐静脉内皮细胞ICAM-1和VCAM-1的表达，促单核细胞-人脐静脉内皮细胞粘附。SiRNA-RPS27a和SiRNA-STBD1均抑制Apelin-13诱导的人脐静脉内皮细胞ICAM-1、VCAM-1的表达，同时抑制Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附。过表达RPS27a和STBD1均促进Apelin-13诱导的人脐静脉内皮细胞 ICAM-1、VCAM-1的表达。10.点突变STBD1184位赖氨酸抑制Apelin-13诱导的人脐静脉内皮细胞ICAM-1、VCAM-1的表达，同时抑制Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附。以上结果揭示了RPS27A类泛素化修饰STBD1锚定内质网膜诱导糖自噬介导apelin-13促单核细胞-血管内皮细胞粘附的新机制，为apelin/APJ系统成为抗AS治疗新靶点提供理论依据。