寡肽/组氨酸转运体介导细菌肽聚糖降解产物跨溶酶体转运致炎症性肠病研究

Inflammatory bowel disease ( IBD) is a nonspecific intestinal inflammatory disease, the underlying basis of the pathogenesis is not yet clear but may involve persistent bacterial infection, a defective mucosal barrier, or an imbalance in the regulation of immune response. Due to the contribution of NODs to the risk of IBD, it was deduced that degradation products of peptidoglycan of bacteria cell wall, such as muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-meso-diaminopimelic acid (tri-DAP) , transported into cytosol, recognized by NODs and subsequently activated the inflammatory response. However, the mechanism of MDP, tri-DAP transport has not been clarified. Proton-coupled oligopeptide transporters (POTs) can transport the di-/tri-peptides and mimic peptide compounds, due to the similarity of the structures of MDP and tri-DAP, it implies that they might be transported by POTs. Our previous study showed that the distal colon and lamina propia mononuclear cells (LPMCs) of the colon from normal mice expressed oligopeptide and histidine transporter 1 and 2 (PHT1 and PHT2) protein, and the PHT2 was significantly up-regulated in IBD mice, but not expressed PEPT1 and PEPT2 proteins. Since PHT1 and PHT2 are localized on lysosomal membrane but not cellular membrane, our hypothesis is that PHT1 and PHT2 may transport MDP, tri-DAP from lysosomes to cytosol, after the bacteria was endocytosed by immune cells and degraded in lysosomes, and subsequently interact with NODs. The present proposal will emply normal/IBD mice, transfected cells/mice and clinical samples, to delineate the protein expression of PEPT1/2 and PHT1/2 in mouse/human colonic tissues and immune cells, to elucidate the role of PHT1/2 in the transport of MDP, tri-DAP, and in the mechanism of IBD. The study will provide the useful information for elucidation of IBD mechanism and discovery of new drug target.

IBD是一组病因不十分明确的非特异性肠道炎症性疾病，无有效治疗措施。IBD与细胞质NODs密切相关，推测细菌肽聚糖降价产物MDP/tri-DAP进入细胞质，被NODs识别，激活炎症反应。但MDP/DAP进入细胞质机制不清。寡肽转运体家族(POTs)可转运二/三肽及拟肽物质，而MDP/DAP结构与POTs底物类似。我们的研究显示，小鼠远端结肠及结肠粘膜固有层单个核细胞寡肽/组氨酸转运体1，2（PHT1/2）蛋白表达高，且IBD时PHT2显著上调。基于PHT1/2位于溶酶体膜，推测细菌被肠道免疫细胞内吞后，在溶酶体内消化，肽聚糖降解产物MDP/DAP经PHT1/2转运入细胞质，与NODs作用。本课题拟在前期研究基础上，应用正常/IBD小鼠、转基因细胞/小鼠以及临床样本，考察PHT1/2在肠道及免疫细胞中的表达，探究其在MDP/DAP转运及IBD中的作用，为阐明IBD发病机制、靶点发现提供依据。

IBD是一组病因不十分明确的非特异性肠道炎症性疾病，目前无有效治疗措施。已发现IBD与细胞质NODs密切相关，故推测溶酶体内的细菌肽聚糖降价产物MDP/tri-DAP进入细胞质，被NODs识别，激活炎症反应。因MDP/tri-DAP为拟肽类物质，而寡肽转运体家族（POTs）可介导二/三肽及拟肽物质的跨膜转运，且PHT2定位于溶酶体膜。因此，探究PHT2在介导MDP/DAP跨溶酶体膜的转运以及与IBD的关系，对阐明其作为潜在的IBD治疗靶点具有重要意义。本项目阐明了小鼠肠道中寡肽转运体的表达，发现了DSS诱导的溃疡性结肠炎小鼠结肠中PHT2表达（mRNA及蛋白水平）显著高于正常小鼠结肠，临床IBD患者的结肠中也发现了同样的现象（mRNA 水平）；应用基因突变及重组表达技术，构建将人源PHT1、PHT2（hPHT1、hPHT2）稳定表达于细胞膜的转基因细胞模型，并应用上述模型，阐明了MDP、tri-DAP的底物特性，筛选获得了一系列hPHT1、hPHT2底物/抑制剂，获得了hPHT1、hPHT2经典底物Gly-sar、组氨酸的摄取动力学参数。此外，我们从基因及蛋白水平阐明了巨噬细胞中PHT2的表达可被TLR2、TLR4、TLR7和TLR9配体，经NF-κB、MAPK和IRF3通路上调；应用基因沉默/过表达技术，发现了PHT2在TLR4配体LPS诱导IL-6和TNF-α生成中的抑制/促进作用。上述研究提示，PHT2/1可介导MDP/tri-DAP由溶酶体转运至细胞质，IBD的发生发展与PHT2的表达水平相关，PHT2在IBD及炎症发生发展中可能具有重要作用。