Relationship between periodontal disease and systemic diseases: molecular mechanisms and novel therapy discovery

为了建立牙周病与多种全身性疾病（如动脉粥样硬化、肥胖、糖尿病、早产和胎儿死亡）之间可能联系的分子基础，我们使用了多种动物模型和不同的细胞类型，并将内溶酶体天冬氨酸蛋白酶组织蛋白酶E （CatE）作为宿主衍生的蛋白酶，将牙龈蛋白酶（GP）作为牙周病源衍生的蛋白酶。在本研究中，我们首次发现CatE通过巨噬细胞中toll样受体TLR4等的适当转运和细胞表面表达，在宿主防御成人牙周病的主要病原细菌牙龈卟啉单胞菌感染中起着至关重要的作用。其次，GP通过选择性地水解LDL胆固醇中的载脂蛋白B-100，介导牙龈假单胞菌感染加速动脉粥样硬化进程。第三，GP通过增强妊娠小鼠感染牙龈卟啉卟啉菌的免疫应答，作为强毒力因子诱导早产和低出生体重。第四，通过对以高脂饮食喂养的CatE-/-小鼠作为肥胖小鼠模型的生化分析，发现CatE在脂肪组织的正常发育中起着至关重要的作用。第四，利用cDNA展示、功能选择、γ-连接阻滞洗牌等技术，合成了针对CatE和GP的肽抑制剂。新开发的抑制剂对每种酶的IC50均为nM级，具有较高的选择性。因此，该方法有望广泛应用于开发各种蛋白酶的肽抑制剂。

To establish the molecular basis of the possible linkage between periodontal diseases and a variety of systemic diseases such as atherosclerosis, obesity, diabetes, and preterm birth and fetal death, we used various animal models and different cell types and manipulated the endolysosomal aspartic proteinase cathepsin E (CatE) as a host-derived protease and gingipains (GP) as a periodontopathogen-derived protease. In this study, we first found that CatE plays a crucial role in host defense against infection with Porphyromonas gingivalis, a major etiological bacterium of adult periodontal disease through proper trafficking and cell surface expressionof Toll-like receptors such as TLR4 in macrophages. Second, GP mediates atherosclerosis progression accelerated by P. gingivalisinfection through selective proteolysis of apolipoprotein B-100 in LDL cholesterol. Third,GP acts as a strong virulence factor to induce preterm birth and low birth weight through the enhancement of immune responses to pregnant mice infected with P. gingivalis. Fourth, CatE plays a crucial role in normal development of adipose tissues through biochemical analysis of CatE-/-mice that were fed a high-fat diet as an obesity mouse model. Forth, using techniques of cDNA display, selection-by-function, γ-ligation-based block shuffling and others, we generated peptide inhibitors for CatE and GP. The newly developed inhibitors for each enzyme showed an IC50 of nM order and high selectivity. This method is thus expected to be widely applicable for the development of peptide inhibitors of various proteases.