Studies on Production of Human Salivary Type Cystatins by Genetic Engineering and their Application to Dental Medicine

这位首席研究员将重点放在了人类半胱氨酸蛋白酶的非竞争性抑制剂--半胱氨酸蛋白酶的非竞争性抑制物--半胱氨酸蛋白酶的抑制剂在本研究项目的第一阶段，开发了用于大规模生产半胱氨酸菌素S.SN和SA的大肠杆菌系统。该系统产生的工程半胱氨酸菌素被认为等同于天然半胱氨酸菌素。该体系还用于生产半胱氨酸蛋白酶S和半胱氨酸蛋白酶C之间的半胱氨酸异构体和嵌合型半胱氨酸半胱氨酸酶。对上述半胱氨酸酶和小肽的动力学研究表明，半胱氨酸蛋白酶的第一和第二发夹环在抑制半胱氨酸蛋白酶中起重要作用。嵌合半胱氨酸菌素的成功制造表明，通过蛋白质工程设计和生产人工半胱氨酸菌素是可能的。最后，人们尝试寻找利用重组人半胱氨酸菌素的方法。结果发现，半胱氨酸菌素能抑制牙龈卟啉单胞菌的生长，并能诱导人牙龈成纤维细胞产生白介素IL-6和IL-8。此外，胱抑素被清楚地证明是组织蛋白酶K的一种强有力的抑制剂，而组织蛋白酶K与破骨细胞性先天性吸收激素有关。这些发现强调了半胱氨酸氨基转移酶具有用于治疗骨质疏松症和牙周病的可能性。

The head investigator took the focus to human cystatins (cystatin S, cystatin SA, systatin SN, cystatin C, and cystatin D), noncompetitive inhibitors for cysteine proteinases of the papain family, in order to utilize their physiological functions. In the first stage of this research project, the E. coli system for the large-scale production of cystatins S. SN, and SA was developed. The engineered cystatins created by this system were considered to be equivalent to the natural cystatins. This system was also employed for the production of cystatin variants and chimeric cystatins between cystatin S and cystatin C. The kinetic study with above cystatins and minicking peptides showed that the first and second hairpin loops of the cystatins are important in the inhibition of cysteine proteinases. The success in creation of chimeric cystatins suggested that it is possible to design and produce artificial cystatins by protein engineering.The attempts for seeking ways to utilize recombinant human cystatins were finally made. As the results, the cystatins were found to inhibit the growth of Porphyromonas gingivalis and to induce interleukins (IL-6 and IL-8) productions by human gingival fibroblasts. In additions, the cystatins were clearly demonstrated to be a strong inhibitor for cathepsin K, which is associated with osteoclastic born resorpiton. These findings emphasize that the cystatins possess possibility of use in therapy for osteoporosis and periodontal disease.