Interaction of apohtosis inhibitor PI-9 with cellular factors

颗粒酶B （Granzyme B, GraB）由细胞毒性T细胞（CTL）和自然杀伤细胞（NK）分泌，在靶细胞的凋亡反应中起关键作用。蛋白酶-9 （PI-9）已知与GrB相互作用并阻止细胞受到CTL和NK细胞的凋亡攻击。最近的研究揭示了PI-9与其他细胞分子如caspase的相互作用。I和弹性。有研究表明，这种丝氨酸蛋白酶具有与多种细胞内分子结合的潜力，并具有其他未知的生理作用。我们已经引入了一个系统，通过该系统，使用HepG2肝细胞在四环素存在下调节PI-9的表达水平。我们观察到PI-9的上调可以阻止GrB引起的细胞凋亡反应。PI-9的诱导也能抑制NK细胞YT的凋亡。这些观察结果使我们能够利用该系统来观察其他分子与PI-9相互作用对细胞生理的影响。我们建立了一个利用大肠杆菌生产重组PI-9的体系。我们开发了一种噬菌体展示系统，以获得与PI-9蛋白高亲和力结合的寡肽。从一个12 mer随机寡核苷酸文库中，我们得到了一个一致的序列（LLADTTHHRPWT）。通过BLAST数据库检索，我们获得了几个已知和未知的蛋白质，其中包含与共识序列同源的氨基酸序列。这些结果鼓励我们进一步研究单个蛋白与PI-9的相互作用是否可能具有显著的生理效应。

Granzyme B (GraB) is secreted from cytotoxic T cells (CTL) and ntural killer (NK) cells and plays a key role in the apoptotic reaction of the target cells. Proteinase-9 (PI-9) is known to interact with GrB and prevent cells from apoptotic attack from CTL and NK cells. Recent investigation by others has revealed the interaction of PI-9 with other cellular molecules such as caspase. I and elastase. It has been suggested that this serine proteinase has the potential of binding to several sets of intracellular molecules and has other unknown physiological roles.We have introduced a system by which PI-9 expression levels were regulated in the presence of tetracycline using HepG2 liver cells. We observed that the upregulation of PI-9 prevent the apoptotic reaction caused by GrB. Induction of PI-9 also prevented the apoptosis by the attack of human NK cell line YT. These observations enable us to utilize this system to observe the effect of the interaction of other molecule with PI-9 on cellular physiology.We have established a system by which we can produce biotinated recombinant PI-9 by using E. Coli. We have developed a phage display system to obtain oligopeptides with high affinity binding to PI-9 protein. From a library of 12-mer random oligonucleotides, we have obtained a consensus sequence (LLADTTHHRPWT). By employing a data base search (BLAST), we have obtained several known and unknown proteins in which contain homologous amino acid sequence with the consensus sequence.These results encourage us to further investigate weatherthe interaction of the individual protein with PI-9 may have significant physiological effect.