Interaction of apohtosis inhibitor PI-9 with cellular factors

2003 Fiscal Year Final Research Report Summary

• Project/Area Number: 13670314
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Immunology
• Research Institution: The University of Tokyo
• Principal Investigator: KANAMORI Hiroshi The University of Tokyo, Faculty of Medicine, Research Associate, 医学部附属病院, 助手 (60211851)
• Co-Investigator(Kenkyū-buntansha): OHNISHI Shin The University of Tokyo, Faculty of Medicine, Lecturer, 医学部附属病院, 講師 (00183236)
• Project Period (FY): 2001 – 2003
• Keywords: Proteinase inhibitor-9 / Phage display

Research Abstract

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.