A Study On Subcellular Localization And Anti-tumor Domain Of Ganoderma Lucidum Immunoregulatory Protein

Malignant tumor is a great threat to human health and a frequently-occurring disease, there is no particular effective control strategies so far. In recent years, with the development of cell biology, biophysics, molecular pharmacology, as well as clarification to the nature of tumor, large-scale, rapid screening of combinatorial chemistry, genetic engineering and other advanced technologies speed up the study and application of drug development process. In the field of Cell Biology, start or activate the tumor cell apoptosis mechanism in different ways to influence the different gene expression in cells to induce apoptosis of tumor cells via specific organelle. In the biophysics research, crystal structures of many key protein factor in cell death process have been revealed and some anti-tumor domains have been identified, investigating on relationship of crystal structure and function has become the most hot and important biological field.This study project will show and discuss all the experiment results about Fungal Immunoregulatory Protein family, nuclear medicated cell death and the relationship of crystal structure and function of Ganoderma lucidum Immunoregulatory Protein.1. The study of recombinant Ganoderma Lucidum Immunoregulatory Protein (rLZ-8)Ganoderma lucidum Immunoregulatory Protein, the separation and purification of small-molecule protein from the extractive of Ganoderma mycelium was done by a Japanese Kino in 1989 (Kohsuke Kino et al., J. Bio. Chem. 1989, 1:472-478), it was named LZ-8, its amino acid sequence and physiological activity of immunity was also tested which indicates that the sequence of protein of LZ-8 is made up of 110 amino acid, serine acetylation on N-terminal, the molecular weight is 12.4 KD, and the isoelectric point is 4.4. In this study, LZ-8 was recombinantly expressed in Pichia pastoris, and production of rLZ-8 was larged to fermentation scale. The major function of Ganoderma Lucidum Immune Regulatory Protein lies in that it promotes the hyperplasia of peripheral lymphocytes and spleen cells, induces the macrophage both in human and animals to secrete various kinds of cell factors (as in interleukin, necrosis factor in tumors and interferon, etc.), and defending and dispelling the infringement of the causative agent, safeguards and maintains the health and to achieve the immune regulatory function.2. The study of rLZ-8's anti-tumor effects and subcellular localizationPrevious studies have shown that, LZ-8 achieves the anti-tumor effect mainly through immune regulation. However, the positive effects of this study is that it discussed the rLZ-8 through specific binding with the tumor cell membrane for the first time, it induces apoptosis direct destruction or kills tumor cells to achieve the anti-tumor effects, at the same time, unlike reducing leukocytes chemotherapy drugs, without affecting normal cells.It is found that rLZ-8 could localize in nuclear of Human Leukemia cells (NB4) and trigger cell death. Observing under Laser Scan Confocal Microscope (LSCM), FITC-RLZ-8 and nuclear occupied the same relative position or the same area in space of the cells, it means that nuclear may be subcellular binding site of rLZ-8.In our research, it also was checked that rLZ-8 behaved as a Ca²⁺ release inducer, Ca²⁺ stores were mainly found in nuclear and ER, which all supported our previous studies. As we known, Ca²⁺ signal is one of important pathways in the cell death.3. The study of rLZ-8's crystal structure and anti-tumor domainTo elucidate mechanism of rLZ-8 inducing tumor cells death further, some crystal structure researches were carried out in this study, and designed and expressed mutants of LZ-8 on the basis of pharmacology and structure again, then examine the activities of mutants. All kinds of work were to search and identify the most important domain of rLZ-8 for killing tumor cells. In 2003 and 2008, Paaventhan and our group presented 1.7 ? X-ray structure of FIP-fve and 1.8 ? X-ray structure of LZ-8, determined by single anomalous diffraction (SAD) using the anomalous signal of bromide ions present in the crystal for phasing.The overall fold of LZ-8 resembles the structure of FIP-fve from F. velutipes, which consists of an N-terminal dimerization domain and a C-terminal FNIII domain. The N-terminal domain is composed of anα-helix and aβ-strand that sustains the dimerization via domain swapping, forming a dumb-bell-shaped dimer. The C-terminal FNIII domain belongs to the immunoglobulin-like-sandwich fold and comprises a sandwich structure of twoβ-sheets (I and II) formed byβ-strands A-B-E and G-F-C-D, respectively To our knowledge, the crystal structure of LZ-8 is the second structure of fungal immunomodulatory proteins to date.Whether the formation of homodimers would also attributed to LZ-8's anti-tumor activity is worth to discuss, because FIP-fve has nearly the same N-terminalα-helix as LZ-8, but has no anti-tumor active. To survey the'hotspots'for the anti-tumor activity, a structure-based multi-alignment was performed. The N-terminalα-helix of LZ-8 and FIP-fve are quite closely similar from alignment results, it prompts us that the N-terminalα-helix may not associate with anti-tumor. The conformations of the loops in the FNIII domain of LZ-8 may be a starting point for elucidating the mechanism of specific residues responsible it's the anti-tumor activity. The comparative structural analysis also provided us some clues to improve.Dealing with the relationship of structure and bio-chemistry have been widely studied and used, but associating structure with cell biology is still a problem. In further researches, we hope that more functional domain of FIPs will be found, it will lay a solid foundation for future studies.