Identification of a Novel Anti-cancer Protein, FIP-bbo, from Botryobasidium botryosum and Protein Structure Analysis using Molecular Dynamic Simulation

Abstract

Fungal immunoregulatory proteins (FIP) are effective small molecule proteins with broad-spectrum immunomodulatory and anti-cancer activities and can be potential agents for the development of clinical drugs and health food additives. In this study, a new member of FIP named FIP-bbo was obtained through Botryobasidium botryosum genome mining. FIP-bbo has the typical characteristics of FIP but is genetically distant from other FIPs. Recombinant FIP-bbo (rFIP-bbo) was produced in an optimized E. coli expression system, and the pure protein was isolated using a Ni-NTA column. Antineoplastic experiments suggested that FIP-bbo is similar to LZ-8 in inhibiting various cancer cells (Hela, Spac-1, and A549) at lower concentrations, but it is not as potent as LZ-8. The molecular mechanism by which FIP-bbo, FIP-fve, and LZ-8 are cytotoxic to cancer cells has been discussed based on molecular dynamics simulation. Point mutations that may improve the thermal stability of FIP-fve and FIP-bbo were predicted. These results not only present a new candidate protein for the development of anticancer adjuvants, but also provide an approach for designing FIPs with high anticancer activity.

Figure 1

Phylogenetic tree and the sequence alignment of FIP.

Figure 2

SDS-PAGE analysis of purified recombinant FIP expressed by E. coli. The cropped images originate from one gel. The full-length gel is presented in Supplementary Fig. S4.

Figure 3

Proliferation inhibition by rFIP on Hela (a), Spac-1 (b) and A549 (c) cells. These cells were treated with 1, 2, 4, 8, 16, 32, and 64 μg/mL rFIPs for 16 h, compared with negative control. *p < 0.05, 0.05 > **p > 0.001, 0.001 > ***p > 0.0001, ****p < 0.0001. Each experiment was repeated at least thrice.

Figure 4

Apoptosis induced by FIP. 8 μg/mL of rLZ–8, rFIP-fve and rFIP-bbo were added into the Hela, Spca-1 and A549 cells, incubated for 24 h and analysed using a flow cytometer to detect the rate of apoptosis.

Figure 5

Cytotoxicity assay of FIP. Hela, Spca-1 and A549 cells were treated with 8 μg/mL of rLZ–8, rFIP-fve and rFIP-bbo and apoptosis was detected using Hoechst Staining Kit.

Figure 6

Effect of FIP on migration of Hela, Spca-1 and A549 cells by wound healing assay. Hela Spca-1 and A549 cells were treated with 8 μg/mL rLZ-8, rFIP-fve, and rFIP-bbo for 36 h. PBS was used as a negative control (NC). Cell migration was observed by microscopy.

Figure 7

Root-mean-square deviations (RMSD) for all the atoms of FIP monomers (A) and dimers (B) from the initial structure.

Figure 8

3D structures of FIP ((A) LZ-8 monomer, (B) FIP-fve monomer, (C) FIP-bbo monomer, (D) LZ-8 dimer, (E) FIP-fve dimer and (F) FIP-bbo dimer) with B-factor. The B-factor value of an amino acid is obtained by taking an average of all a B-factor atoms that make up the amino acid. The B-factor values are represented by red, yellow, green, and blue in descending order.

Figure 9

The structures of the three simulation systems by cluster analysis. The conformational changes of LZ-8, FIP-fve and FIP-bbo from cluster analysis. Their formation of homo-dimers is decided by the directions of N-terminal α-helix. If N-terminal α-helix turns to the left, the FIP becomes the closed structure, and when it turns to the right, it becomes the opened structure.

Figure 10

The proportion of the open structure in the molecular dynamics trajectory.

Figure 11

Radius of gyrations for all the Cα atoms of FIP dimers during the molecular dynamics simulation.