ヒト癌抑制タンパク質101F6の分子機能に関する研究

概要

101F6 protein is a heme-containing transmembrane protein which can be found to be expressed in the membranes of endoplasmic reticulum and vesicles of human cells. It is a member of cytochrome 6501 protein family. The predicted structure of human 101F6 protein, consisting of222 amino acid residues, had six transmembrane α-helices and four fully-conserved His residues for the binding of two hemes. In this study, recombinant human 101F6 protein was expressed in the methylotrophic yeast Pichia pastoris cells transformed with the expression vector pPICZB-101 F6-Hiss. Purification of 101F6 was conducted by using a Ni-NTA-Sepharose affinity column. Better purification results were obtained when DDM instead of β-OG was used as a solubilizing detergent.

101F6 protein was successfully incorporated into phospholipid bilayer nanodiscs with different sizes by using three MSP constructs in different lengths (MSP1D1, MSP1D1AH5, MSP1E3D1). These different sizes of MSP proteins were each produced by E. coli BL21(DE3) strain after transformation with plasmids such as pMSPIDl, pET28a-MSP 1D1ΔΗ5, or pMSPlE3Dl, respectively. The expressed MSPs were each purified by Ni-Sepharose affinity chromatographic techniques. Reconstitutions of 101F6 protein into nanodiscs were conducted by the method of self-assembly by mixing at an optimizing ratio of MSP: lipids: 101F6 protein. There is a possibility that 101F6 may be incorporated as a monomer or a homodimer into these nanodiscs.

The data for the nanodisc reconstitution obtained in the case of 101F6-MSP1D1 nanodisc showed that best results were obtained at a mixing ratio of 2:160:2. From SDS- PAGE analysis with silver staining, two protein bands for MSP1D1 and 101F6 appeared on the gel at their expected positions inferred from those of purified samples.

For 101F6-MSP1D1AH5 nanodisc better results were obtained in the case of (2:160:1) ratio. Also, using of pre-formed empty nanodisc (MSP1D1AH5: DMPC) increase the percentage of reconstitution. Furthermore, 101F6 was reconstituted into large-sized MSP1E3D1 nanodisc. Reconstitution was enhanced by using pre-formed empty nanodisc (MSP1E3D1: DMPC) to incorporate 101F6.

The nanodisc environments were found to maintain the reconstituted 101F6 protein in native-like membrane environments without detergents, resulting in increased stability at room temperatures. This would be very suitable for various functional and biochemical measurements of 101F6 protein.

We studied the human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity. We found for the first time that purified 101F6 both in detergent micelle state and in phospholipid bilayer nanodisc state has an authentic ferric reductase activity by single turnover kinetic analyses. The kinetic analysis on the ferrous heme oxidation of reduced 101F6 upon the addition of a ferric substrate, ferric ammonium citrate (FAC), showed concentration­ dependent accelerations of its reaction with reasonable values of and Vmax. We further verified the authenticity of the ferric reductase activity of 101F6 using nitroso-PSAP as a Fe2+-specific colorimetric chelator.

101F6 in nanodisc state had much higher efficiency for FAC (Vmax/KM = 0.185 min1 /μΜ) than 101F6 in detergent micelle state (Vmax/KM = 0.032 min-1/μΜ). Based on these results, we concluded thatく the structure of the active site of 101F6 in detergent micelle state may be slightly changed from its native one in the cell membranes due to the complete substitution of the phospholipids moiety characterized by the ordered charged head groups and hydrophobic tails with less ordered detergent molecules having a shorter hydrophobic tail and a sugar head group. Therefore, the Fe3+ substrate FAC can bind to the active site of 101F6 in nanodisc in a higher affinity than in detergent micelle state. Thus, nanodiscs can provide native-like membrane environments suitable for maintaining the protein stability for a longer time even at room temperature and enabling the fine-tuning of the enzymatic specificity of 101F6.

Furthermore, we tried to study the effect of expression of 101F6 on cell proliferation of A549 cancer cells with/or without the addition of ascorbic acid as an electron donor to 101F6. The human 101F6 protein was successfully expressed inside the A549 cells after the transfection with pcDNA3.1-hl01F6 plasmid. We found that the growth of A549 cancer cells was inhibited when 101F6 protein was forced expressed with/or without the addition of ascorbic acid (AsA).

Further, I focused on the in vitro protein-protein interactions by the expression of 101F6-His protein inside A549 cancer cell lines to determine if some protein molecules can interact with 101F6. This interaction might be affecting the function of 101F6 as tumor suppressor protein where 101F6 can kill and suppress cancer cells, but the molecular mechanism of the tumor suppressor activities is still unknown. I succeeded in partial purification of 101F6 protein expressed in the cultured A549 cancer cells by using a Ni-membrane column for the first time.

Some candidate protein molecules possibly interacting with 101F6 were found around 14 and 20 kDa in SDS-PAGE (silver staining).

Based on our findings, we considered to connect between the function of 101F6 as a ferric reductase activity and its possible function as a tumor suppressor protein to clarify the mechanism by which 101F6 protein suppresses and inhibits the growth of cancer cells. We propose that, inside mammalian cells such as A549 cancer cells, 101F6 can reduce ferric ion to ferrous ion which in turn can induce cancer cell death via ferroptosis mechanism.

Therefore, the following scenario to clarify the mechanism of action of 101F6 protein may be considered. The 101F6 protein located at endosome membranes would transfer electrons from cytosolic ascorbate (AsA) (as a physiological electron donor) to the intravesicular side to reduce ferric ions (Fe3+) to ferrous ions (Fe2+). Then, the ferrous ion in the intravesicular side can be transported into the cytoplasm through DMT 1(divalent metal transporter 1).

If we assumed the efficient ferric reductase activity of 101F6 protein in the presence of enough amounts of AsA in the cells, a significant accumulation of ferrous ion in the cytoplasm would occur. This would be followed by the reaction of Fe2+ with hydrogen peroxide H202 resulting in the formation of lethal ROS by Fenton reaction, which would induce cell death via ferroptosis, a non-apoptotic form of cell death. It is well known that significant amounts of H202 are produced as byproducts from the respiratory chain and from other cellular metabolic pathways. In addition, superoxide anion radical (O2··) can be converted to H2O2 by superoxide dismutase enzymes (SODs). It is well known that the cellular concentration of H2O2 increased in cancer cells compared to normal cells.

From this view, we thought that hl01F6 protein and ascorbate (AsA) may have an important role for the regulation of iron-uptake and iron homeostasis in human cells and hl01F6 protein directly or indirectly might be included as a part of the ferroptosis (iron-dependent cell death process) mechanism by employing its ferric reductase function.

On the other hand, for further clarification of the function of 101F6 protein as ferric reductase, the role of 101F6 protein may be similar to the function of Steap3 being expressed in endosomal membranes of the cells and has a ferric reductase activity. Indeed, Steap3 was identified to be induced by the tumor-suppressing protein p53 and to induce apoptosis via caspase-3 activation pathway in cancer cells. Further, p53 was found to induce ferroptosis by inhibiting cystine uptake. It is also known that depletion of reduced glutathione (GSH) levels due to inhibiting cystine uptake increases the levels of ROS accumulation which induce ferroptosis.

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