We tried to identify more specific ligands to HECAT5 which are able to modulate the activity of HECAT5. During literature investigation, we came to know that J. D. Mainland et al conducted high-throughput screen of 73 odorants against a clone library of 511 human olfactory receptors (J. D. Mainland, 2015). We found out HECAT5 variant form with one amino acid difference in this screening dataset, and it responded to six candidate odorants.
It has been well known that the sequences in crucial motifs for ligand-receptor binding such as toggle switch and ionic lock are conserved between members of OR family (de March et al, 2015). Comparison of sequence between HECAT5 and other ORs showed that those motifs are conserved in HECAT5.
This leads us to harness the interaction of HECAT5 variant with those odorants as a docking models (J. D. Mainland, 2015) to aim at identification of strong ligands to modulate HECAT5 activity. Based on comparison of amino acid sequence similarity between HECAT5 with other OR members which their 3D sequence already have been analyzed, we selected one OR with the highest similarity with HECAT5. By using homology modeling program from Schrodinger, HECAT5 sequence was co-aligned with the 3D structure of selected OR protein, which designated the homology model of HECAT5. Then, androstenone, a candidate odorant, was merged into the homology model to establish ligand docking molecular model of HECAT5 protein. Using this docking model, more than 140 chemicals were selected from ‘GPCR ligand focus library’ as well as various commercial chemical libraries. The candidate chemicals need to be analyzed their functions to act as agonist or antagonist.
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Figure 10. Construction of homology model of HECAT5. Virtual structure of HECAT5 when different ligands bind.
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Ⅳ. DISCUSSION
HECAT5 has great potential to increase self-renewal property in vitro. It is a novel observation that olfactory receptor can regulate cell cycle even when only overexpressed.
And it appears that HECAT5 possesses different action in different cell types. We are struggle for finding modulators of HECAT5 because controlling the effect of HECAT5 with external molecules can be applied for therapeutic uses. However, there are some limitations to be addressed.
Above all, we need to define the biological function of HECAT5 in human embryonic stem cells. It can be figured out whether HECAT5 works in hESCs as proliferation regulator by using siRNA of HECAT5. This will support our hypothesis that hESC-specific HECAT5 regulates cell proliferation and give a hint how olfactory receptor has functioned in embryonic stem cells as a gatekeeper receiving external signals.
Furthermore, protein level of HECAT5 has not been detected by Western blot and Immunocytochemistry. This is because sole isolation of membrane protein was very difficult.
According to a research group, they used Rho tag to promote olfactory receptor protein expression on the cell surface as well as accessary factor constructs such as RTP1S, Ric8b (Hanyi Zhuang and Hiroaki Matsunami, 2008). Thus, tagging Rho to HECAT5 is under progress. We hope this would make it possible that we can check protein expression level of HECAT5 and its location within the cell.
GPCRs mediate many physiological processes by transmitting a variety of extracellular signals into intracellular responses (Xavier et al, 2007). In case of olfactory receptors, it is generally known that OR sends intracellular signaling through Gαs protein. And we were able to exclude other possible pathways such as Gαq pathway and ERK pathway as we witnessed that neither Gq inhibitor blocked the effect of HECAT5 nor phosphorylated ERK level was affected by HECAT5. Still, the assay to confirm whether HECAT5 follows cAMP-PKA downstream pathway starting from Gαs is required.
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When it comes to therapeutic use of HECAT5, we are aware of the fact that GPCRs are targeted by approximately 25% of the drugs on the market today, which testifies the significant physiological roles GPCRs play in transducing signals from outside of cells to the inside (KA. Jacobson, 2015). However, knowledge about GPCR structure, conformation that bias receptor signaling, and each downstream signaling should be prior to finding the target of GPCRs. To be concrete, very first step which ligands bind may determine which signals they actually send to. In this respect, it is considered very significant to better understand the dynamic range and diverse mechanisms of GPCR activation (DB Staus, 2016). Based on the OR structure, active ligand binding site of HECAT5 from molecular modeling, and candidate ligands, we plan to explore each effect of chemical on cell proliferation. To list up of candidate chemicals is actually not enough because binding pocket of OR is spacious than that of other GPCR, resulting in both agonist and antagonist are able to bind. This makes it hard to distinguish which chemical acts as agonist or antagonist. Thus, we will figure out how outcome of HECAT5, increased cell proliferation, has been changed after treatment of each candidate chemicals and determine their mode of action as well as drugability.
Further, robust and efficient iPSCs generation without genomic insertion of transgenes has emerged as a challenge to be solved. Many research groups are developing iPSC induction methods including miRNA, small molecules, and nonintegrating virus in order with a minimum number of transgenes (Nasir Malik and Mahendra S. Rao, 2013). In this sense, we anticipate that while HECAT5 may act with other reprogramming factors, agonist of HECAT5 may play a role to increase the efficiency of iPSC generation (Dharmendra Kumar et al, 2017), on the other hand, antagonist of HECAT5 may act as a cancer cell blocker (Jane E. Visvader et al, 2012).
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Ⅴ. CONCLUSION
Using digital differential display (DDD), we identified HECAT5 gene which is expressed both in hES cell and cancer cell, not in normal tissues. HECAT5 is a member of olfactory receptor and has 7-transmembrane structure. Overexpression of HECAT5 dramatically increased cell proliferation and this effect was enhanced by treatment of HECAT5’s candidate ligand, nonanoic acid. Of three major pathways in GPCR downstream signaling, we confirmed that neither Gαq pathway nor ERK pathway are specific to HECAT5. Next, going to upper stage where ligand and HECAT5 encounters, we expect to reveal the mechanism of HECAT5 activation according to conformational change in HECAT5 and the chemical screening.
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