Preparation of conophylline affinity nano-beads and identification of a target protein
Graphical abstract
We prepared conophylline-linked nano-beads and conophylline was found to directly interact with ARL6IP.
Introduction
Conophylline (1) isolated from the leaves of Tavertaemontana divaricata of Malaysia is a low-molecular-weight compound having a vinca alkaloid structure.1, 2 Also, we isolated it as a Ras function inhibitor from the leaves of Ervatamia microphylla collected in Thailand.3 Moreover, recently we also found that conophylline showed antidiabetic activity in vivo, as it decreased the blood glucose level in diabetic model mice. Its mechanism of action is unique, since conophylline induces the differentiation of insulin-producing cells from precursor cells in culture4 and in animals.5 So it may be useful for the regeneration therapy of type-2 diabetes mellitus. Recently, conophylline was also shown to induce the differentiation of insulin-producing cells from mouse bone marrow cells when added together with betacellulin-d4,6 thus suggesting its usefulness for transplantation therapy of type-1 diabetes. However, its molecular mechanism of action and the target molecule have not yet been well documented. Especially, identification of the target molecule(s) of conophylline should give us new insights into the function of the identified protein, and also into the molecular mechanism of its β-cell differentiation-promoting activity.
Latex nano-beads, termed SG beads, are a new innovative tool for affinity purification-based identification of the target molecules of bioactive compounds.7 SG beads are composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, and have several advantages over conventional affinity-purification supports; for example, their extremely large surface area results in a relatively high binding capacity, and their lack of pores facilitates the efficient removal of residual proteins. SG beads have been used successfully to purify various proteins, including transcription factors and several target proteins for various chemical compounds. DNA affinity latex beads were used to purify sequence-specific DNA-binding proteins directly from crude cell extracts.8 The target proteins of several bioactive compounds have been identified using this device, as in the example of the anti-NF-κB agent E3330.7 The beads were also used to show that deoxycytidine kinase, an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of methotrexate.9 Moreover, by use of affinity latex beads, the mitochondrial transporter of 2-oxoglutarate, was shown to bind to PdTCPP, a phosphorescent porphyrin derivative; and PdTCPP was found to inhibit 2-oxoglutarate uptake into mitochondria.10
Thus, we prepared conophylline-ligated latex nano-beads and used them to screen for and identify the target protein of conophylline.
Section snippets
Synthesis of conophylline amino-derivative
In screening for target proteins by affinity-purification using nano-beads, the bait for the target protein should be immobilized. Either a carboxyl or amino-group of the bioactive compound under study can be employed for the immobilization on the surface of SG beads through amide-bond formation. Therefore, we synthesized an amino derivative of conophylline according to the reactions depicted in Figure 1. We introduced an amino function at the site of the oxirane ring by performing the ring
Discussion
Conophylline shows antidiabetic and anticancer activities in vivo; however, its molecular target has not been elucidated. In the present research, we identified ARL6IP as the target protein of conophylline by affinity-purification from both cytosolic and nuclear extracts derived from conophylline-sensitive cells. Subsequent immuno-blotting analysis revealed that ARL6IP tended to be present more in the nuclear extract than in the cytosolic one, which suggests that the protein was localized in
Materials
Conophylline was isolated in our laboratory from the leaves of E. microphylla,2 and stored at –20 °C in benzene at 1 mg/ml. For use, the solution was dried; and the conophylline was dissolved in MeOH at 10 μg/ml.
Recombinant human HGF was purchased from Sigma–Aldrich, Inc. (St. Louis, MO). Recombinant human activin A was obtained from R&D Systems (Minneapolis, MN). Western lightning™ chemiluminescence reagents came from PerkinElmer, Norwalk, CT. Horseradish peroxidase (HRP)-conjugated anti-rabbit
Acknowledgments
This work was financially supported in part by grants from the programs Grants-in-Aid for Scientific Research on Priority Areas and Grants-in-Aid for the Global Centers of Excellence (GCOE) Program (2007–2012 to Keio University) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT). This work was also supported by the High-Tech Research Center Project for Private Universities: matching fund subsidy from MEXT, 2006−2010.
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