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Chasing Endogenous Receptor Dynamics by Chemical Protein Labeling
POTENTIAL ENERGY
Chasing Endogenous Receptor Dynamics by Chemical Protein Labeling Shohei Uchinomiya1,2,*
Dr. Shohei Uchinomiya obtained his PhD in March 2014 from Kyoto University in Japan, where he worked on chemistry-based protein labeling with functional molecules under the supervision of Prof. Itaru Hamachi. Thereafter, he joined Prof. Young-Tae Chang’s group at the National University of Singapore as a postdoctoral researcher from 2014 to 2015 (the group is currently at Pohang University of Science and Technology in Korea). He began his academic career in 2015 as an assistant professor in Prof. Akio Ojida’s group at Kyushu University in Japan. His current research focuses on live-cell imaging of biomolecules with synthetic fluorescent probes. Cytokine and growth factor receptors, such as receptor tyrosine kinases and G-protein-coupled receptors, are crucial for signaling cascades that modulate cell survival, proliferation,
and migration.1 Because these receptors are also deeply involved in the growth of various cancer cells, a number of them have been investigated as important therapeutic targets.2 Therefore, a deep understanding of their functions is expected to facilitate the development of new anti-cancer drugs. For this goal, fluorescence imaging that enables the detection of biomolecule dynamics in live cells is a powerful tool because the functions of cytokine and growth factor receptors are deeply involved in their dynamics, such as lateral movement on the cell surface, internalization, and recycling. In this issue of Chem, we report a new method for selective and covalent labeling of ‘‘endogenous’’ cytokine and growth factor receptors and fluorescence imaging of their dynamic behavior in live cells (Figure 1).3 In this method, 4-dimethylaminopridine (DMAP) with a Ni(II)-nitrilotriacetate (Ni-NTA) moiety is tethered to a cytokine or growth factor fused with an oligohistidine tag (His-tag) through a selective interaction between the NiNTA moiety and the His-tag. This DMAP-tethered cytokine or growth factor, named a reactive cytokine or growth factor, enables the selective binding of DMAP to the target receptor through an interaction between the receptor and the cytokine or growth factor, which facilitates the DMAP-catalyzed acyl transfer reaction from an acyl donor to a nucleophilic amino acid on the surface of the target receptor. Using this method, we successfully achieved the selective labeling of endogenous epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor type 4 (CXCR4) on the surface of a live cell. More importantly, we were also able to fluorescently visualize the dynamics of EGFR and CXCR4 upon stimulations with corresponding ligands. For example, when EGFR labeled with fluorophore was stimu-
lated with EGF, fluorescent particles were observed in the cytosol after 1 hr by confocal laser scanning microscopy, indicating successful imaging of the internalization of endogenous EGFR in live cells. Moreover, when the labeled EGFR was stimulated with another ligand, transforming growth factor a (TGF-a), fluorescence intensity on the cell surface decreased after 10 min and then transiently recovered after an additional 30 min. This fluorescence recovery on the membrane was not observed after EGF stimulation, indicating that EGFR recycling induced by TGF-a stimulation was successfully observed. We also performed fluorescence imaging of CXCR4 dynamics upon stimulation with stromal-cell-derived 1a (SDF1a), a ligand of CXCR4. The labeled CXCR4 was predominantly observed at the plasma membrane before stimulation with SDF1a. In contrast, the labeled CXCR4 was mainly observed in the cytoplasm of migrating cells after SDF1a stimulation. It has been reported that cell-surface CXCR4 localizes to the leading edge after ligand stimulation, rapidly internalizes to the cytoplasm, and localizes to the back of the migrating cells.4,5 However, these behaviors were detected by anti-CXCR4 antibodies or fluorescent proteins that were not able to distinguish between endocytosed CXCR4 and newly synthesized CXCR4 inside the cells. In contrast, our method was able to selectively label and chase the CXCR4 that localized on the cell membrane before SDF1a stimulation without detecting the newly synthesized CXCR4. The key points of our method are (1) the simple and flexible construction of reactive cytokines and growth factors in an in situ manner and (2) the direct and covalent labeling of the target receptors with detection probes.
Chem 4, 1191–1193, June 14, 2018
1191
Figure 1. Selective and Covalent Labeling of Endogenous Cytokine and Growth Factor Receptors on Live-Cell Surfaces with Reactive Cytokines and Growth Factors
Generally, probe-conjugated cytokines and growth factors are used for fluorescence imaging of endogenous receptors on the surface of a live cell. However, chemical modification of cytokines and growth factors with synthetic probes sometimes causes a loss of function of ligands as a result of the fragile and sensitive features of their structures. Moreover, the receptors are indirectly labeled with the probes on the basis of non-covalent interactions between the receptors and their ligands because the probes are conjugated to the ligands and not the receptors themselves. Therefore, there is always a concern that the probes will dissociate from the receptors during analysis of receptor dynamics. By contrast, our method enables the construction of various reactive cytokines and growth factors by simply mixing DMAP with Ni-NTA and His-tagfused ligands, whereby endogenous receptors can be directly and covalently labeled with detection probes on the surface of the live cell. This method has the potential to be used for fluorescence imaging of various receptors dy-
1192 Chem 4, 1191–1193, June 14, 2018
namics in live cells, as shown for EGFR and CXCR4. I have been very fortune to have the opportunities to join Prof. Hamachi’s group in Japan during my PhD study and join Prof. Young-Tae Chang’s group in Singapore during my postdoctoral training. The research topics of Prof. Hamachi’s group are very wide and include protein chemical labeling, fluorescence imaging, and hydrogel-based sensors. They are based on not only organic chemistry but also analytical chemistry, coordination chemistry, supermolecular chemistry, and biology. Indeed, the technique that we report in this issue of Chem was developed through a combination of organic chemistry, coordination chemistry, and biology. Prof. YoungTae Chang’s group includes chemists and biologists who come from several countries. Although they have different research and even cultural backgrounds, they work together, discuss the same project from various viewpoints, and encourage each other.
My PhD and postdoctoral training thus gave me great opportunities to realize how important it is for a good researcher to have communication skills and knowledge of various fields. In research, it is important to learn about various research fields in addition to our own and collaborate with other researchers. This is also essential in chemical biology, in which chemists develop tools for understanding highly complicated biological systems. To date, various chemical tools developed for analyses of biological events have contributed to our understanding of complex biological systems. However, it is still challenging to detect many biological phenomena with chemical tools. Now, I’m starting my academic career as an assistant professor in Prof. Ojida’s group at Kyushu University, where I work on live-cell imaging of biomolecules with synthetic fluorescent probes. During my academic career, I want to contribute to the understanding of biological systems through the
development of new chemical tools as a chemical biologist. 1. Lemmon, M.A., and Schlessinger, J. (2010). Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134.
Shimada, I., and Hamachi, I. (2018). Endogenous membrane receptor labeling by reactive cytokines and growth factors to chase their dynamics in live cells. Chem 4, this issue, 1451–1464.
2. Cohen, P. (2002). Protein kinases–The major drug targets of the twenty-first century? Nat. Rev. Drug Discov. 1, 309–315.
4. Pelletier, A.J., van der Laan, L.J., Hildbrand, P., Siani, M.A., Thompson, D.A., Dawson, P.E., Torbett, B.E., and Salomon, D.R. (2000). Presentation of chemokine SDF-1 a by fibronectin mediates directed migration of T cells. Blood 96, 2682–2690.
3. Takaoka, Y., Uchinomiya, S., Kobayashi, D., Endo, M., Hayashi, T., Fukuyama, Y., Hayasaka, H., Miyasaka, M., Ueda, T.,
5. van Buul, J.D., Voermans, C., van Gelderen, J., Anthony, E.C., van der Schoot, C.E., and Hordijk, P.L. (2003). Leukocyte-endothelium
interaction promotes SDF-1-dependent polarization of CXCR4. J. Biol. Chem. 278, 30302–30310. 1Department
of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan
2Present
address: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan *Correspondence: [email protected] https://doi.org/10.1016/j.chempr.2018.05.023
Chem 4, 1191–1193, June 14, 2018 1193
Chasing Endogenous Receptor Dynamics by Chemical Protein Labeling Shohei Uchinomiya1,2,*
Dr. Shohei Uchinomiya obtained his PhD in March 2014 from Kyoto University in Japan, where he worked on chemistry-based protein labeling with functional molecules under the supervision of Prof. Itaru Hamachi. Thereafter, he joined Prof. Young-Tae Chang’s group at the National University of Singapore as a postdoctoral researcher from 2014 to 2015 (the group is currently at Pohang University of Science and Technology in Korea). He began his academic career in 2015 as an assistant professor in Prof. Akio Ojida’s group at Kyushu University in Japan. His current research focuses on live-cell imaging of biomolecules with synthetic fluorescent probes. Cytokine and growth factor receptors, such as receptor tyrosine kinases and G-protein-coupled receptors, are crucial for signaling cascades that modulate cell survival, proliferation,
and migration.1 Because these receptors are also deeply involved in the growth of various cancer cells, a number of them have been investigated as important therapeutic targets.2 Therefore, a deep understanding of their functions is expected to facilitate the development of new anti-cancer drugs. For this goal, fluorescence imaging that enables the detection of biomolecule dynamics in live cells is a powerful tool because the functions of cytokine and growth factor receptors are deeply involved in their dynamics, such as lateral movement on the cell surface, internalization, and recycling. In this issue of Chem, we report a new method for selective and covalent labeling of ‘‘endogenous’’ cytokine and growth factor receptors and fluorescence imaging of their dynamic behavior in live cells (Figure 1).3 In this method, 4-dimethylaminopridine (DMAP) with a Ni(II)-nitrilotriacetate (Ni-NTA) moiety is tethered to a cytokine or growth factor fused with an oligohistidine tag (His-tag) through a selective interaction between the NiNTA moiety and the His-tag. This DMAP-tethered cytokine or growth factor, named a reactive cytokine or growth factor, enables the selective binding of DMAP to the target receptor through an interaction between the receptor and the cytokine or growth factor, which facilitates the DMAP-catalyzed acyl transfer reaction from an acyl donor to a nucleophilic amino acid on the surface of the target receptor. Using this method, we successfully achieved the selective labeling of endogenous epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor type 4 (CXCR4) on the surface of a live cell. More importantly, we were also able to fluorescently visualize the dynamics of EGFR and CXCR4 upon stimulations with corresponding ligands. For example, when EGFR labeled with fluorophore was stimu-
lated with EGF, fluorescent particles were observed in the cytosol after 1 hr by confocal laser scanning microscopy, indicating successful imaging of the internalization of endogenous EGFR in live cells. Moreover, when the labeled EGFR was stimulated with another ligand, transforming growth factor a (TGF-a), fluorescence intensity on the cell surface decreased after 10 min and then transiently recovered after an additional 30 min. This fluorescence recovery on the membrane was not observed after EGF stimulation, indicating that EGFR recycling induced by TGF-a stimulation was successfully observed. We also performed fluorescence imaging of CXCR4 dynamics upon stimulation with stromal-cell-derived 1a (SDF1a), a ligand of CXCR4. The labeled CXCR4 was predominantly observed at the plasma membrane before stimulation with SDF1a. In contrast, the labeled CXCR4 was mainly observed in the cytoplasm of migrating cells after SDF1a stimulation. It has been reported that cell-surface CXCR4 localizes to the leading edge after ligand stimulation, rapidly internalizes to the cytoplasm, and localizes to the back of the migrating cells.4,5 However, these behaviors were detected by anti-CXCR4 antibodies or fluorescent proteins that were not able to distinguish between endocytosed CXCR4 and newly synthesized CXCR4 inside the cells. In contrast, our method was able to selectively label and chase the CXCR4 that localized on the cell membrane before SDF1a stimulation without detecting the newly synthesized CXCR4. The key points of our method are (1) the simple and flexible construction of reactive cytokines and growth factors in an in situ manner and (2) the direct and covalent labeling of the target receptors with detection probes.
Chem 4, 1191–1193, June 14, 2018
1191
Figure 1. Selective and Covalent Labeling of Endogenous Cytokine and Growth Factor Receptors on Live-Cell Surfaces with Reactive Cytokines and Growth Factors
Generally, probe-conjugated cytokines and growth factors are used for fluorescence imaging of endogenous receptors on the surface of a live cell. However, chemical modification of cytokines and growth factors with synthetic probes sometimes causes a loss of function of ligands as a result of the fragile and sensitive features of their structures. Moreover, the receptors are indirectly labeled with the probes on the basis of non-covalent interactions between the receptors and their ligands because the probes are conjugated to the ligands and not the receptors themselves. Therefore, there is always a concern that the probes will dissociate from the receptors during analysis of receptor dynamics. By contrast, our method enables the construction of various reactive cytokines and growth factors by simply mixing DMAP with Ni-NTA and His-tagfused ligands, whereby endogenous receptors can be directly and covalently labeled with detection probes on the surface of the live cell. This method has the potential to be used for fluorescence imaging of various receptors dy-
1192 Chem 4, 1191–1193, June 14, 2018
namics in live cells, as shown for EGFR and CXCR4. I have been very fortune to have the opportunities to join Prof. Hamachi’s group in Japan during my PhD study and join Prof. Young-Tae Chang’s group in Singapore during my postdoctoral training. The research topics of Prof. Hamachi’s group are very wide and include protein chemical labeling, fluorescence imaging, and hydrogel-based sensors. They are based on not only organic chemistry but also analytical chemistry, coordination chemistry, supermolecular chemistry, and biology. Indeed, the technique that we report in this issue of Chem was developed through a combination of organic chemistry, coordination chemistry, and biology. Prof. YoungTae Chang’s group includes chemists and biologists who come from several countries. Although they have different research and even cultural backgrounds, they work together, discuss the same project from various viewpoints, and encourage each other.
My PhD and postdoctoral training thus gave me great opportunities to realize how important it is for a good researcher to have communication skills and knowledge of various fields. In research, it is important to learn about various research fields in addition to our own and collaborate with other researchers. This is also essential in chemical biology, in which chemists develop tools for understanding highly complicated biological systems. To date, various chemical tools developed for analyses of biological events have contributed to our understanding of complex biological systems. However, it is still challenging to detect many biological phenomena with chemical tools. Now, I’m starting my academic career as an assistant professor in Prof. Ojida’s group at Kyushu University, where I work on live-cell imaging of biomolecules with synthetic fluorescent probes. During my academic career, I want to contribute to the understanding of biological systems through the
development of new chemical tools as a chemical biologist. 1. Lemmon, M.A., and Schlessinger, J. (2010). Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134.
Shimada, I., and Hamachi, I. (2018). Endogenous membrane receptor labeling by reactive cytokines and growth factors to chase their dynamics in live cells. Chem 4, this issue, 1451–1464.
2. Cohen, P. (2002). Protein kinases–The major drug targets of the twenty-first century? Nat. Rev. Drug Discov. 1, 309–315.
4. Pelletier, A.J., van der Laan, L.J., Hildbrand, P., Siani, M.A., Thompson, D.A., Dawson, P.E., Torbett, B.E., and Salomon, D.R. (2000). Presentation of chemokine SDF-1 a by fibronectin mediates directed migration of T cells. Blood 96, 2682–2690.
3. Takaoka, Y., Uchinomiya, S., Kobayashi, D., Endo, M., Hayashi, T., Fukuyama, Y., Hayasaka, H., Miyasaka, M., Ueda, T.,
5. van Buul, J.D., Voermans, C., van Gelderen, J., Anthony, E.C., van der Schoot, C.E., and Hordijk, P.L. (2003). Leukocyte-endothelium
interaction promotes SDF-1-dependent polarization of CXCR4. J. Biol. Chem. 278, 30302–30310. 1Department
of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan
2Present
address: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan *Correspondence: [email protected] https://doi.org/10.1016/j.chempr.2018.05.023
Chem 4, 1191–1193, June 14, 2018 1193