Laboratory of Protein Synthesis and Expression

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Professor
Junichi TAKAGI
mail takagi@protein.osaka-u.ac.jp
Associate Professor
Kenji IWASAKI
mail ikenji@protein.osaka-u.ac.jp
Assistant Professor
Yu KITAGO
mail kitago@protein.osaka-u.ac.jp
Fields

Signal Transduction Biology

Belong

test

Location

location

Research Theme

Cellular response to the extracellular environment depends on the “sensing” the extracellular cues by use of the receptor-ligand system. Binding of ligands to the extracellular domain of the receptors transduce signals into cells that initiates various cellular events, ultimately changing the cell fate. Most of the “signal transduction researches” deal with cytoplasmic events such as phosphorylation/dephosphorylation of signaling molecules and subsequent recruitment of adapter molecules, but mechanism for the “signal transmission across the membrane”, the very first step in the signaling pathway is poorly understood. Our study focuses on questions such as how receptors recognize their specific ligands, how this recognition leads to structural change in the receptor complex, and how the information cross the plasma membrane without transporting chemical entity. Using structural as well as chemical approach, we would tackle on this difficult problem to obtain insights into the mechanism of transmembrane signaling.

Structural Determination of Extracellular Proteins

Extracellular segment of receptors is often comprised of multiple domains. As biologically important ligand-binding site resides in this modular structure, structural information is critical for the development of drugs. We use various approaches (e.g., X-ray crystallography, and electron microscopy) to obtain structural/conformational information on the receptors and other biologically important molecules.

Biochemical/biophysical Analysis of Receptor-ligand Interactions

Through functional mapping and mutational analysis, structural determinants on receptor molecule important for the ligand binding are analyzed. Obtained structures are used to design mutant receptors with altered affinity and/or specificity.

Ultrastructural analysis of proteins using electron microscopy/electron tomography

Dynamic nature of protein structures (i.e., conformational change upon lgand binding) can be analyzed by single-particle analysis strategy using electron microscopic methods. We are also developing electron tomography, a technology that enables visualization of protein complexes in cells or tissures. Ultimately, these techniques will unravel poorly understood molecular mechanism underlying the transmembrane signaling.

Establishment of an efficient, large-scale recombinant protein production system using mammalian cells

In order to facilitate structural analysis of extracellular proteins that have intrinsic difficulty in recombinant production, high-capacity production system using mammalian cells is established. Use of a unique cell line with carbohydrate-processing deficiency enables production of recombinant glycoproteins with homogeneous sugar chains, which would be ideal for crystallization/structure determination.

0X-ray crystal structure of a headpiece fragment of integrin α5β1, the fibronectin receptor. The binding mode of the ligand (RGD peptide) is shown in the inset.

1Artificial "synapse" at cell-cell junction visualized using correlation light-electron microscopy (CLEM).

Bibliography

Nagae, M., Re, S., Mihara, E., Nogi, T., Sugita, Y., and Takagi, J. Crystal structure of α5β1 integrin ectodomain: Atomic details of the fibronectin receptor. J Cell Biol 197 , 131 - 140 (2012)

Kishimoto-Okada, A., Murakami, S., Ito, Y., Horii, N., Furukawa, H., Takagi, J., Iwasaki, K. Comparison of the envelope architecture of E. coli using two methods: CEMOVIS and cryo-electron tomography J. Electron Microsc. 59 , 419 - 426 (2010)

Nogi, T., Yasui, N., Mihara, E., Matsunaga, Y., Noda, M., Yamashita, N., Toyofuku, T., Goshima, Y., Kumanogoh, A, and Takagi, J. Structural basis for semaphorin signaling through the plexin receptor. Nature 467 , 1123 - 1127 (2010)

Contact

http://www.protein.osaka-u.ac.jp/rcsfp/synthesis/

http://www.protein.osaka-u.ac.jp/rcsfp/synthesis/