Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells

Shuya Kasai; Shinji Kajimoto; Yuma Ito; Tomo Saito; Ken Ichi Yasumoto; Makio Tokunaga; Kumiko Sakata-Sogawa; Hiroshi Fukumura; Kazuhiro Sogawa

doi:10.1093/jb/mvw068

Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-x_L in living cells

Shuya Kasai, Shinji Kajimoto, Yuma Ito, Tomo Saito, Ken Ichi Yasumoto, Makio Tokunaga, Kumiko Sakata-Sogawa, Hiroshi Fukumura, Kazuhiro Sogawa

Research output: Contribution to journal › Article › peer-review

3 Citations (Scopus)

Abstract

Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color singlemolecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1a or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-x_L, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

Original language	English
Pages (from-to)	291-296
Number of pages	6
Journal	Journal of Biochemistry
Volume	161
Issue number	3
DOIs	https://doi.org/10.1093/jb/mvw068
Publication status	Published - 2017

Keywords

Conformational change
Dual-color single molecule imaging
FLIM-FRET analysis

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10.1093/jb/mvw068

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title = "Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells",

abstract = "Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color singlemolecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1a or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.",

keywords = "Conformational change, Dual-color single molecule imaging, FLIM-FRET analysis",

author = "Shuya Kasai and Shinji Kajimoto and Yuma Ito and Tomo Saito and Yasumoto, {Ken Ichi} and Makio Tokunaga and Kumiko Sakata-Sogawa and Hiroshi Fukumura and Kazuhiro Sogawa",

note = "Publisher Copyright: {\textcopyright} 2016 The Authors.",

year = "2017",

doi = "10.1093/jb/mvw068",

language = "English",

volume = "161",

pages = "291--296",

journal = "Journal of Biochemistry",

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T1 - Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-xL in living cells

AU - Kasai, Shuya

AU - Kajimoto, Shinji

AU - Ito, Yuma

AU - Saito, Tomo

AU - Yasumoto, Ken Ichi

AU - Tokunaga, Makio

AU - Sakata-Sogawa, Kumiko

AU - Fukumura, Hiroshi

AU - Sogawa, Kazuhiro

PY - 2017

Y1 - 2017

N2 - Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color singlemolecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1a or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

AB - Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color singlemolecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1a or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET. Reduced lifetimes of the donor were partially restored by coexpression of HIF-1α or Bcl-xL, binding proteins of IPAS in the nucleus and mitochondria, respectively. This alteration in lifetimes demonstrates that conformational changes occurred in IPAS by their binding.

KW - Conformational change

KW - Dual-color single molecule imaging

KW - FLIM-FRET analysis

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DO - 10.1093/jb/mvw068

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C2 - 28003430

AN - SCOPUS:85019539290

SN - 0021-924X

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JO - Journal of Biochemistry

JF - Journal of Biochemistry

IS - 3

ER -

Conformational changes in inhibitory PAS domain protein associated with binding of HIF-1α and Bcl-x_L in living cells

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Keywords

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