<i>In Cellulo</i> Mapping of Subcellular Localized Bilirubin

Park, Jong‐Seok; Nam, Eunju; Lee, Hye-Kyeong; Lim, Mi Hee; Rhee, Hyun‐Woo

doi:10.1021/acschembio.6b00017

Cited by 21 publications

(21 citation statements)

References 45 publications

(77 reference statements)

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“…In this context, the discovery of UnaG protein from freshwater eel (Kumagai et al, 2013), a protein belonging to FABPs that selectively binds bilirubin and generates a fluorescent signal, improved the detection of endogenously generated bilirubin enabling studies of its subcellular localization and transport. Endogenous UCB seems to be mainly located at ER cytosolic membranes, associated with HMOX1/BLVRA activities and has also been detected in the nucleus and mitochondria (Park et al, 2016). However, intracellular transport mechanisms of endogenous UCB and its role in subcellular compartments remain to be explored.…”

Section: Biosynthesis Metabolism Bioavailability and Activitymentioning

confidence: 99%

Heme Oxygenase Dependent Bilirubin Generation in Vascular Cells: A Role in Preventing Endothelial Dysfunction in Local Tissue Microenvironment?

2020

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Among antioxidants in the human body, bilirubin has been recognized over the past 20 years to afford protection against different chronic conditions, including inflammation and cardiovascular disease. Moderate increases in plasma concentration and cellular bilirubin generation from metabolism of heme via heme oxygenase (HMOX) in virtually all tissues can modulate endothelial and vascular function and exert antioxidant and antiinflammatory roles. This review aims to provide an up-to-date and critical overview of the molecular mechanisms by which bilirubin derived from plasma or from HMOX1 activation in vascular cells affects endothelial function. Understanding the molecular actions of bilirubin may critically improve the management not only of key cardiovascular diseases, but also provide insights into a broad spectrum of pathologies driven by endothelial dysfunction. In this context, therapeutic interventions aimed at mildly increasing serum bilirubin as well as bilirubin generated endogenously by endothelial HMOX1 should be considered.

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Section: Biosynthesis Metabolism Bioavailability and Activitymentioning

confidence: 99%

Heme Oxygenase Dependent Bilirubin Generation in Vascular Cells: A Role in Preventing Endothelial Dysfunction in Local Tissue Microenvironment?

2020

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“…Moreover, UnaG has gained attention as a fluorescent probe because of its high fluorescence QE and the features of instantaneous and oxygen-independent fluorophore formation, as compared to GFP-family FPs in general, which require several hours for formation of fluorophores by post-transcriptional modification involving a molecular oxygen. Indeed, by employing these advantages, UnaG already has been applied to live-cell fluorescence imaging in a number of studies, as a fluorescent indicator for protein-protein interactions by bi-molecular fluorescence complementation (13), as a fluorescence sensor for hypoxic conditions in tissues (14), and as a fluorescent probe for BR distribution in cells (15).…”

Section: Introductionmentioning

confidence: 99%

Two Distinct Fluorescence States of the Ligand-Induced Green Fluorescent Protein UnaG

Shitashima

Togo

Kumagai

et al. 2017

Biophysical Journal

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UnaG is a recently discovered ligand-induced fluorescent protein that utilizes bound bilirubin (BR) as its fluorophore. The fluorescence of the UnaG-BR complex (holoUnaG) compares in quantum efficiency to that of enhanced green fluorescent protein, but it is superior in that the fluorophore formation is instantaneous and not dependent on oxygen; hence, much attention has been paid to UnaG as a new fluorescent probe. However, many important molecular properties of fluorescent probes remain unknown, such as the association/dissociation rates of BR, which determine the stability thereof, and the dispersibility of UnaG in aqueous solutions, which influence the functions of labeled proteins. In this study, we found, in the process of investigating the association rate, that the holoUnaG takes two distinct fluorescence states, which we named holoUnaG and holoUnaG. The holoUnaG initially forms after binding BR and then changes to the brighter holoUnaG by a reversible intra-molecular reaction, thereby finally reaching an equilibrium between the two states. Spectroscopic analysis indicated that the intra-molecular reaction was associated not with a chemical change of BR but with a change in the environmental conditions surrounding BR. We also revealed that the molecular brightness ratio and equilibrium population ratio of the two states (holoUnaG/holoUnaG) were 1:3.9 and 6:4, respectively, using photon number counting analysis. From these results, we have suggested a novel schema, to our knowledge, for the formation of the UnaG and BR complex system and have determined the various rate constants associated therein. Additionally, using analytical ultracentrifugation, we established that UnaG in the apo-state (apoUnaG) and the holoUnaG are monomeric in aqueous solution. These findings provide not only key information for the practical use of UnaG as a fluorescent probe, but also the possibility for development of a brighter UnaG mutant by genetic engineering to constitutive holoUnaG.

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“…The fluorogenic center of ligand-bound UnaG (holoUnaG) is considered as BR rather than the protein, because the protein without the ligand (apoUnaG) is not fluorescent. The high contrasts between holoUnaG and apoUnaG and between holoUnaG and BR, as well as the highly specific binding between BR and UnaG, allowed efficient sensing of BR in vitro and in cells 32,33 . The application area of UnaG was further extended to monitor the activity of membrane transporters 34 , to regulate the activity of a POI 35 , to report the protein-protein interactions 36 , to investigate hypoxia states of cells 37,38 , and to monitor calcium and BR simultaneously 39 .…”

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confidence: 99%

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

et al. 2020

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We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of highquality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on-and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for highperformance super-resolution imaging.

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In Cellulo Mapping of Subcellular Localized Bilirubin

Cited by 21 publications

References 45 publications

Heme Oxygenase Dependent Bilirubin Generation in Vascular Cells: A Role in Preventing Endothelial Dysfunction in Local Tissue Microenvironment?

Heme Oxygenase Dependent Bilirubin Generation in Vascular Cells: A Role in Preventing Endothelial Dysfunction in Local Tissue Microenvironment?

Two Distinct Fluorescence States of the Ligand-Induced Green Fluorescent Protein UnaG

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

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