A new anticancer toxin based on HER2/neu-specific DARPin and photoactive flavoprotein miniSOG

Прошкина, Г. М.; Shilova, O N; Ryabova, Anastasia V.; Stremovskiy, Oleg A.; Deyev, Sergey M.

doi:10.1016/j.biochi.2015.08.013

Cited by 46 publications

(37 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The construction of the plasmid encoding DARPin-miniSOG and the expression and purification procedures were previously described in detail (16). The construction of the plasmid encoding DARPin-miniSOG and the expression and purification procedures were previously described in detail (16).…”

Section: Expression and Purification Of Darpin-minisogmentioning

confidence: 99%

“…The increase in fluorescence after aldehyde fixation is considered to be common and is thought to be caused by Schiff acid-base reactions with amine groups generating adducts that are intensely fluorescent (13). This recombinant protein was originally designed as a targeted photosensitizer miniSOG (15) specifically binding to the receptor ErbB2 (HER2) on the cell surface because of DARPin_9-29 module (16). Another commonly used fixative is ethanol.…”

mentioning

confidence: 99%

“…As a model of a problematic system, we used SK-BR-3 cells, which have relatively high autofluorescence, stained with the dim green fluorophore DARPin-miniSOG. This recombinant protein was originally designed as a targeted photosensitizer miniSOG (15) specifically binding to the receptor ErbB2 (HER2) on the cell surface because of DARPin_9-29 module (16). The fluorescent properties of DARPin-miniSOG are provided by flavin mononucleotide (FMN) cofactor of miniSOG.…”

mentioning

confidence: 99%

See 2 more Smart Citations

The effect of trypan blue treatment on autofluorescence of fixed cells

2017

View full text Add to dashboard Cite

Controlling background fluorescence remains an important challenge in flow cytometry, as autofluorescence can interfere with the detection of chromophores. Furthermore, experimental procedures can also affect cellular fluorescence in certain regions of the emission spectrum. In this work, the effects of fixation, permeabilization, and heating on cellular autofluorescence are analyzed in various spectral regions, along with the influence of trypan blue as a quenching dye for these treatments. The impact of these procedures on the staining of SK-BR-3 cells with a dim green fluorophore, a miniSOG (mini Singlet Oxygen Generator) flavoprotein in the form of the recombinant protein DARPin-miniSOG, is also evaluated. The data presented here indicate that fixation of certain types of cells leads to noticeable increase of the autofluorescence. Our results also suggest that trypan blue should be used as an autofluorescence quencher only with bright green emitters since it interferes with the fluorescent signal in a longer-wavelength region of the spectrum and as a result causes reduction of the signal from dim green fluorescent agents. © 2017 International Society for Advancement of Cytometry.

show abstract

Section: Expression and Purification Of Darpin-minisogmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

The effect of trypan blue treatment on autofluorescence of fixed cells

2017

View full text Add to dashboard Cite

show abstract

“…The coding sequence of miniSOG was amplified from the pDARP-miniSOG plasmid [22] using the specific primers oGP13 nucleases AvaI and XbaI and cloned into a pNL1.1.CMV vector digested with the same restriction enzymes. As a result, a pNanoLuc-miniSOG plasmid was obtained containing NanoLuc - and miniSOG -encoding sequences within the same reading frame connected by a linker region under the control of the constitutive promoter CMV.…”

Section: Methodsmentioning

confidence: 99%

Flavoprotein miniSOG Cytotoxisity Can Be Induced By Bioluminescence Resonance Energy Transfer

et al. 2016

View full text Add to dashboard Cite

In this study, we investigated the possibility of phototoxic flavoprotein miniSOG (photosensitizer) excitation in cancer cells by bioluminescence occurring when luciferase NanoLuc oxidizes its substrate, furimazine. We have shown that the phototoxic flavoprotein miniSOG expressed in eukaryotic cells in fusion with NanoLuc luciferase is activated in the presence of its substrate, furimazine. Upon such condition, miniSOG possesses photoinduced cytotoxicity and causes a 48% cell death level in a stably transfected cell line.

show abstract

“…MiniSOG localized to the mitochondria, plasma membrane, or chromatin was shown to mediate blue lightinduced death of mammalian cells in vitro (13). Also, externally added miniSOG (fused to an antibody or DARPin for cell typespecific targeting) possessed high phototoxicity against tumor cells in vitro (14,15). …”

mentioning

confidence: 99%

Lysosome-Associated MiniSOG as a Photosensitizer for Mammalian Cells

et al. 2016

View full text Add to dashboard Cite

Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7–mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis.

show abstract

A new anticancer toxin based on HER2/neu-specific DARPin and photoactive flavoprotein miniSOG

Cited by 46 publications

References 19 publications

The effect of trypan blue treatment on autofluorescence of fixed cells

The effect of trypan blue treatment on autofluorescence of fixed cells

Flavoprotein miniSOG Cytotoxisity Can Be Induced By Bioluminescence Resonance Energy Transfer

Lysosome-Associated MiniSOG as a Photosensitizer for Mammalian Cells

Contact Info

Product

Resources

About