Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

  title={Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching},
  author={Ilka Starke and Kathryn Marie Johnson and Jan Petersen and Peter Gr{\"a}ber and Anthony Opipari and Gary D Glick and Michael B{\"o}rsch},
  booktitle={SPIE BiOS},
Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C… 

Visualizing Mitochondrial FoF1-ATP Synthase as the Target of the Immunomodulatory Drug Bz-423

It is reported how Förster resonance energy transfer can be employed to colocalize the enzyme and the fluorescently tagged Bz-423 within the mitochondria of living cells with nanometer resolution.

Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

Their mitochondrial nano-environment was investigated by FLIM and superresolution microscopy in living human cells and different lifetimes and anisotropy values were found.

Ligand-induced oligomerization of the human GPCR neurotensin receptor 1 monitored in living HEK293T cells

Oligomerization of NTSR1 was monitored before and after stimulation of the receptor with its ligand by FLIM and homoFRET microscopy and by time-lapse imaging using structured illumination microscopy (SIM).

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

G protein-coupled receptor 1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET, and agonist binding to NTSR1 was demonstrated by time-resolved single-molecule Förster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

Observing monomer: dimer transitions of neurotensin receptors 1 in single SMALPs by homoFRET and in an ABELtrap

The oligomerization state of the human NTSR1 tagged with mRuby3 is reported by dissolving the plasma membranes of living HEK293T cells into 10 nm-sized soluble lipid nanoparticles by addition of styrene-maleic acid copolymers (SMALPs).



Mechanistic basis for therapeutic targeting of the mitochondrial F1F0-ATPase.

The data support a model in which the interplay of these features underlies the favorable properties of Bz-423, and represent key criteria for the development of therapeutic F 1 F o -ATPase inhibitors, which should have utility across a range of areas.

The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP Synthase: Emerging New Roles in Mitochondrial Pathophysiology

Findings are consistent with the demonstration that dimers of ATP synthase generate Ca2+-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synth enzyme is directly involved in PTP formation, although the underlying mechanism remains to be established.

Targeting cytochrome C oxidase in mitochondria with Pt(II)-porphyrins for photodynamic therapy

Electron microscopy revealed the disruption of the mitochondrial christae as a primary step of Photodynamic Therapy (PDT), and time resolved phosphorescence measurements identified COX as the binding site for Pt(II)-TMPyP in living HeLa cells.

Dimers of mitochondrial ATP synthase form the permeability transition pore

It is shown that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), which sensitizes the PTP to Ca2+.

The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL trap

This work labels F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer and compares FRET changes in single F1 and FRET histograms for different biochemical conditions to evaluate the proposed regulatory mechanism.

NMR studies of an immunomodulatory benzodiazepine binding to its molecular target on the mitochondrial F(1)F(0)-ATPase.

Data suggest that 1 might inhibit the enzyme through an allosteric mechanism where binding results in conformational changes that perturb the OSCP-F(1) interface resulting in disrupted communication between the peripheral stalk and the F(1)-domain of the enzyme.

Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

Progress is reported of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

The immunomodulatory benzodiazepine Bz-423 inhibits B-cell proliferation by targeting c-myc protein for rapid and specific degradation.

It is shown that the basis for the antiproliferative effects of Bz-423 is the rapid and specific depletion of c-myc protein, which is coupled to growth-suppressing effects on key regulators of proliferation and cell cycle progression.

Apoptotic Signaling Activated by Modulation of the F0F1-ATPase: Implications for Selective Killing of Autoimmune Lymphocytes

The apoptotic mechanism elucidated in Jurkat cells provides important clues into the cell-type- and disease-selective effects of Bz-423 in MRL-lpr mice.