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Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

Viard M, Garg H, Blumenthal R, Raviv Y - BMC Cell Biol. (2009)

Bottom Line: Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced.Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nanobiology Program, Center of Cancer Research, National Cancer Institute, Frederick, Maryland, USA. viardm@mail.ncifcrf.gov

ABSTRACT

Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.

Results: INA treatment of various cell lines followed by irradiation with UV light (310-380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.

Conclusion: INA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.

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INA-UV treatment affects the translational diffusion of proteins in the cell membrane. Fluorescence recovery after photobleaching (FRAP) of CCR5-GFP expressed in Hela cells treated or not with 20 μM INA-UV. A. Fluorescence signal recovery as a function of time after photobleaching. B. Computed mobile fraction of CCR5-GFP in control cells and cells treated with 20 μM INA-UV. Data are mean ± S.D of seven measurements.
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Figure 9: INA-UV treatment affects the translational diffusion of proteins in the cell membrane. Fluorescence recovery after photobleaching (FRAP) of CCR5-GFP expressed in Hela cells treated or not with 20 μM INA-UV. A. Fluorescence signal recovery as a function of time after photobleaching. B. Computed mobile fraction of CCR5-GFP in control cells and cells treated with 20 μM INA-UV. Data are mean ± S.D of seven measurements.

Mentions: Our efforts to identify the mechanism of action of INA-UV mediated apoptosis induction suggest a direct effect on membrane proteins. Although CXCR4 and MRP1 functions were inhibited by INA-UV treatment, this was not the case with IGF1R function. Photoactivation of INA results in the covalent binding of the probe to membrane proteins in the lipidic bilayer with no particular specificity. This may alter the function of proteins via a variety of mechanisms including the translational mobility of receptors in the plasma membrane. Hence we sought to assess the effects of INA modifications on the mobility of membrane proteins using CCR5-GFP as a reporter. FRAP (fluorescence recovery after photobleaching) is a commonly used method to detect the mobility of fluorescently tagged protein and lipids in cells. We transiently expressed CCR5-GFP in Hela cells and performed FRAP measurements with or without INA-UV treatment. As shown in figure 9 there is a dramatic impact of INA-UV treatment on fluorescence recovery of CCR5-GFP. Curve-fitting recovery after photobleaching shows that INA-UV treatment greatly affects the mobile fraction of CCR5-GFP molecules (figure 9B). INA-UV treatment results in the immobilization of CCR5-GFP while the effect on the mobility of lipids is not significant [17]. These results suggest that INA via direct interaction with transmembrane protein alters the function and mobility of various cellular receptors.


Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

Viard M, Garg H, Blumenthal R, Raviv Y - BMC Cell Biol. (2009)

INA-UV treatment affects the translational diffusion of proteins in the cell membrane. Fluorescence recovery after photobleaching (FRAP) of CCR5-GFP expressed in Hela cells treated or not with 20 μM INA-UV. A. Fluorescence signal recovery as a function of time after photobleaching. B. Computed mobile fraction of CCR5-GFP in control cells and cells treated with 20 μM INA-UV. Data are mean ± S.D of seven measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666636&req=5

Figure 9: INA-UV treatment affects the translational diffusion of proteins in the cell membrane. Fluorescence recovery after photobleaching (FRAP) of CCR5-GFP expressed in Hela cells treated or not with 20 μM INA-UV. A. Fluorescence signal recovery as a function of time after photobleaching. B. Computed mobile fraction of CCR5-GFP in control cells and cells treated with 20 μM INA-UV. Data are mean ± S.D of seven measurements.
Mentions: Our efforts to identify the mechanism of action of INA-UV mediated apoptosis induction suggest a direct effect on membrane proteins. Although CXCR4 and MRP1 functions were inhibited by INA-UV treatment, this was not the case with IGF1R function. Photoactivation of INA results in the covalent binding of the probe to membrane proteins in the lipidic bilayer with no particular specificity. This may alter the function of proteins via a variety of mechanisms including the translational mobility of receptors in the plasma membrane. Hence we sought to assess the effects of INA modifications on the mobility of membrane proteins using CCR5-GFP as a reporter. FRAP (fluorescence recovery after photobleaching) is a commonly used method to detect the mobility of fluorescently tagged protein and lipids in cells. We transiently expressed CCR5-GFP in Hela cells and performed FRAP measurements with or without INA-UV treatment. As shown in figure 9 there is a dramatic impact of INA-UV treatment on fluorescence recovery of CCR5-GFP. Curve-fitting recovery after photobleaching shows that INA-UV treatment greatly affects the mobile fraction of CCR5-GFP molecules (figure 9B). INA-UV treatment results in the immobilization of CCR5-GFP while the effect on the mobility of lipids is not significant [17]. These results suggest that INA via direct interaction with transmembrane protein alters the function and mobility of various cellular receptors.

Bottom Line: Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced.Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nanobiology Program, Center of Cancer Research, National Cancer Institute, Frederick, Maryland, USA. viardm@mail.ncifcrf.gov

ABSTRACT

Background: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated.

Results: INA treatment of various cell lines followed by irradiation with UV light (310-380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor.

Conclusion: INA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.

Show MeSH
Related in: MedlinePlus