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(Cell Chemical Biology 24, 1259–1275; October 19, 2017) During the cropping of Figure 2H, the image of the p62 row was deleted in error and then remaining labels were shifted down by one row and were therefore out of registration with the images. Figure 2H has now been corrected in the article online and in print; the corrected Figure 2H is also shown below. The authors apologize for this labeling error.

Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.

Abstract: While cytochrome P450 enzymes (CYPs) are implicated in tumor angiogenesis through biosynthesis of epoxyeicosatrienoic acids (EETs), little is known about breast cancer cell-intrinsic CYPs that exhibit epoxygenase activity, such as CYP3A4. In an orthotopic breast cancer model, silencing of epithelial CYP3A4 suppressed angiogenesis-related escape of ER+ breast tumors from dormancy. While the diabetes drug metformin inhibits mitochondrial complex I and inhibits tumor growth, how it does so is unknown. Metformin inhibited CYP epoxygenase activity and co-crystallized in the active site of CYP3A4, hydrogen bonding with arginine 212, allowing the development of hexyl-benzyl-biguanide (HBB) as a CYP3A4 inhibitor using molecular modeling. HBB exhibited more than 10-fold greater potency than metformin for inhibition of ER+ mammary tumor growth and inhibited associated tumor angiogenesis. HBB inhibited EET biosynthesis ∼40-fold more potently than metformin and was ∼40-fold more potent for activation of AMPK phosphorylation. EETs suppressed and CYP silencing promoted AMPK phosphorylation, linking CYPs with AMPK regulation in breast cancer. HBB depolarized mitochondria, reduced oxygen consumption rates and suppressed the Warburg effect, while EETs restored the mitochondrial membrane potential. CYP3A4 silencing and HBB treatment increased reactive oxygen species (ROS) production, suggesting that CYPs suppress cancer cell death, in part, through suppression of ROS. CYP3A4 silencing sensitized breast cancer cells to hormonal therapy and chemotherapy, abrogated by EETs. Because EETs are autocrine, paracrine and endocrine, these results implicate CYPs in tumor growth, in part, through cell-cell mediation of mitochondrial homeostasis and demonstrate the potential of CYP3A4 as a therapeutic target in breast cancer. Citation Format: Zhijun Guo, Irina F. Sevrioukova, Eric Hanse, Ilia Denisov, Xia Zhang, Ting-Lan Chiu, Daniel Swedien, Justin Stamschror, Juan Alvarez, William Marerro Ortiz, Monique Morgan, Michael Maher, Kathryn J. Chavez, Dafydd Thomas, Young Kyung Bae, Jonathan Henriksen, Beverly Norris, Robert J. Schumacher, Henry Wang, Robin Bliss, Haitao Chu, Rebecca Cuellar, Thomas L. Poulos, Stephen G. Sligar, William Atkins, Stephen Schmechel, Jorge Capdevila, John Falck, Ian Blair, Jeffrey P. Jones, Gunda Georg, Kalpna Gupta, Ameeta Kelekar, Elizabeth Amin, David A. Potter. CYP3A4 epoxygenase activity mediates ER+ mammary tumor growth and angiogenesis, in part, through EET biosynthesis and is inhibited by biguanides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3568. doi:10.1158/1538-7445.AM2015-3568

Abstract: Metformin is a widely used biguanide diabetes drug that is associated with decreased breast cancer risk and is currently being studied for treatment and prevention of breast cancer. While metformin and biguanides buformin and phenformin exhibit inhibitory activity against breast cancer in vitro and in vivo, they lack potency (IC50=5-20 mM) and their mechanisms of action remain unclear. More potent biguanides may provide insights into biguanide anti-cancer activity and we therefore studied the novel biguanide N1-hexyl-N5-benzyl-biguanide mesylate (HBB), which potently inhibits the MCF-7 and MDA-MB-231 breast cancer lines (IC50=20 uM for both lines). HBB induces AMPK phosphorylation in both lines at 10 uM concentration, whereas similarly dosed metformin, buformin or phenformin exhibits no activity. HBB also inhibits STAT3 phosphorylation at 10 uM concentration, whereas metformin dosed at 10 uM exhibits no activity. HBB reduced the mitochondrial membrane potential of both lines, but the effect was more prominent in the MDA-MB-231 line. HBB also induced ROS within 2.5 hours of exposure in the MCF-7 and MDA-MB-231 lines and caused rapid necrosis, but not apoptosis. N-acetylcysteine provides partial protection from HBB for MDA-231 line, but not the MCF-7 line. HBB provides proof of principle that highly potent biguanides can be synthesized with at least 250-fold greater potency than metformin, which can provide insights into the cancer inhibitory mechanisms of biguanide drugs. R01 CA113570, Randy Shaver Foundation, CTSI University of Minnesota Citation Format: Zhijun Guo, Kathryn J. Chavez, Juan Alvarez, Xia Zhang, Beverly Norris, Michael Maher, Monique Morgan, Robert J. Schumacher, Rebecca Cuellar, Irina F. Sevrioukova, Thomas L. Poulos, Ilia Denisov, Stephen G. Sligar, Kalpna Gupta, Ian A. Blair, Jorge Capdevila, Ameeta Kelekar, Elizabeth Amin, Gunda Georg, David A. Potter. Breast cancer inhibition by a novel and potent biguanide, N1-hexyl-N5-benzyl-biguanide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2014-2689

Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.