Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs 1 . Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)-a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions N.Y. designed and performed the RNAi and VDAC overexpression, quantitative PCR, erastin analogue viability and chemical characterization experiments. E.Z. performed two-dimensional western analysis, PARP-1 and pro-caspase-3 cleavage, and cytochrome c release experiments. E.Z. and N.Y. performed transmission electron microscopy experiments. A.J.B., D.J.F. and N.Y. performed the NADH oxidation and direct binding experiments. W.S.Y. characterized sensitivity to erastin in the BJderived cell series. A.J.W. performed the MEK1/2 inhibitor experiment. I.S. and A.J.B. synthesized erastin analogues. R.S. and S.L.L. provided BRAF shRNAs, analysis of BRAF knockdown and the phospho-ERK western analysis. J.M.P., J.J.B. and S.S. were responsible for setting up the technology platform to pull down proteins binding to small molecule compounds. M.v.R. and J.M.P. performed the pull-down experiments. J.J.B., J.M.P. and S.S. designed, reviewed and supervised the pull-down experiments, and contributed to the analysis of the data. B.R.S. conceived of and supervised the project, designed and analysed experiments, and performed the anti-oxidant studies. B.R.S. and N.Y. prepared the manuscript. (Fig. 1a , Supplementary Fig. 1 and ref. 3 ). This cell death was not dependent on the rate of cell division, nor was it idiosyncratic to these cells ( Fig. 1a and Supplementary Fig. 2), because cell lines engineered in a similar way responded similarly. Author InformationWe found th...
SummaryGrowth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/ LpoB and their PBP docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth.
SummaryThe murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylasetranspeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.
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