The mitochondrial protein, the voltage-dependent anion channel (VDAC), is implicated in the control of apoptosis, including via its interaction with the pro- and antiapoptotic proteins. We previously demonstrated the direct interaction of Bcl2 with VDAC, leading to reduced channel conductance. VDAC1-based peptides interacted with Bcl2 to prevent its antiapoptotic activity. Here, using a variety of approaches, we show the interaction of the antiapoptotic protein, Bcl-xL, with VDAC1 and reveal that this interaction mediates Bcl-xL protection against apoptosis. C-terminally truncated Bcl-xL(Δ21) interacts with purified VDAC1, as revealed by microscale thermophoresis and as reflected in the reduced channel conductivity of bilayer-reconstituted VDAC1. Overexpression of Bcl-xL prevented staurosporine-induced apoptosis in cells expressing native VDAC1 but not certain VDAC1 mutants. Having identified mutations in VDAC1 that interfere with the Bcl-xL interaction, certain peptides representing VDAC1 sequences, including the N-terminal domain, were designed and generated as recombinant and synthetic peptides. The VDAC1 N-terminal region and two internal sequences were found to bind specifically, and in a concentration- and time-dependent manner, to immobilized Bcl-xL(Δ21), as revealed by surface plasmon resonance. Moreover, expression of the recombinant peptides in cells overexpressing Bcl-xL prevented protection offered by the protein against staurosporine-induced apoptosis. These results point to Bcl-xL acting as antiapoptotic protein, promoting tumor cell survival via binding to VDAC1. These findings suggest that interfering with Bcl-xL binding to the mitochondria by VDAC1-based peptides may serve to induce apoptosis in cancer cells and to potentiate the efficacy of conventional chemotherapeutic agents.
"mitochondrial membrane, VDACs interact directly with a plethora of pro-and anti-apoptotic factors, either members of the Bcl-2 protein family such as Bcl-xL (Arbel et al., 2012; Malia and Wagner, 2007), Bid/tBid(Rostovtseva et al., 2004), Bax/Bak, (Shimizu et al., 2001), hexokinases (Godbole et al., 2013) or Bnip3 (Chaanine et al., 2013). The precise molecular functions and stoichiometries of these VDAC/apoptotic factor complexes are poorly understood, although tentative structural predictions for several VDAC/effector complexes have been suggested (G.Veresov and Davidovskii, 2014). "
[Show abstract][Hide abstract] ABSTRACT: Abstract Voltage dependent anion channels (VDAC) are the most abundant proteins in the outer mitochondrial membrane. Although they are essential in metabolite exchange, cell defense and apoptosis, the molecular mechanism of these VDAC-mediated processes remains elusive. Here we review recent progress in terms of VDACs' structure and regulation with a special focus on the molecular aspects of gating and the interaction with effector proteins.
"Measuring the thermophoretic behaviour of a protein in the presence of differing ligand concentrations by MST allows quantitative analysis of molecular interactions in solution on the microlitre scale. The MST technique has previously been used to investigate protein-protein2122, small organic molecule-protein232425 and antibody-protein interactions26. "
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.
"Alternatively, dimers and higher oligomers of VDAC1 might form the conduit for the efflux of cyt c (Shoshan-Barmatz et al., 2010). Binding of anti-apoptotic Bcl-2 and BclxL to VDAC1 (with resulting inhibition of porin) (Shimizu et al., 2000) has an anti-apoptotic action (e.g., Arbel et al., 2012). In contrast, block of VDAC1 by the phosphorothioate oligonucleotide G3139 (Tan, 2012) or by avicins (plant saponins with anticancer activity) is pro-apoptotic, presumably by reducing flux of metabolites across the OMM (Haridas et al., 2007). "
[Show abstract][Hide abstract] ABSTRACT: Several types of channels play a role in the maintenance of ion homeostasis in subcellular organelles including endoplasmatic reticulum, nucleus, lysosome, endosome, and mitochondria. Here we give a brief overview of the contribution of various mitochondrial and other organellar channels to cancer cell proliferation or death. Much attention is focused on channels involved in intracellular calcium signaling and on ion fluxes in the ATP-producing organelle mitochondria. Mitochondrial K(+) channels (Ca(2+)-dependent BKCa and IKCa, ATP-dependent KATP, Kv1.3, two-pore TWIK-related Acid-Sensitive K(+) channel-3 (TASK-3)), Ca(2+) uniporter MCU, Mg(2+)-permeable Mrs2, anion channels (voltage-dependent chloride channel VDAC, intracellular chloride channel CLIC) and the Permeability Transition Pore (MPTP) contribute importantly to the regulation of function in this organelle. Since mitochondria play a central role in apoptosis, modulation of their ion channels by pharmacological means may lead to death of cancer cells. The nuclear potassium channel Kv10.1 and the nuclear chloride channel CLIC4 as well as the endoplasmatic reticulum (ER)-located inositol 1,4,5-trisphosphate (IP3) receptor, the ER-located Ca(2+) depletion sensor STIM1 (stromal interaction molecule 1), a component of the store-operated Ca(2+) channel and the ER-resident TRPM8 are also mentioned. Furthermore, pharmacological tools affecting organellar channels and modulating cancer cell survival are discussed. The channels described in this review are summarized on Figure 1. Overall, the view is emerging that intracellular ion channels may represent a promising target for cancer treatment.
Frontiers in Physiology 09/2013; 4:227. DOI:10.3389/fphys.2013.00227 · 3.53 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.