VDAC1: from structure to cancer therapy

Abstract

Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to cancer. Found at the outer mitochondrial membrane, VDAC1 assumes a crucial position in the cell, controlling the metabolic cross-talk between mitochondria and the rest of the cell. Moreover, its location at the boundary between the mitochondria and the cytosol enables VDAC1 to interact with proteins that mediate and regulate the integration of mitochondrial functions with other cellular activities. As a metabolite transporter, VDAC1 contributes to the metabolic phenotype of cancer cells. This is reflected by VDAC1 over-expression in many cancer types, and by inhibition of tumor development upon silencing VDAC1 expression. Along with regulating cellular energy production and metabolism, VDAC1 is also a key protein in mitochondria-mediated apoptosis, participating in the release of apoptotic proteins and interacting with anti-apoptotic proteins. The involvement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space is discussed, as is VDAC1 oligomerization as an important step in apoptosis induction. VDAC also serves as an anchor point for mitochondria-interacting proteins, some of which are also highly expressed in many cancers, such as hexokinase (HK), Bcl2, and Bcl-xL. By binding to VDAC, HK provides both metabolic benefit and apoptosis-suppressive capacity that offers the cell a proliferative advantage and increases its resistance to chemotherapy. VDAC1-based peptides that bind specifically to HK, Bcl2, or Bcl-xL abolished the cell's abilities to bypass the apoptotic pathway. Moreover, these peptides promote cell death in a panel of genetically characterized cell lines derived from different human cancers. These and other functions point to VDAC1 as a rational target for the development of a new generation of therapeutics.

Keywords: VDAC protein; anti-apoptotic Bcl-2; apoptosis; cancer metabolism; hexokinase; mitochondrial porin.

FIGURE 1

Schematic representation of VDAC1 as a multi-functional channel and convergence point for a variety of cell survival and cell death signals. The various functions of VDAC1 include control of the metabolic cross-talk between the mitochondria and the rest of the cell, cellular energy homeostasis by transporting ATP/ADP and NADH between the inter-membrane space and the cytosol and by binding HK, signaling by transporting Ca²⁺, ROS release to the cytosol and apoptosis, both by binding to the apoptosis regulatory proteins, Bcl-2 family and HK. Also presented are the Ca²⁺ influx and efflux transport systems in the outer and inner mitochondrial membranes and Ca²⁺-mediated regulation of the tricarboxylic acid (TCA) cycle. The activation of pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH) and α-ketoglutarate dehydrogenase (αKGDH) by intra-mitochondrial Ca²⁺, leading to enhanced activity of the TCA cycle, is shown. The electron transport chain (ETC) and the ATP synthase (F_oF₁) are also presented. VDAC in the OMM is presented as a Ca²⁺ channel. In the IMM, the uptake of Ca²⁺ into the matrix is mediated by a Ca²⁺-selective transporter, the mitochondrial Ca²⁺ uniporter (MCU), regulated by a calcium-sensing accessory subunit (MICU1). Ca²⁺ efflux is mediated by NCLX, a Na⁺/Ca²⁺ exchanger. High levels of matrix Ca²⁺ accumulation trigger the opening of the PTP, a fast Ca²⁺ release channel. Molecular fluxes are indicated by arrows.

FIGURE 2

Proposed three-dimensional structure of VDAC1. (A) Side-view of the X-ray crystal structure of mouse VDAC1 (Ujwal et al., 2008) in a ribbon representation. The β-barrel is formed by 19 β-strands and the N-terminal helix is folded into the pore interior. β-strands 1 and 19 are parallel and colored blue. The C- and N-termini are annotated as C and N, respectively. Loops and unstructured regions are colored green. (B) Top-view of VDAC1 with the N-terminal inside the pore. (C) VDAC1 in a proposed conformation with the N-terminal outside the channel where it can interact with other proteins (PDB code: 3EMN).

FIGURE 3

Channel properties of bilayer-reconstituted purified VDAC1. Bilayer-reconstituted VDAC single and multi-channel activity was assayed as described previously (Gincel et al., 2000). Purified VDAC (1–5 ng) was reconstituted into a PLB. In (A), a typical activity recording of VDAC incorporated into a PLB is presented as current traces obtained in response to voltage steps from 0 mV to either -10 or -60 mV. In symmetric solution (1 M NaC), when the voltage was changed from -0 to 10 mV, the channel opens and remains stable in this conformation for up to 2 h. However, when the voltage was changed from 0 to -60 mV, the current first increased, due to a greater driving force. However, within less than 1 s, channel conductance decreased and VDAC assumed multiple conductance states. The dashed line indicates the zero current level, while the sub-states of the channel are indicated by arrowheads. In (B), Multi-channel recordings of the average steady-state conductance of VDAC are presented as a function of voltage. The conductance (G_o) at a given voltage was normalized to the conductance at -10 mV (G_max). Each point is the average of three experiments. This voltage-dependent behavior is well known for VDAC.

FIGURE 4

Schematic representation of proposed models for the release of apoptogenic proteins from the mitochondrial inter-membrane space mediating the mitochondrial death decision. Different models explaining how OMM permeability changes during apoptosis induction, allowing the release of apoptogenic factors, such as Cyto c. (A) A permeability transition pore (PTP) provides the apoptogenic proteins release pathway. It is proposed that a large conductance pore-forming complex, the PTP, composed of VDAC at the OMM, ANT at the IMM and CypD in the matrix, allows apoptogenic protein release. (B) VDAC closure and OMM rupture serves as the cytochrome c release pathway. Prolonged VDAC closure leads to mitochondrial matrix swelling, OMM rupture, and hence, the appearance of a non-specific release pathway for apoptogenic proteins. (C) Bax activation followed by its oligomerization resulting in OMM permeabilization. Upon apoptosis induction, Bax became associated with mitochondria as a large oligomer/complex, forming a Cyto c-conducting channel in the OMM. (D) A pore formed by oligomerized forms of Bax and Bak after their activation by tBID. BH3-only proteins (e.g., Bid) induce oligomerization of Bax/Bak on the OMM, resulting in Bax activation and OMM permeabilization. (E) MAC as the release pathway. The mitochondrial apoptosis-induced channel, MAC, is a high-conductance channel that forms during early apoptosis and is a putative cytochrome c release channel. MAC formation occurs without loss of outer membrane integrity and depolarization. Members of the Bcl-2 family of proteins regulate apoptosis by controlling the formation of MAC. (F) A Bax- and VDAC-based hetero-oligomer mediates cytochrome c release. The interaction of pro-apoptotic proteins (Bax/Bak) with VDAC forms a cytochrome c release pathway. (G) A lipid channel formed by the lipid, ceramide. Ceramides were shown to induce apoptosis via direct action on mitochondria. A self-assembled ceramide channel is proposed to act as the apoptotic protein release pathway. (H) Oligomeric VDAC1 as a channel for the release of apoptotic proteins. A protein-conducting channel is formed within a VDAC1 homo-oligomer. VDAC1 oligomerization thus functions in mitochondria-mediated apoptosis (see VDAC1 Oligomerization and Release of Cytochrome c). The dynamic equilibrium between VDAC monomeric and oligomeric states can be regulated by various factors, such as Ca²⁺, oxidative stress and cytochrome c.

FIGURE 5

Apoptosis induction and VDAC1 oligomerization. VDAC1 oligomerization as induced by apoptosis inducers and revealed by EGS-based cross-linking. HeLa cells were incubated for 16 h with STS (0.2 μM) or selenite (8 μM), as described previously (Keinan et al., 2010), washed with PBS and incubated with EGS (300 μM) at 30^°C for 10 min, followed by SDS-PAGE (10% acrylamide) and Western blotting using anti-VDAC1 antibodies. VDAC1 monomers, dimers, trimers, tetramers, and multimers are indicated. The arrow indicates an anti-VDAC1 antibody-labeled protein band migrating below the position of monomeric VDAC1. The positions of molecular weight protein standards are also provided.

FIGURE 6

Detachment of mitochondrial-bound HK-I-GFP induced by STS. To demonstrate HK-I binding to mitochondria (i.e., VDAC) as well as detachment, a HK-I–GFP fusion protein was expressed in HEK–T cells. Confocal fluorescence microscopy showed that in control cells expressing HK-I–GFP, the fluorescence is punctuated, as expected for mitochondrial distribution. On the other hand, induction of apoptosis by STS detaches mitochondrial-bound HK-I–GFP. HEK–T cells were transfected to express HK-I–GFP and after 48 h were exposed to STS (0.8 μM), stained with 25 nM of the mitochondrial marker MitoTracker Red and visualized with confocal microscopy. The punctuated HK-I–GFP fluorescence, originally co-localized with mitochondria (control), as also reflected in the co-localization with MitoTracker Red (Merge panel), was converted to diffuse labeling of the cytosol after exposure to STS. Images are representative microscopic fields from one of three similar experiments (scale bar = 5 μm).

FIGURE 7

Reactive oxygen species occlusion and degradation in mitochondria can prevent cell damage. A schematic presentation summarizing current knowledge and proposed control mechanisms for ROS production, neutralization, and cellular effects (see VDAC1 Function in ROS Release, ROS-mediated Apoptosis and Interaction with NO). Free radicals generated by the electron transport chain are generally regarded as toxic metabolites and, as such, are degraded by specialized enzymes, namely catalases, peroxidases, superoxide dismutases, and glutathione peroxidase. Mitochondrial ROS cross the OMM via VDAC. The ROS that escapes catalytic removal can cause oxidative damage () to mitochondria, and when released from the mitochondria, can damage cellular proteins, lipids, and DNA. In addition, ROS can induce apoptosis. ROS is proposed to induce Cyto c release by oxidizing the mitochondria-specific phospholipid, cardiolipin (CL). ROS-oxidized CL has a markedly lower affinity for Cyto c, thus rendering Cyto c free in the inter-membrane space. ROS can also induce VDAC oligomerization to yield a mega-channel mediating Cyto c release. VDAC inhibitors (e.g., RuR, AzRu, DIDS) and hexokinase (HK) can prevent ROS release to the cytosol. CuZnSOD, cytosolic (copper–zinc-containing) superoxide dismutases; MnSOD, mitochondrial (manganese-containing) superoxide dismutases; GPx, glutathione peroxidase.