Nurit Keinan

VDAC, a multi-functional mitochondrial protein regulating cell life and death

Research over the past decade has extended the prevailing view of the mitochondrion to include functions well beyond the generation of cellular energy. It is now recognized that mitochondria play a crucial role in cell signaling events, inter-organellar communication, aging, cell proliferation, diseases and cell death. Thus, mitochondria play a central role in the regulation of apoptosis (programmed cell death) and serve as the venue for cellular decisions leading to cell life or death. One of the mitochondrial proteins controlling cell life and death is the voltage-dependent anion channel (VDAC), also known as mitochondrial porin. VDAC, located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, thereby controlling cross-talk between mitochondria and the rest of the cell. VDAC is also a key player in mitochondria-mediated apoptosis. Thus, in addition to regulating the metabolic and energetic functions of mitochondria, VDAC appears to be a convergence point for a variety of cell survival and cell death signals mediated by its association with various ligands and proteins. In this article, we review what is known about the VDAC channel in terms of its structure, relevance to ATP rationing, Ca(2+) homeostasis, protection against oxidative stress, regulation of apoptosis, involvement in several diseases and its role in the action of different drugs. In light of our recent findings and the recently solved NMR- and crystallography-based 3D structures of VDAC1, the focus of this review will be on the central role of VDAC in cell life and death, addressing VDAC function in the regulation of mitochondria-mediated apoptosis with an emphasis on structure-function relations. Understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of functions, all important for cell life and death. This review also provides insight into the potential of VDAC1 as a rational target for new therapeutics.

The VDAC1 N-terminus is essential both for apoptosis and the protective effect of anti-apoptotic proteins

The release of mitochondrial-intermembrane-space pro-apoptotic proteins, such as cytochrome c, is a key step in initiating apoptosis. Our study addresses two major questions in apoptosis: how are mitochondrial pro-apoptotic proteins released and how is this process regulated? Accumulating evidence indicates that the voltage-dependent anion channel (VDAC) plays a central role in mitochondria-mediated apoptosis. Here, we demonstrate that the N-terminal domain of VDAC1 controls the release of cytochrome c, apoptosis and the regulation of apoptosis by anti-apoptotic proteins such as hexokinase and Bcl2. Cells expressing N-terminal truncated VDAC1 do not release cytochrome c and are resistant to apoptosis, induced by various stimuli. Employing a variety of experimental approaches, we show that hexokinase and Bcl2 confer protection against apoptosis through interaction with the VDAC1 N-terminal region. We also demonstrate that apoptosis induction is associated with VDAC oligomerization. These results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the mechanism of cytochrome c release and how anti-apoptotic proteins regulate apoptosis and promote tumor cell survival.

Oligomerization of the mitochondrial protein voltage-dependent anion channel is coupled to the induction of apoptosis.

ABSTRACT Accumulating evidence implicates that the voltage-dependent anion channel (VDAC) functions in mitochondrion-mediated apoptosis and as a critical player in the release of apoptogenic proteins, such as cytochrome c, triggering caspase activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c-conducting channel remain unknown. Here the relationship between VDAC oligomerization and the induction of apoptosis was examined. We demonstrated that apoptosis induction by various stimuli was accompanied by highly increased VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using bioluminescence resonance energy transfer technology. VDAC oligomerization was induced in all cell types and with all apoptosis inducers used, including staurosporine, curcumin, As2O3, etoposide, cisplatin, selenite, tumor necrosis factor alpha (TNF-α), H2O2, and UV irradiation, all acting through different mechanisms yet all involving mitochondria. Moreover, correlation between the levels of VDAC oligomerization and apoptosis was observed. Furthermore, the apoptosis inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) inhibited VDAC oligomerization. Finally, a caspase inhibitor had no effect on VDAC oligomerization and cytochrome c release. We propose that VDAC oligomerization is involved in mitochondrion-mediated apoptosis and may represent a general mechanism common to numerous apoptogens acting via different initiating cascades. Thus, targeting the oligomeric status of VDAC, and hence apoptosis, offers a therapeutic strategy for combating cancers and neurodegenerative diseases.

Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c, AIF and Smac/Diablo

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H2O2 or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.

The role of calcium in VDAC1 oligomerization and mitochondria-mediated apoptosis

The voltage-dependent anion channel (VDAC), located at the outer mitochondria membrane (OMM), mediates interactions between mitochondria and other parts of the cell by transporting anions, cations, ATP, Ca(2+), and metabolites. Substantial evidence points to VDAC1 as being a key player in apoptosis, regulating the release of apoptogenic proteins from mitochondria, such as cytochrome c, and interacting with anti-apoptotic proteins. Recently, we demonstrated that VDAC1 oligomerization is a general mechanism common to numerous apoptogens acting via different initiating cascades and proposed that a protein-conducting channel formed within a VDAC1 homo/hetero oligomer mediates cytochrome c release. However, the molecular mechanism responsible for VDAC1 oligomerization remains unclear. Several studies have shown that mitochondrial Ca(2+) is involved in apoptosis induction and that VDAC1 possesses Ca(2+)-binding sites and mediates Ca(2+) transport across the OMM. Here, the relationship between the cellular Ca(2+) level, [Ca(2+)]i, VDAC1 oligomerization and apoptosis was studied. Decreasing [Ca(2+)]i using the cell-permeable Ca(2+) chelating reagent BAPTA-AM was found to inhibit VDAC1 oligomerization and apoptosis, while increasing [Ca(2+)]i using Ca(2+) ionophore resulted in VDAC1 oligomerization and apoptosis induction in the absence of apoptotic stimuli. Moreover, induction of apoptosis elevated [Ca(2+)]i, concomitantly with VDAC1 oligomerization. AzRu-mediated inhibition of mitochondrial Ca(2+) transport decreased VDAC1 oligomerization, suggesting that mitochondrial Ca(2+) is required for VDAC1 oligomerization. In addition, increased [Ca(2+)]i levels up-regulate VDAC1 expression. These results suggest that Ca(2+) promotes VDAC1 oligomerization via activation of a yet unknown signaling pathway or by increasing VDAC1 expression, leading to apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Ca2 +-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca(2+) across the OMM and because Ca(2+) has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca(2+) levels ([Ca(2)(+)]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca(2+) and induce VDAC1 over-expression. Direct elevation of [Ca(2+)]i by the Ca(2+)-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca(2+)]i using the cell-permeable Ca(2+)-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca(2+)]i levels and apoptosis induction, as induced by H2O2 or As2O3, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca(2+)-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca(2+)]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca(2+) promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Oligomerization of the Mitochondrial Protein VDAC1: From Structure to Function and Cancer Therapy

The voltage-dependent anion channel (VDAC1), lying in the mitochondrial outer membrane (OMM), mediates the transport of ions and metabolites, thus controlling the cross talk between mitochondria and the rest of the cell. VDAC1 has also been recognized as a key protein in mitochondria-mediated apoptosis, is the proposed target for the pro- and antiapoptotic Bcl-2-family of proteins and is involved in apoptotic protein release from the mitochondria. Questions, however, remain as to if and how VDAC1 mediates the transfer of apoptotic proteins across the OMM. Our recent studies suggest that upon apoptosis induction, VDAC1 oligomerizes to form a new large pore allowing the passage of a folded protein, like cytochrome c. This review provides insight into the central role of VDAC1 in mammalian cell life and death and emphasizes VDAC1 function in apoptosis, focusing on VDAC1 oligomerization as a key step in mitochondria-mediated apoptosis and key structural features of VDAC1 that mediate its apoptotic function.