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, As 2 O 3 , etoposide, cisplatin, selenite, tumor necrosis factor alpha (TNF-␣), H 2 O 2 , 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.It is well accepted that mitochondria serve as integrators and amplifiers of programmed cell death through the regulation of apoptosis, mediating the release of proapoptotic proteins and/or disrupting cellular energy metabolism (25). During the transduction of an apoptotic signal into the cell, an alteration in mitochondrial membrane permeability occurs, facilitating the release of apoptogenic proteins, such as cytochrome c (Cyto c), apoptosis-inducing factor (AIF), and Smac/DIABLO, from the intermembrane space into the cytosol (45). These proteins participate in complex processes resulting in the activation of proteases and nucleases, leading to protein and DNA degradation and, ultimately, cell death (25). However, it remains unclear how these apoptotic initiators cross the outer mitochondrial membrane (OMM) and are released into the cytosol. While some models predict that such release is facilitated by swelling of the mitochondrial matrix and subsequent rupture of the OMM, other models predict the formation of protein-conducting channels that are large enough to allow the passage of Cyto c and other proteins into the cytosol without compromising OMM integrity (15,44,47). The voltage-dependent anion channel (VDAC) offers such a route (45).Located in the OMM, the VDAC forms the main interface between the mitochondrial and cellular metabolisms by medi...

Structure-based Analysis of VDAC1 Protein

“…VDAC1 Oligomeric State and Conformational Changes Realized upon Apoptosis Induction-Recently, using chemical cross-linking and BRET2, we demonstrated that the oligomeric assembly of VDAC1 in cultured cells is highly enhanced upon induction of apoptosis by various inducers, all of which act via mitochondria but through different mechanisms (28,29). These and other findings led us to propose that VDAC1 exists in a dynamic equilibrium between monomeric and oligomeric forms that shifts toward oligomerization upon apoptosis induction.…”

“…Indeed, using chemical cross-linking, we demonstrated that the oligomeric assembly of VDAC1 in cultured cells is highly enhanced (up to 20-fold) upon induction of apoptosis by various agents (e.g. STS, curcumin, As 2 O 3 , etoposide, cisplatin, TNF-␣, H 2 O 2 , UV irradiation, or VDAC1 overexpression), all of which act via mitochondria but through different mechanisms (28,29). Moreover, when we employed bioluminescence resonance energy transfer (BRET2) to monitor VDAC1 oligomerization in living cells, we noted that incubation with apoptosis inducers enhanced the BRET2 signal, whereas exposure to apoptosis inhibitors prevented this enhancement (29).…”

“…STS, curcumin, As 2 O 3 , etoposide, cisplatin, TNF-␣, H 2 O 2 , UV irradiation, or VDAC1 overexpression), all of which act via mitochondria but through different mechanisms (28,29). Moreover, when we employed bioluminescence resonance energy transfer (BRET2) to monitor VDAC1 oligomerization in living cells, we noted that incubation with apoptosis inducers enhanced the BRET2 signal, whereas exposure to apoptosis inhibitors prevented this enhancement (29). These findings support the hypothesis that VDAC oligomerization is involved in cytochrome c release and, therefore, in apoptosis.…”

Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells

“…Short hairpins (sh) targeting two nonoverlapping sequences within the coding region of human VDAC1 were designed based on previously published sequences (62,63). The shRNA was created using the following complementary sets of PAGE-purified oligonucleotides (Integrated DNA Technologies): VDAC1 sh1 forward (5′-TCACTAGGCACCGAGA-TTATTTCAAGAGAATAATCTCGGTGCCTAGTGTTTTTTC-3′), VDAC1 sh1 reverse (5′-TCGAGAAAAAACACTAGGCACCGAGATTATTCTCTTGAAATAATCTCGGTG-CCTAGTGA-3′); VDAC1 sh2 forward (5′-TGTGACGGGCAGTCTGGAATTTCAA-GAGAATTCCAGACTGCCCGTCACTTTTTTC-3′), VDAC1 sh2 reverse (5′-TCGA-GAAAAAAGTGACGGGCAGTCTGGAATTCTCTTGAAATTCCAGACTGCCCGTC-ACA-3′).…”

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

“…Interestingly, VDAC1 was previously identified as a TG2 substrate in PMA-stimulated H9c2 cells [27]. It may be that TG2-mediated amine incorporation into HK1 and VDAC interferes with the ability of these proteins to interact with each other and modulate cardioprotection and/or cell survival [64]. It is also notable that ANP32A functions as an inhibitor of protein phosphatase 2A and thus may regulate ERK1/2 and PKC; therefore it is conceivable that its modification by TG2…”