Hexokinase-I protection against apoptotic cell death is mediated via interaction with the voltage-dependent anion channel-1: mapping the site of binding

In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents.     

Mapping the ruthenium red-binding site of the voltage-dependent anion channel-1

We have previously shown that ruthenium red (RuR) binds to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, decreasing channel conductance and protecting against apoptotic cell death. In this report, we define the murine and yeast VDAC1 amino acid residues involved in the interaction with RuR. Binding of RuR to bilayer-reconstituted mVDAC1 and the resulting channel closure was inhibited upon mutation of specific VDAC1 residues. RuR protection against cell death, as induced by overexpression of native or mutated mVDAC1, was also diminished upon mutation of these amino acids. Moreover, RuR-mediated inhibition of cytochrome c release normally induced by staurosporine was not observed in cells expressing mutants VDAC1. We found that four glutamate residues, two each located in the first and third mVDAC1 cytosolic loops, are required for the interaction of VDAC1 with RuR and subsequent protection against cell death. Similar results were obtained with Q72E-yeast VDAC1, except that only three glutamate residues, located in two cytosolic loops were required. As a hexavalent reagent, RuR is expected to bind to more than one negatively charged group. Our results thus clearly indicate that RuR protects against cell death via a direct interaction with VDAC1 to inhibit cytochrome c release and subsequent cell death.     

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.     

Key regions of VDAC1 functioning in apoptosis induction and regulation by hexokinase

The voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, and thus controls cross-talk between mitochondria and the cytosol. VDAC also serves as a site for the docking of cytosolic proteins, such as hexokinase, and is recognized as a key protein in mitochondria-mediated apoptosis. The role of VDAC in apoptosis has emerged from various studies showing its involvement in cytochrome c release and apoptotic cell death as well as its interaction with proteins regulating apoptosis, including the mitochondria-bound isoforms of hexokinase (HK-I, HK-II). Recently, the functional HK-VDAC association has shifted from being considered in a predominantly metabolic light to the recognition of its major impact on the regulation of apoptotic responsiveness of the cell. Here, we demonstrate that the HK-VDAC1 interaction can be disrupted by mutating VDAC1 and by VDAC1-based peptides, consequently leading to diminished HK anti-apoptotic activity, suggesting that disruption of HK binding to VDAC1 can decrease tumor cell survival. Indeed, understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of differing functions, all important for cell life and death. By expressing VDAC1 mutants and VDAC1-based peptides, we have identified VDAC1 amino acid residues and domains important for interaction with HK and protection against apoptosis. These include negatively- and positively-charged residues, some of which are located within β-strands of the protein. The N-terminal region of VDAC1 binds HK-I and prevents HK-mediated protection against apoptosis induced by STS, while expression of a VDAC N-terminal peptide detaches HK-I-GFP from mitochondria. These findings indicate that the interaction of HK with VDAC1 involves charged residues in several β-strands and in the N-terminal domain. Displacing HK, serving as the ‘guardian of the mitochondrion’, from its binding site on VDAC1 may thus be exploited as an approach to cancer therapy.     

Voltage-dependent Anion Channel 1-based Peptides Interact with Bcl-2 to Prevent Antiapoptotic Activity

The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance. Expression of Bcl-2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl-2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized Bcl-2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl-2 prevented Bcl-2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl-2-GFP-mediated protection against apoptosis. These results point to Bcl-2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl-2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.     

Localization of the voltage-dependent anion channel-1 Ca2+-binding sites

Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca(2+)-binding proteins and to strongly inhibit their Ca(2+)-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca(2+)-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca(2+), in the presence of 1M NaCl, but not Mg(2+), prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [(103)Ru]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca(2+)-binding site(s). AzRu protected against apoptosis induced by over-expression of native but not E72Q- or E202Q- murine VDAC1 in T-REx-293 cells depleted of endogenous hVDAC1. Chymotrypsin and trypsin digestion of AzRu-labeled VDAC followed by MALDI-TOF analysis revealed two AzRu-bound peptides corresponding to E72- and E202-containing sequences. These results suggest that the VDAC Ca(2+)-binding site includes E72 and E202, located, according to a proposed VDAC1 topology model, on two distinct cytosolic loops. Furthermore, AzRu protection against apoptosis involves interaction with these residues. Photoreactive AzRu represents an important tool for identifying novel Ca(2+)-binding proteins and localizing their Ca(2+)-binding sites.     

Molecular model of hexokinase binding to the outer mitochondrial membrane porin (VDAC1): Implication for the design of new cancer therapies

A key feature of many cancers is the capacity and the propensity to metabolize glucose to lactic acid at a very high rate even in the presence of oxygen. This characteristic was first discovered in 1924 by Otto Heinrich Warburg. Hexokinase, the first enzyme in the glycolytic pathway, not only improves the cell's energy supply in malignant cells, but also protects cancer cells against apoptosis through direct interaction with mitochondria and with the Voltage Dependent Anion Channel 1 (VDAC1). The rupture of HK:VDAC1 protein complex provides a therapeutic opportunity, as this association appears to protect tumor cells from mitochondrial outer membrane permeabilization, an event that marks the point of no return in multiple pathways leading to cell death. In the absence of a crystallographic structure and in order to perform an in silico screening of possible small molecules able to inhibit the protein association, we are presenting a computational model of HK-I:VDAC1 complex. It appears as evident how the first 15 N-terminal residues of HK-I interact with the inner part of the barrel of VDAC1 and not with the outside walls, within the mitochondrial membrane as previously believed. This finding is in agreement with the existence of a secondary ATP binding site in the same N-terminal region of HK-I which seems to have a crucial role in HK-I interaction with VDAC1. This evidence appears to be in accord also with the high levels of ATP that are found in cancer cells. Eventually such arrangements may contribute to stabilize the tertiary structure of VDAC1 while shielding from pro-apoptotic factor binding, protecting in a synergic way the tumoral cell from programmed death.     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein

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.     

Increased expression of cyclin A1 protein is associated with all-trans retinoic acid-induced apoptosis

Deregulated cell growth and inhibition of apoptosis are hallmarks of cancer. All-trans retinoic acid induces clinical remission in patients with acute promyelocytic leukemia by inhibiting cell growth and inducing differentiation and apoptosis of the leukemic blasts. An important role of the cell cycle regulatory protein, cyclin A1, in the development of acute myeloid leukemia has previously been demonstrated in a transgenic mouse model. We have recently shown that there was a direct interaction between cyclin A1 and a major all-trans retinoic acid receptor, RAR alpha, following all-trans retinoic acid treatment of leukemic cells. In the present study, we investigated whether cyclin A1 might be involved in all-trans retinoic acid-induced apoptosis in U-937 leukemic cells. We found that all-trans retinoic acid-induced apoptosis was associated with concomitant increase in cyclin A1 expression. However, there was no induction of cyclin A1 mRNA expression following the all-trans retinoic acid-induced apoptosis. Treatment of cells with a caspase inhibitor was not able to prevent all-trans retinoic acid-induced up-regulation of cyclin A1 expression. Interestingly, induced cyclin A1 expression in U-937 cells led to a significant increase in the proportion of apoptotic cells. Further, U-937 cells overexpressing cyclin A1 appeared to be more sensitive to all-trans retinoic acid-induced apoptosis indicating the ability of cyclin A1 to mediate all-trans retinoic acid-induced apoptosis. Induced cyclin E expression was not able to initiate cell death in U-937 cells. Our results indicate that cyclin A1 might have a role in apoptosis by mediating all-trans retinoic acid-induced apoptosis.     

Possible participation of the Rho/Rho-associated coiled-coil-forming kinase pathway in the cell death of Cryptococcus neoformans caused by Staphylococcus aureus adherence

We here report the apoptotic death of a fungus, Cryptococcus neoformans (C. neoformans), in response to adherence of the pathogenic bacterium Staphylococcus aureus (S. aureus). In co-culture, cryptococcal actin was visibly aggregated. To investigate the mechanism of death, the participation of small GTP(guanosine triphosphate)-binding proteins belonging to the Rho subfamily, which regulate the actin cytoskeleton, was explored. C. neoformans was cultured with S. aureus in the presence of N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide (Y-27632), an inhibitor of Rho-associated coiled-coil forming kinase (ROCK), a downstream effector of Rho. Death of C. neoformans was significantly reduced by the inhibitor. Concomitantly, Y-27632 prevented the aggregation of actin. Therefore, it was concluded that the Rho/ROCK pathway is involved in cell death induced by adherence stress. Increased expression of the voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, has previously been observed in the apoptosis-like death of C. neoformans in the presence of hydrogen peroxide. Ruthenium red (RuR), which binds to VDAC and inhibits cytochrome c release, was used to determine the involvement of VDAC following adherence stress caused by S. aureus. RuR treatment increased the viability of C. neoformans co-cultured with S. aureus in a dose dependent manner. These findings suggest that Rho-ROCK signaling could be involved, via a mitochondrial pathway, in the apoptosis-like death of C. neoformans induced by the adherence of S. aureus.     

Increased sensitivity to cis-diamminedichloroplatinum induced apoptosis with mitochondrial DNA depletion

Malignant cells harbor mechanisms which allow escape from drug-induced apoptosis, and the drug-resistance phenotype can be significantly associated with resistance to programmed cell death. There is accumulating evidence that mitochondria play a role in the tumorigenic phenotype, including the relative resistance to apoptosis. Whether changes at the mitochondrial level per se, would impact on the relative sensitivity of malignant cells to undergo drug-induced apoptosis, is not know. Accordingly, we determined if depleting mitochondrial DNA (mtDNA) would change the susceptibility of U937 cells to undergo apoptosis. With depletion, increases in sensitivity to cis-diamminedichoroplatinum (cisplatin)-induced apoptosis was observed. This sensitivity could be reverted to the parental phenotype by transforming the depleted cells with normal platelet mitochondria. mRNA expression of BAX, BCL2, MDR1, MRP, ERCC1 and ERCC2, putatively associated with cisplatin resistance to apoptotic death was unchanged. Inhibition of mitochondrial ATP production by oligomycin did not result in a change in ATP levels, indicating energetics were not playing a role in the observed phenotype changes. All U937 cells (with/without mtDNA) continued to respond to cisplatin by an apoptotic death. MtDNA-encoded molecules may be playing a role in the relative sensitivity of cells to undergo a cisplatin-induced apoptotic death, but may not be required for cells to undergo apoptosis per se.     

LPS-induced apoptosis is dependent upon mitochondrial dysfunction

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.     

Mouse uterine epithelial apoptosis is associated with expression of mitochondrial voltage-dependent anion channels,release of cytochrome C from mitochondria,and the ratio of Bax to Bcl-2 or Bcl-X

The release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members and is considered to take place through voltage-dependent anion channels (VDACs) on the outer membranes of mitochondria, results in activation of effector caspases, such as caspase-3, which induce apoptosis. We studied the involvement of the mitochondrial apoptosis pathway in uterine epithelial apoptosis. Estradiol-17beta pellets were implanted into ovariectomized mice and removed 4 days later (Day 0). The apoptotic index (percentage of apoptotic cells) of the luminal epithelium increased markedly, peaking on Day 2, whereas that of the glandular epithelium increased much less. Expression of VDAC1, 2, and 3 mRNAs increased in the luminal epithelium in correlation with the apoptotic index of the luminal epithelium. No increases in VDAC1, 2, and 3 mRNA levels were observed in the stroma or muscle, where no apoptosis occurs. VDAC1 protein levels in the uterus also correlated well with the apoptotic index of the luminal epithelium. In addition, the apoptotic index showed good correlation with the release of cytochrome c from mitochondria, activation of caspase-3, which was immunohistochemically detected only in the epithelium, and the mRNA and protein ratios of Bax:Bcl-2 and Bax:Bcl-X in the uterus. The present results suggest that the release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members, plays a role in uterine epithelial apoptosis after estrogen deprivation. The increase in VDAC expression may facilitate the release of cytochrome c during apoptosis.     

Menadione-induced apoptosis in U937 cells involves Bid cleavage and stefin B degradation

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.     

Mitochondrial targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of the stress-activated protein kinase (SAPK) and induction of terminal monocytic differentiation. The present studies demonstrate that TPA targets SAPK to mitochondria by a mechanism dependent on activation of protein kinase C (PKC) beta. Translocation of SAPK to mitochondria in response to TPA is associated with release of cytochrome c, caspase-3 activation and induction of apoptosis. The results show that TPA induces the association of SAPK with the mitochondrial anti-apoptotic Bcl-x(L) protein. Overexpression of Bcl-x(L) attenuated the apoptotic response to TPA treatment. Moreover, expression of Bcl-x(L) mutated at sites of SAPK phosphorylation (Thr-47, -115) was more effective than wild-type Bcl-x(L) in abrogating TPA-induced cytochrome c release and apoptosis. By contrast, expression of Bcl-x(L) had little effect on induction of the monocytic phenotype. These findings indicate that myeloid leukemia cells respond to TPA with targeting of SAPK to mitochondria and that this response contributes to terminal differentiation through the release of cytochrome c and induction of apoptosis.     

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.     

The c-Jun N-terminal kinase 1 activity is differentially regulated by specific mechanisms during apoptosis

We show here that JNK1 activity is rapidly up-regulated and prolonged by specific mechanisms during apoptosis induced by paclitaxel- or ginsenoside-Rh2 in SK-HEP-1 cells. The early phase of JNK1 activation is prevented in cells expressing the dominant negative SEK1 mutant, although this JNK1 perturbation does not prevent apoptotic cell death. The later phase of JNK1 activation, which is temporally coincided with caspase-dependent cleavage of JNK1-associated p21(WAF1/CIP1), is efficiently prevented by expressing p21D112N, an uncleavable mutant of p21(WAF1/CIP1) and this perturbation of JNK1 activation results in prevention of apoptosis. The later JNK1 activation and apoptotic progression are also prevented by co-treatments of cells with rottlerin, a PKC-delta inhibitor or z-VAD-fmk, a pan caspase inhibitor. We also provide evidence that apoptotic cell death is significantly promoted in cells expressing JNK1, while this apoptotic cell death is effectively suppressed in cells expressing the dominant negative JNK1 mutant (DN-JNK1) or JBD, a JNK inhibitor protein. Thus, the later phase of JNK1 activation, which is linked to a caspase-dependent mechanism that requires PKC-delta activity, is associated with the induction of apoptosis, while the early JNK1 activation that is associated with a SEK1-mediated mechanism is not directly involved in apoptotic progression.     

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.