Fluoxetine (Prozac) interaction with the mitochondrial voltage-dependent anion channel and protection against apoptotic cell death

Fluoxetine (Prozac) is a potent antidepressant compound inhibiting serotonin reuptake, but also Na+, K+ and Ca2+ channels and reported to both trigger and prevent apoptosis. Recently, fluoxetine was found to increase the voltage sensitivity of the mitochondrial voltage-dependent anion channel (VDAC). VDAC which functions in transporting metabolites across the mitochondria also plays a crucial role in apoptosis. Here, we demonstrate that fluoxetine interacted with VDAC and decreased its conductance. Fluoxetine inhibited the opening of the mitochondrial permeability transition pore, the release of cytochrome c, and protected against staurosporine-induced apoptotic cell death. These findings may explain some of the reported fluoxetine side effects.     

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC)

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided -strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 Å resolution. In mitochondrial porins the N-terminal region forms an amphipathic -helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.     

The mitochondrial benzodiazepine receptor: Evidence for association with the voltage-dependent anion channel (VDAC)

Specific, high-affinity receptors for numerous drugs have recently been localized to mitochondrial membrane proteins. This review discusses the association of the mitochondrial receptor for benzodiazepines (mBzR) with the voltage-dependent anion channel (VDAC), indicating a possible auxiliary role for VDAC as a putative drug binding protein. The proposed subunit composition of the purified mBzR complex isolated from rat kidney mitochondria includes VDAC, which functions as a recognition site for benzodiazepines (e.g., flunitrazepam), the adenine nucleotide carrier (ADC), and an 18 kDa outer membrane protein identified by covalent labelling with the mBzR antagonists isoquinoline carboxamides (e.g., PK 14105).Abbreviations and chemical names: Ro5-4864: 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepin-2-one; Ro15-1788: ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-]-[1,4]benzodiazepine-3-carboxylate; AHN-086: (1-(2-isothiocyanatoethyl-7-chloro-1,3-dihydro-5-(4-chlorophenyl)-2H-1,4-benzodiazepin-2-one hydrochloride;) PK11195: 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-isoquinoline-3-carboxamide; PK14105: 1-(2-fluoro-5-nitrophenyl)-3-isoquinoline-carboxylic acid.     

Phosphorylation of rat brain mitochondrial voltage-dependent anion as a potential tool to control leakage of cytochrome c

Apoptosis is a controlled form of cell death that participates in development, elimination of damaged cells and maintenance of cell homeostasis. Also, it plays a role in neurodegenerative disorders like Alzheimer's disease. Recently, mitochondria have emerged as being pivotal in controlling apoptosis. They house a number of apoptogenic molecules, such as cytochrome c, which are released into the cytoplasm at the onset of apoptosis. When rat brain mitochondrial voltage-dependent anion channel (VDAC), an outer mitochondrial membrane protein, interacts with Bcl-2 family proteins Bax and tBid, its pore size increases, leading to the release of cytochrome c and other apoptogenic molecules into the cytosol and causing cell death. Regulation of this tBid- and Bax-induced increase in pore size of VDAC is a significant step to control cell death induced by cytochrome c. In this work, we have shown, through bilayer electrophysiological experiments, that the increase in VDAC conductance as a result of its interaction with Bax and tBid is reduced because of the action of cyclic AMP-dependent protein kinase A (PKA) in the presence of ATP. This indicates that the increase in the pore size of VDAC after its interaction with Bax and tBid is controlled via phosphorylation of this channel by PKA. This, we believe, could be a mechanism of controlling cytochrome c-mediated cell death in living cells.     

Induction of mitochondrial dysfunction by poly(ADP-ribose) polymer: Implication for neuronal cell death

Poly(ADP-ribose) polymerase-1 (PARP-1) mediates neuronal cell death in a variety of pathological conditions involving severe DNA damage. Poly(ADP-ribose) (PAR) polymer is a product synthesized by PARP-1. Previous studies suggest that PAR polymer heralds mitochondrial apoptosis-inducing factor (AIF) release and thereby, signals neuronal cell death. However, the details of the effects of PAR polymer on mitochondria remain to be elucidated. Here we report the effects of PAR polymer on mitochondria in cells in situ and isolated brain mitochondria in vitro. We found that PAR polymer causes depolarization of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore early after injury. Furthermore, PAR polymer specifically induces AIF release, but not cytochrome c from isolated brain mitochondria. These data suggest PAR polymer as an endogenous mitochondrial toxin and will further our understanding of the PARP-1-dependent neuronal cell death paradigm.     

Probing the structure of the mitochondrial channel,VDAC, by site-directed mutagenesis: A progress report

The voltage-dependent anion-selective channel (VDAC) of the mitochondrial outer membrane is formed by a small ( 30 kDa) polypeptide, but shares with more complex channels the properties of voltage-dependent gating and ion selectivity. Thus, it is a useful model for studying these properties. The molecular biology techniques available in yeast allow us to construct mutant versions of the cloned yeast VDAC genein vitro, using oligonucleotide-directed mutagenesis, and to express the mutant genes in yeast cells in the absence of wild-type VDAC. We find that one substitution mutation (lys 61 to glu) alters the selectivity of VDAC.     

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.     

LvHemB1, a novel cationic antimicrobial peptide derived from the hemocyanin of Litopenaeus vannamei,induces cancer cell death by targeting mitochondrial voltage-dependent anion channel 1

Cell Biology and Toxicology - Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells....     

Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface

The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.     

Voltage-dependent anion-selective channel 1 (VDAC1)--a mitochondrial protein, rediscovered as a novel enzyme in the plasma membrane

The eukaryotic porin or voltage-dependent anion-selective channel (VDAC1) is a pore-forming protein discovered twenty five years ago in the mitochondrial outer membrane. Its gene in eukaryotes is known, but its tertiary structure has never been solved. Structure predictions highlight the presence of several amphipathic beta-strands possibly organised in a beta-barrel. VDAC1 has recently been described as being a NADH:ferricyanide reductase in the plasma membrane. There it affects the regulation of cell growth and death. Physiological cell death (apoptosis) has become a major research focus of biomedical research. Regulation of the enzyme will have impacts on cancer and autoimmune diseases (insufficient apoptosis) as well as neurodegenerative diseases (excessive apoptosis). VDAC1 in the plasma membrane establishes a novel level of apoptosis regulation putatively via its redox activity.     

Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.     

Dorfin prevents cell death by reducing mitochondrial localizing mutant superoxide dismutase 1 in a neuronal cell model of familial amyotrophic lateral sclerosis

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.     

In EXOG‐depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain

Depletion of mitochondrial endo/exonuclease G‐like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease G‐like‐depleted cells, and this is marked by a decrease in mitochondrial reserve capacity. Neurohormonal stimulation with phenylephrine (PE) did not have an additive effect on the hypertrophic response induced by endo/exonuclease G‐like depletion. Interestingly, PE‐induced atrial natriuretic peptide (ANP) gene expression was completely abolished in endo/exonuclease G‐like‐depleted cells, suggesting a reverse signaling function of endo/exonuclease G‐like. Endo/exonuclease G‐like depletion initially resulted in increased mitochondrial OCR, but this declined upon PE stimulation. In particular, the reserve capacity of the mitochondrial respiratory chain and maximal respiration were the first indicators of perturbations in mitochondrial respiration, and these marked the subsequent decline in mitochondrial function. Although pathological stimulation accelerated these processes, prolonged EXOG depletion also resulted in a decline in mitochondrial function. At early stages of endo/exonuclease G‐like depletion, mitochondrial ROS production was increased, but this did not affect mitochondrial DNA (mtDNA) integrity. After prolonged depletion, ROS levels returned to control values, despite hyperpolarization of the mitochondrial membrane. The mitochondrial dysfunction finally resulted in cell death, which appears to be mainly a form of necrosis. In conclusion, endo/exonuclease G‐like plays an essential role in cardiomyocyte physiology. Loss of endo/exonuclease G‐like results in diminished adaptation to pathological stress. The decline in maximal respiration and reserve capacity is the first sign of mitochondrial dysfunction that determines subsequent cell death.     

MCL-1ES, a novel variant of MCL-1, associates with MCL-1L and induces mitochondrial cell death

Myeloid cell leukemia-1 (MCL-1L) is a pro-survival member of the BCL-2 family that promotes cell survival. In this study, we identify a new splicing variant of human MCL-1 that encodes MCL-1ES (extra short). Sequence analysis indicates that this variant results from splicing within the first coding exon of MCL-1 at a non-canonical GC-AG donor-acceptor pair. The deduced sequence of MCL-1ES encodes a protein of 197 amino acids, and the PEST (proline, glutamic acid, serine, and threonine) motifs present in MCL-1L are absent. MCL-1ES interacts with MCL-1L and induces mitochondrial cell death, suggesting that alternative splicing of MCL-1 may control the fate of cells.

Structured summary

MINT-7255705, MINT-7255718, MINT-7255731, MINT-7255743:MCL1-ES (uniprotkb:Q07820-2) physically interacts (MI:0914) with MCL1-1L (uniprotkb:Q07820-1) by anti tag coimmunoprecipitation (MI:0007)MINT-7255771:MCL1-ES (uniprotkb:Q07820-2) physically interacts (MI:0914) with Beta actin (uniprotkb:P60709) by anti tag coimmunoprecipitation (MI:0007)MINT-7255781:MCL1-ES (uniprotkb:Q07820-2) physically interacts (MI:0914) with GAPDH (uniprotkb:P04406) by anti tag coimmunoprecipitation (MI:0007)MINT-7255756:MCL1-ES (uniprotkb:Q07820-2) physically interacts (MI:0914) with COX IV (uniprotkb:P13073) by anti tag coimmunoprecipitation (MI:0007)     

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death,is necessary for developmental cell death in Drosophila midgut

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported¹ Dasari SK, Bialik S, Levin-Zaidman S, Levin-Salomon V, Merrill AH, Jr., Futerman AH, Kimchi A. Signalome-wide rnai screen identifies gba1 as a positive mediator of autophagic cell death. Cell Death Differ. 2017;24(7):1288-1302. https://doi.org/10.1038/cdd.2017.80. PMID:28574511[Crossref], [PubMed], [Web of Science ®] [Google Scholar] on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.     

Biphasic effect of nitric oxide on the cardiac voltage-dependent anion channel

Nitric oxide (NO) effects on the cardiac mitochondrial voltage-dependent anion channel (VDAC) are unknown. The effects of exogenous NO on VDAC purified from rat hearts were investigated in this study. When incorporated into lipid bilayers, VDAC was inhibited directly by an NO donor, PAPA NONOate, in a concentration-dependent biphasic manner. This was prevented by an NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The effect paralleled that of NO in delaying the opening of the mitochondrial permeability transition (PT) pore. These biphasic effects on the cardiac VDAC and the mitochondrial PT pore reveal a tandem impact of NO on the two mitochondrial entities.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.