TRIM8 promotes ovarian cancer proliferation and migration by targeting VDAC2 for ubiquitination and degradation.

BACKGROUND: Ovarian cancer is a common gynecological tumor with high malignant potential and poor prognosis. TRIM8, is involved in the development of various tumors, but its precise regulatory role in ovarian cancer is still unknown. AIMS: The aim of this study was to explore the specific mechanism by which TRIM8 regulates ovarian cancer. MATERIALS AND METHODS: We used bioinformatics analysis to screen for high expression of TRIM8 in ovarian cancer. The expression of TRIM8 in healthy and cancerous ovarian tissues was assessed by immunofluorescence. TRIM8 was silenced or overexpressed in ovarian cancer cell lines, with cell proliferation and migration evaluated by CCK8, transwell and clonal formation assays. The effect of TRIM8 on ovarian cancer cells in vivo was assessed by subcutaneous tumor formation experiments in nude mice. The potential interacting protein VDAC2 was identified by mass spectrometry. The mechanism underlying TRIM8 regulation of VDAC2 was evaluated by co-immunoprecipitation and western blotting. RESULTS: TRIM8 was overexpressed in ovarian cancer. TRIM8 promoted the proliferation and migration of ovarian cancer cells in vitro and the growth of subcutaneous tumors in mice in vivo. TRIM8 interacted with VDAC2, weakened the stability of the protein, and promoted its polyubiquitination and subsequent degradation. Knockdown of VDAC2 increased the resistance of ovarian cancer cells to iron death, whereas overexpression of VDAC2 attenuated ovarian cancer progression induced by TRIM8 overexpression. DISCUSSION: TRIM8 promotes ovarian cancer proliferation and migration by targeting VDAC2 for ubiquitination and degradation, these finding may provide new targets for the treatment of ovarian cancer. CONCLUSION: TRIM8 degraded VDAC2 through the ubiquitination pathway, increased the resistance of ovarian cancer cells to iron death, and promoted the proliferation and migration of ovarian cancer.

Intramolecular Disulfide Bridges in Voltage-Dependent Anion Channel 2 (VDAC2) Protein from Rattus norvegicus Revealed by High-Resolution Mass Spectrometry.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

Key residues in the VDAC2-BAK complex can be targeted to modulate apoptosis.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.

Lamprey VDAC2: Suppressing hydrogen peroxide-induced 293T cell apoptosis by downregulating BAK expression.

The voltage-dependent anion channel 2 (VDAC2) is the abundant protein in the outer mitochondrial membrane. Opening VDAC2 pores leads to the induction of mitochondrial energy and material transport, facilitating interaction with various mitochondrial proteins implicated in essential processes such as cell apoptosis and proliferation. To investigate the VDAC2 in lower vertebrates, we identified Lr-VDAC2, a homologue of VDAC2 found in lamprey (Lethenteron reissneri), sharing a sequence identity of greater than 50 % with its counterparts. Phylogenetic analysis revealed that the position of Lr-VDAC2 aligns with the lamprey phylogeny, indicating its evolutionary relationship within the species. The Lr-VDAC2 protein was primarily located in the mitochondria of lamprey cells. The expression of the Lr-VDAC2 protein was elevated in high energy-demanding tissues, such as the gills, muscles, and myocardial tissue in normal lampreys. Lr-VDAC2 suppressed H2O2 (hydrogen peroxide)-induced 293 T cell apoptosis by reducing the expression levels of Caspase 3, Caspase 9, and Cyt C (cytochrome c). Further research into the mechanism indicated that the Lr-VDAC2 protein inhibited the pro-apoptotic activity of BAK (Bcl-2 antagonist/killer) protein by downregulating its expression at the protein translational level, thus exerting an anti-apoptotic function similar to the role of VDAC2 in humans.

BmNPV p35 regulates apoptosis in Bombyx mori via a novel target of interaction with the BmVDAC2-BmRACK1 complex.

Voltage-dependent anion channel 2 (VDAC2) is an important channel protein that plays a crucial role in the host response to viral infection. The receptor for activated C kinase 1 (RACK1) is also a key host factor involved in viral replication. Our previous research revealed that Bombyx mori VDAC2 (BmVDAC2) and B. mori RACK1 (BmRACK1) may interact with Bombyx mori nucleopolyhedrovirus (BmNPV), though the specific molecular mechanism remains unclear. In this study, the interaction between BmVDAC2 and BmRACK1 in the mitochondria was determined by various methods. We found that BmNPV p35 interacts directly with BmVDAC2 rather than BmRACK1. BmNPV infection significantly reduced the expression of BmVDAC2, and activated the mitochondrial apoptosis pathway. Overexpression of BmVDAC2 in BmN cells inhibited BmNPV-induced cytochrome c (cyto c) release, decrease in mitochondrial membrane potential as well as apoptosis. Additionally, the inhibition of cyto c release by BmVDAC2 requires the involvement of BmRACK1 and protein kinase C. Interestingly, overexpression of p35 inhibited cyto c release during mitochondrial apoptosis in a RACK1 and VDAC2-dependent manner. Even the mutant p35, which loses Caspase inhibitory activity, could still bind to VDAC2 and inhibit cyto c release. In summary, our results indicated that BmNPV p35 interacts with the VDAC2-RACK1 complex to regulate apoptosis by inhibiting cyto c release. These findings confirm the interaction between BmVDAC2 and BmRACK1, the interaction between p35 and the VDAC2-RACK1 complex, and a novel target that BmNPV p35 regulates apoptosis in Bombyx mori via interaction with the BmVDAC2-BmRACK1 complex. The result provide an initial exploration of the function of this interaction in the BmNPV-induced mitochondrial apoptosis pathway.

Neuronal Protection by Ha-RAS-GTPase Signaling through Selective Downregulation of Plasmalemmal Voltage-Dependent Anion Channel-1.

The small GTPase RAS acts as a plasma membrane-anchored intracellular neurotrophin counteracting neuronal degeneration in the brain, but the underlying molecular mechanisms are largely unknown. In transgenic mice expressing constitutively activated V12-Ha-RAS selectively in neurons, proteome analysis uncovered a 70% decrease in voltage-dependent anion channel-1 (VDAC-1) in the cortex and hippocampus. We observed a corresponding reduction in the levels of mRNA splicing variant coding for plasma membrane-targeted VDAC-1 (pl-VDAC-1) while mRNA levels for mitochondrial membrane VDAC-1 (mt-VDAC-1) remained constant. In primary cortical neurons derived from V12-Ha-RAS animals, a decrease in pl-VDAC-1 mRNA levels was observed, accompanied by a concomitant reduction in the ferricyanide reductase activity associated with VDAC-1 protein. Application of MEK inhibitor U0126 to transgenic cortical neurons reconstituted pl-VDAC-1 mRNA to reach wild-type levels. Excitotoxic glutamate-induced cell death was strongly attenuated in transgenic V12-Ha-RAS overexpressing cortical cultures. Consistently, a neuroprotective effect could also be achieved in wild-type cortical cultures by the extracellular application of channel-blocking antibody targeting the N-terminus of VDAC-1. These results may encourage novel therapeutic approaches toward blocking pl-VDAC-1 by monoclonal antibody targeting for complementary treatments in transplantation and neurodegenerative disease.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Deubiquitinase UCHL1 regulates estradiol synthesis by stabilizing voltage-dependent anion channel 2.

Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The ubiquitin-proteasome system plays a critical role in maintaining protein homeostasis and stability of the estrous cycle, but knowledge of deubiquitination enzyme function in estradiol synthesis is limited. Here, we observe that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is more significant in estrous sows and high litter-size sows than in nonestrous sows and low-yielding sows. Overexpression of UCHL1 promotes estradiol synthesis in granulosa cells, and interference with UCHL1 has the opposite effect. UCHL1 binds, deubiquitinates, and stabilizes voltage-dependent anion channel 2 (VDAC2), promoting the synthesis of the estradiol precursor pregnenolone. Cysteine 90 (C90) of UCHL1 is necessary for its deubiquitination activity, and Lys45 and Lys64 in VDAC2 are essential for its ubiquitination and degradation. In vivo, compared with WT and sh-NC-AAV groups, the estrus cycle of female mice is disturbed, estradiol level is decreased, and the number of antral follicles is decreased after the injection of sh-UCHL1-AAV into ovarian tissue. These findings suggest that UCHL1 promotes estradiol synthesis by stabilizing VDAC2 and identify UCHL1 as a candidate gene affecting reproductive performance.

Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway.

IMPORTANCE: Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.

Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.

Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.

STING Suppresses Mitochondrial VDAC2 to Govern RCC Growth Independent of Innate Immunity.

STING is an innate immune sensor for immune surveillance of viral/bacterial infection and maintenance of an immune-friendly microenvironment to prevent tumorigenesis. However, if and how STING exerts innate immunity-independent function remains elusive. Here, the authors report that STING expression is increased in renal cell carcinoma (RCC) patients and governs tumor growth through non-canonical innate immune signaling involving mitochondrial ROS maintenance and calcium homeostasis. Mitochondrial voltage-dependent anion channel VDAC2 is identified as a new STING binding partner. STING depletion potentiates VDAC2/GRP75-mediated MERC (mitochondria-ER contact) formation to increase mitochondrial ROS/calcium levels, impairs mitochondria function, and suppresses mTORC1/S6K signaling leading to RCC growth retardation. STING interaction with VDAC2 occurs through STING-C88/C91 palmitoylation and inhibiting STING palmitoyl-transferases ZDHHCs by 2-BP significantly impedes RCC cell growth alone or in combination with sorafenib. Together, these studies reveal an innate immunity-independent function of STING in regulating mitochondrial function and growth in RCC, providing a rationale to target the STING/VDAC2 interaction in treating RCC.

Voltage-dependent anion channel 2 (VDAC2) facilitates the accumulation of rice stripe virus in the vector Laodelphax striatellus.

Rice stripe virus (RSV) causes enormous losses in rice production and is transmitted by the small brown planthopper, Laodelphax striatellus, in a persistent-propagative manner. RSV accumulation within the gut lumen of the vector is indispensable for the successful transmission to rice and insects. In this study, we obtained a 1464 bp full-length cDNA of a voltage-dependent anion channel 2 from L. striatellus (LsVDAC2), which encodes a 283 amino acid protein. RSV infection increased the expression of LsVDAC2 in the midguts and ovaries of L. striatellus by 260% and 228%, respectively. Silencing of LsVDAC2 resulted in a 88% reduction of RSV loads at 24 h after RNAi, indicating that LsVDAC2 facilitates RSV accumulation in the vector. Yeast two-hybrid and GST pulldown assays demonstrated that LsVDAC2 interacted with RSV RNA-dependent RNA polymerase, RdRp. Furthermore, experiments in vivo and in vitro showed that LsVDAC2 induced the apoptotic response in RSV-infected insects and tissues. Silencing of LsVDAC2 via RNAi significantly reduced the expression of genes for apoptosis-related caspases 1a and 1c by 62% and 78%, respectively, in RSV-infected vectors. Whether LsVDAC2-induced RSV accumulation is related to RSV RdRp and LsVDAC2-induced cell apoptosis deserves further investigation.

VDAC2 as a novel target for heart failure: Ca2+ at the sarcomere, mitochondria and SR.

Despite a growing number of successful therapies, heart failure remains the most common cause of death and disability worldwide. Thus, new and novel therapeutic strategies are urgently needed. Mitochondria of cardiomyocytes generate ATP that is needed to power cardiac contraction. Mitochondrial-derived ATP activate myosin ATPase at the sarcomere and the sarcoplasmic reticular (SR) ATPase Ca2+ pump, both which intersect with Ca2+ during contraction. Failure to maintain the relationship between mitochondria and SR can lead to cardiomyocyte dysfunction and heart failure. Here, we discuss recent discoveries that reveal Ca2+ transport via the voltage dependent anion channel (VDAC) into the mitochondria can favorably impact cardiac contraction and prevent cardiac arrhythmias. In a broader view, discussion of the opening of a new era for HF therapeutics that will address the sarcomere, SR and mitochondria as a functional unit.

A conventional immune regulator mitochondrial antiviral signaling protein blocks hepatic steatosis by maintaining mitochondrial homeostasis.

BACKGROUND AND AIMS: Although the prevalence of NAFLD has risen dramatically to 25% of the adult population worldwide, there are as yet no approved pharmacological interventions for the disease because of uncertainty about the underlying molecular mechanisms. It is known that mitochondrial dysfunction is an important factor in the development of NAFLD. Mitochondrial antiviral signaling protein (MAVS) is a critical signaling adaptor for host defenses against viral infection. However, the role of MAVS in mitochondrial metabolism during NAFLD progression remains largely unknown. APPROACH AND RESULTS: Based on expression analysis, we identified a marked down-regulation of MAVS in hepatocytes during NAFLD progression. By using MAVS global knockout and hepatocyte-specific MAVS knockout mice, we found that MAVS is protective against diet-induced NAFLD. MAVS deficiency induces extensive mitochondrial dysfunction during NAFLD pathogenesis, which was confirmed as impaired mitochondrial respiratory capacity and membrane potential. Metabolomics data also showed the extensive metabolic disorders after MAVS deletion. Mechanistically, MAVS interacts with the N-terminal stretch of voltage-dependent anion channel 2 (VDAC2), which is required for the ability of MAVS to influence mitochondrial function and hepatic steatosis. CONCLUSIONS: In hepatocytes, MAVS plays an important role in protecting against NAFLD by helping to regulate healthy mitochondrial function. These findings provide insights regarding the metabolic importance of conventional immune regulators and support the possibility that targeting MAVS may represent an avenue for treating NAFLD.

SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility in mice.

Calcineurin is a calcium-dependent phosphatase that plays roles in a variety of biological processes including immune responses. In spermatozoa, there is a testis-enriched calcineurin composed of PPP3CC and PPP3R2 (sperm calcineurin) that is essential for sperm motility and male fertility. Because sperm calcineurin has been proposed as a target for reversible male contraceptives, identifying proteins that interact with sperm calcineurin widens the choice for developing specific inhibitors. Here, by screening the calcineurin-interacting PxIxIT consensus motif in silico and analyzing the function of candidate proteins through the generation of gene-modified mice, we discovered that SPATA33 interacts with sperm calcineurin via a PQIIIT sequence. Spata33 knockout mice exhibit reduced sperm motility because of an inflexible midpiece, leading to impaired male fertility, which phenocopies Ppp3cc and Ppp3r2 knockout mice. Further analysis reveals that sperm calcineurin disappears from the mitochondria in the Spata33 knockout testis. In addition, immunoprecipitation analysis indicates that sperm calcineurin interacts with not only SPATA33 but also the mitochondrial protein VDAC2. These results indicate that SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility.

Cardiac-specific deletion of voltage dependent anion channel 2 leads to dilated cardiomyopathy by altering calcium homeostasis.

Voltage dependent anion channel 2 (VDAC2) is an outer mitochondrial membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. However, the specific role of VDAC2 in intracellular calcium dynamics and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac ventricular myocyte-specific developmental deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium causes severe impairment in excitation-contraction coupling by altering both intracellular and mitochondrial calcium signaling. We also observed adverse cardiac remodeling which progressed to severe cardiomyopathy and death. Reintroduction of VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype. Activation of VDAC2 by efsevin increased cardiac contractile force in a mouse model of pressure-overload induced heart failure. In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing cellular calcium signaling. Through this unique role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure.

α-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport.

Voltage-dependent anion channel (VDAC) is the most ubiquitous channel at the mitochondrial outer membrane, and is believed to be the pathway for calcium entering or leaving the mitochondria. Therefore, understanding the molecular mechanisms of how VDAC regulates calcium influx and efflux from the mitochondria is of particular interest for mitochondrial physiology. When the Parkinson's disease (PD) related neuronal protein, alpha-synuclein (αSyn), is added to the reconstituted VDAC, it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule VDAC electrophysiology of reconstituted VDAC we now demonstrate that, at CaCl2 concentrations below 150 mM, αSyn reverses the channel's selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by αSyn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. -Our findings with higher calcium concentrations also demonstrate that the phenomenon of "charge inversion" is taking place at the level of a single polypeptide chain. Measurements of ion selectivity of three VDAC isoforms in CaCl2 gradient show that VDAC3 exhibits the highest calcium permeability among them, followed by VDAC2 and VDAC1, thus pointing to isoform-dependent physiological function. Mutation of the E73 residue - VDAC1 purported calcium binding site - shows that there is no measurable effect of the mutation in either open or αSyn-blocked VDAC1 states. Our results confirm VDACs involvement in calcium signaling and reveal a new regulatory role of αSyn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction.

Protein mass spectrometry reveals lycorine exerting anti-multiple-myeloma effect by acting on VDAC2 and causing mitochondrial abnormalities.

OBJECTIVE: Two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry were performed to compare the proteomic alterations of lycorine-treated and control cells to further investigate the anti-multiple myeloma (MM) mechanisms of lycorine. RESULTS: Mass spectrometry results showed that after lycorine treatment of MM cells, 42% of the differentially expressed proteins had subcellular localization, mainly, on mitochondria. Voltage-dependent anion-selective channel protein 2 (VDAC2), the most abundant protein in the outer mitochondrial membrane, was up-regulated after treatment with lycorine and was subsequently verified by western blot analysis. Further studies on mitochondria found that lycorine was able to increase abnormal mitochondria and increase mitochondrial membrane potential. CONCLUSIONS: Lycorine can achieve the effect of resisting multiple myeloma by acting on VDAC2 and causing mitochondrial abnormalities.

SPATA33 is an autophagy mediator for cargo selectivity in germline mitophagy.

Selective autophagic degradation of mitochondria (mitophagy) is important in maintaining proper cellular homeostasis. Here, we found that SPATA33 is a novel autophagy mediator for mitophagy in testis. The SPATA33 protein localizes on mitochondria via its binding of the carboxyl terminal with the outer mitochondrial membrane protein VDAC2. Upon starvation induction, SPATA33 is recruited to autophagosome by binding the autophagy machinery ATG16L1 via its N-terminal along with mitochondria. Notably, Spata33 knockout inhibited autophagy and overexpression can promote autophagosome formation for mitochondrial sequestration. Therefore, SPATA33 confers selectivity for mitochondrial degradation and promotes mitophagy in male germline cells.

Bisindolylpyrrole triggers transient mitochondrial permeability transitions to cause apoptosis in a VDAC1/2 and cyclophilin D-dependent manner via the ANT-associated pore.

Bisindolylpyrrole at 0.1 µM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 µM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.