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.

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.

Single-cell and bulk RNA sequencing data jointly reveals VDAC2's impacts on prognosis and immune landscape of NSCLC.

Non-small cell lung cancer (NSCLC) is characterized by stronger metastatic ability and worse prognosis. In NSCLC, hypoxia is a major cause of invasion and metastasis through promoting angiogenesis. In present study, NSCLC cell clusters were extracted from single cell-sequencing dataset GSE131907, which were combined with hypoxia-related genes to group clusters. qRT-PCR and western blot were used to validate the expression of target gene. Nine NSCLC clusters were extracted, which were divided into two hypoxia-related subgroups, C1 and C2. Totally 101 differentially expressed prognostic genes were identified between subgroups. Of which, VDAC2 showed excellent prognostic value for NSCLC and was selected for further analysis. VDAC2 was upregulated in tumor samples in TCGA and was correlated with advanced stages. In vitro experiments validated this trend. Five crucial immune cells showed differential infiltration proportions between high and low VDAC2 expression groups. VDAC2 knockdown significantly inhibited the proliferation and invasion ability of NSCLC cells. Integrating single cell and bulk sequencing data as well as wet lab experiments, hypoxia-related VDAC2 exhibited important prognostic value and showed the promise of becoming immune-therapy target in NSCLC.

Interactome Analysis Identifies the Role of BZW2 in Promoting Endoplasmic Reticulum-Mitochondria Contact and Mitochondrial Metabolism.

Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins.

Mechanistic engineering of celastrol liposomes induces ferroptosis and apoptosis by directly targeting VDAC2 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of most common and deadliest malignancies. Celastrol (Cel), a natural product derived from the Tripterygium wilfordii plant, has been extensively researched for its potential effectiveness in fighting cancer. However, its clinical application has been hindered by the unclear mechanism of action. Here, we used chemical proteomics to identify the direct targets of Cel and enhanced its targetability and anti-tumor capacity by developing a Cel-based liposomes in HCC. We demonstrated that Cel selectively targets the voltage-dependent anion channel 2 (VDAC2). Cel directly binds to the cysteine residues of VDAC2, and induces cytochrome C release via dysregulating VDAC2-mediated mitochondrial permeability transition pore (mPTP) function. We further found that Cel induces ROS-mediated ferroptosis and apoptosis in HCC cells. Moreover, coencapsulation of Cel into alkyl glucoside-modified liposomes (AGCL) improved its antitumor efficacy and minimized its side effects. AGCL has been shown to effectively suppress the proliferation of tumor cells. In a xenograft nude mice experiment, AGCL significantly inhibited tumor growth and promoted apoptosis. Our findings reveal that Cel directly targets VDAC2 to induce mitochondria-dependent cell death, while the Cel liposomes enhance its targetability and reduces side effects. Overall, Cel shows promise as a therapeutic agent for HCC.

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.

VDAC2 malonylation participates in sepsis-induced myocardial dysfunction via mitochondrial-related ferroptosis.

Sepsis-induced myocardial dysfunction (SIMD) is a prevalent and severe form of organ dysfunction with elusive underlying mechanisms and limited treatment options. In this study, the cecal ligation and puncture and lipopolysaccharide (LPS) were used to reproduce sepsis model in vitro and vivo. The level of voltage-dependent anion channel 2 (VDAC2) malonylation and myocardial malonyl-CoA were detected by mass spectrometry and LC-MS-based metabolomics. Role of VDAC2 malonylation on cardiomyocytes ferroptosis and treatment effect of mitochondrial targeting nano material TPP-AAV were observed. The results showed that VDAC2 lysine malonylation was significantly elevated after sepsis. In addition, the regulation of VDAC2 lysine 46 (K46) malonylation by K46E and K46Q mutation affected mitochondrial-related ferroptosis and myocardial injury. The molecular dynamic simulation and circular dichroism further demonstrated that VDAC2 malonylation altered the N-terminus structure of the VDAC2 channel, causing mitochondrial dysfunction, increasing mitochondrial ROS levels, and leading to ferroptosis. Malonyl-CoA was identified as the primary inducer of VDAC2 malonylation. Furthermore, the inhibition of malonyl-CoA using ND-630 or ACC2 knock-down significantly reduced the malonylation of VDAC2, decreased the occurrence of ferroptosis in cardiomyocytes, and alleviated SIMD. The study also found that the inhibition of VDAC2 malonylation by synthesizing mitochondria targeting nano material TPP-AAV could further alleviate ferroptosis and myocardial dysfunction following sepsis. In summary, our findings indicated that VDAC2 malonylation plays a crucial role in SIMD and that targeting VDAC2 malonylation could be a potential treatment strategy for SIMD.

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.

Ablation of GPR56 Causes ß-Cell Dysfunction by ATP Loss through Mistargeting of Mitochondrial VDAC1 to the Plasma Membrane.

The activation of G Protein-Coupled Receptor 56 (GPR56), also referred to as Adhesion G-Protein-Coupled Ceceptor G1 (ADGRG1), by Collagen Type III (Coll III) prompts cell growth, proliferation, and survival, among other attributes. We investigated the signaling cascades mediating this functional effect in relation to the mitochondrial outer membrane voltage-dependent anion Channel-1 (VDAC1) expression in pancreatic ß-cells. GPR56KD attenuated the Coll III-induced suppression of P70S6K, JNK, AKT, NFκB, STAT3, and STAT5 phosphorylation/activity in INS-1 cells cultured at 20 mM glucose (glucotoxicity) for 72 h. GPR56-KD also increased Chrebp, Txnip, and Vdac1 while decreasing Vdac2 mRNA expression. In GPR56-KD islet ß-cells, Vdac1 was co-localized with SNAP-25, demonstrating its plasma membrane translocation. This resulted in ATP loss, reduced cAMP production and impaired glucose-stimulated insulin secretion (GSIS) in INS-1 and human EndoC ßH1 cells. The latter defects were reversed by an acute inhibition of VDAC1 with an antibody or the VDAC1 inhibitor VBIT-4. We demonstrate that Coll III potentiates GSIS by increasing cAMP and preserving ß-cell functionality under glucotoxic conditions in a GPR56-dependent manner by attenuating the inflammatory response. These results emphasize GPR56 and VDAC1 as drug targets in conditions with impaired ß-cell function.

DNA-PKcs and ATM modulate mitochondrial ADP-ATP exchange as an oxidative stress checkpoint mechanism.

DNA-PKcs is a key regulator of DNA double-strand break repair. Apart from its canonical role in the DNA damage response, DNA-PKcs is involved in the cellular response to oxidative stress (OS), but its exact role remains unclear. Here, we report that DNA-PKcs-deficient human cells display depolarized mitochondria membrane potential (MMP) and reoriented metabolism, supporting a role for DNA-PKcs in oxidative phosphorylation (OXPHOS). DNA-PKcs directly interacts with mitochondria proteins ANT2 and VDAC2, and formation of the DNA-PKcs/ANT2/VDAC2 (DAV) complex supports optimal exchange of ADP and ATP across mitochondrial membranes to energize the cell via OXPHOS and to maintain MMP. Moreover, we demonstrate that the DAV complex temporarily dissociates in response to oxidative stress to attenuate ADP-ATP exchange, a rate-limiting step for OXPHOS. Finally, we found that dissociation of the DAV complex is mediated by phosphorylation of DNA-PKcs at its Thr2609 cluster by ATM kinase. Based on these findings, we propose that the coordination between the DAV complex and ATM serves as a novel oxidative stress checkpoint to decrease ROS production from mitochondrial OXPHOS and to hasten cellular recovery from OS.

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.

SH3BP5-AS1/IGF2BP2/VDAC2 Axis Promotes the Apoptosis and Ferroptosis of Bladder Cancer Cells.

BACKGROUND: Bladder cancer (BC) is the most common malignant tumor in the urinary system with a high incidence, imposing a burden on the healthcare system worldwide. The participation of long non-coding RNAs (lncRNAs) in BC has attracted increasing attention. OBJECTIVE: The aim in the current study was to explore the potential mechanism involving SH3BP5-AS1 in modulating BC cell proliferation, apoptosis and ferroptosis. METHODS: qPCR and WB analysis measured the expression of RNAs and proteins. Functional and mechanism experiments were performed to investigate RNA impacts on cell proliferation, apoptosis and ferroptosis, and explore the correlation between RNA and protein expression. RESULTS: SH3BP5-AS1 was down-regulated in BC cells, and SH3BP5-AS1 overexpression could inhibit BC cell proliferation but facilitate the cell apoptosis. SH3BP5-AS1 was also found to facilitate the ferroptosis of BC cells. Additionally, SH3BP5-AS1 was confirmed to recruit IGF2BP2 to regulate VDAC2 expression in the m6A-dependent manner. VDAC2 was detected to be down-regulated in BC cells and was verified to inhibit BC cell growth. Moreover, it was indicated from rescue assays that SH3BP5-AS1 could modulate VDAC2 expression to promote the ferroptosis of BC cells. CONCLUSION: SH3BP5-AS1 could affect BC cell proliferation, apoptosis and ferroptosis via IGF2BP2/VDAC2, providing a novel molecular perspective for understanding BC.

Hedyotis diffusa injection induces ferroptosis via the Bax/Bcl2/VDAC2/3 axis in lung adenocarcinoma.

BACKGROUND: Lung cancer has the highest mortality rate among all cancer types. In combination with multiple chemotherapeutic options, traditional Chinese medicine has proven indispensable for the comprehensive treatment of lung cancer. PURPOSE: To investigate the effects of Hedyotis diffusa on lung adenocarcinoma cell lines and a BALB/c nude mouse xenograft model, and determine whether HDI could induce ferroptosis in lung adenocarcinoma cells along with the underlying mechanism. METHODS: The anti-tumor activity of HDI was determined in vitro by cell counting kit-8, clonogenic, and transwell assays. Subsequently, electron microscopy, a lipid reactive oxygen species assay, ferrous ion staining, and a malondialdehyde assay were performed to determine the effect on ferroptosis in lung adenocarcinoma cells. The mechanism was then further investigated using small molecule inhibitors, siRNA, and plasmid overexpression in vitro. Finally, the effects of HDI were assessed in tumor-bearing BALB/c nude mice, and HE staining was performed to observe tissue damage after HDI treatment. RESULTS: In vitro experiments showed that HDI could inhibit the viability of lung adenocarcinoma cells and induce lung adenocarcinoma cells ferroptosis via mechanisms independent of GPX4 and PUFA-PLS pathways but closely associated with VDAC2/3. HDI regulated VDAC2/3 activity by promoting Bax via inhibiting Bcl2, thereby inducing ferroptosis in lung adenocarcinoma cells. Furthermore, in vivo experiments showed that HDI significantly inhibited the growth of subcutaneous tumors in BALB/c nude mice with less organ damage and toxicity, and significantly increased the expression of the ferroptosis-related indicators 4HNE, TFR, and HMOX1 in tumor tissue. CONCLUSION: HDI can significantly reduce the survival of lung adenocarcinoma cells in vitro, inhibit the growth of subcutaneously transplanted tumors in BALB/c nude mice in vivo, and induce ferroptosis in lung adenocarcinoma cells via Bcl2 inhibition to promote Bax regulation of VDAC2/3.

VDAC2 and the BCL-2 family of proteins.

The BCL-2 protein family govern whether a cell dies or survives by controlling mitochondrial apoptosis. As dysregulation of mitochondrial apoptosis is a common feature of cancer cells, targeting protein-protein interactions within the BCL-2 protein family is a key strategy to seize control of apoptosis and provide favourable outcomes for cancer patients. Non-BCL-2 family proteins are emerging as novel regulators of apoptosis and are potential drug targets. Voltage dependent anion channel 2 (VDAC2) can regulate apoptosis. However, it is unclear how this occurs at the molecular level, with conflicting evidence in the literature for its role in regulating the BCL-2 effector proteins, BAK and BAX. Notably, VDAC2 is required for efficient BAX-mediated apoptosis, but conversely inhibits BAK-mediated apoptosis. This review focuses on the role of VDAC2 in apoptosis, discussing the current knowledge of the interaction between VDAC2 and BCL-2 family proteins and the recent development of an apoptosis inhibitor that targets the VDAC2-BAK interaction.

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.

Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats / 中国实验方剂学杂志

Objective:To investigate the mechanism of Shugan Bushen Yulin decoction in inhibiting voltage-dependent anion-selective channel protein 2 （VDAC2） gene methylation， affecting sperm mitochondrial function， and improving sperm motility through the cyclic adenosine monophosphate/protein kinase A （cAMP/PKA） pathway. Method:Forty male SD rats were randomly divided into the blank group， model group， high- and low-dose Shugan Bushen Yulin decoction groups， and L-carnitine group， with eight rats in each group. Adenine （0.05 g·kg^-1） was administered by gavage for 14 d for inducing oligospermia and asthenospermia. Rats in the Shugan Bushen Yulin decoction groups were treated with intragastric administration of 32.4， 8.1 g·kg^-1Shugan Bushen Yulin decoction， respectively， while those in the L-carnitine group received 0.27 g·kg^-1 L-carnitine by gavage. Following the measurement of sperm motility using an automatic sperm analyzer， the pathological changes in testicular tissue were observed by hematoxylin-eosin （HE） staining. Sperm mitochondrial membrane potential was detected by flow cytometry. The expression of VDAC2 in the testicular tissue was determined by immunofluorescence assay. Real-time polymerase chain reaction （Real-time PCR） was conducted for detecting VDAC2 mRNA expression in testicular tissue. The methylation of VDAC2 gene was examined using bisulfite sequencing. The cAMP expression in testicular tissue was detected by enzyme-linked immunosorbent assay （ELISA）， and the PKA protein expression in testicular tissue by Western blot. Result:Compared with the blank group， the model group exhibited significantly decreased sperm density and motility （<italic>P</italic><0.01）， increased mitochondrial membrane potential （<italic>P</italic><0.01）， down-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in testicular tissue （<italic>P</italic><0.01）， and elevated VDAC2 gene methylation （<italic>P</italic><0.01）. Compared with the model group， L-carnitine and Shugan Bushen Yulin decoction at the high and low doses all remarkably increased the sperm density and motility and mitochondrial membrane potential （<italic>P</italic><0.01）， up-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue （<italic>P</italic><0.01）， and lowered the methylation of VDAC2 in testicular tissue （<italic>P</italic><0.01）. The comparison with the L-carnitine group showed that the sperm density and motility and mitochondrial membrane potential in the low-dose Shugan Bushen Yulin decoction group declined significantly （<italic>P</italic><0.01）. The VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue were significantly down-regulated （<italic>P</italic><0.01）， while the methylation of VDAC2 was significantly enhanced （<italic>P</italic><0.01）. Conclusion:Shugan Bushen Yulint decoction may inhibit VDAC2 gene methylation， increase VDAC2 expression， regulate cAMP/PKA pathway， and change mitochondrial membrane potential to enhance the sperm motility.

Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER-mitochondria contact sites.

Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.

Inflammatory IFIT3 renders chemotherapy resistance by regulating post-translational modification of VDAC2 in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide and effective therapy remains a challenge. IFIT3 is an interferon-stimulated gene with antiviral and pro-inflammatory functions. Our previous work has shown that high expression of IFIT3 is correlated with poor survival in PDAC patients who receive chemotherapy suggesting a link between IFIT3 and chemotherapy resistance in PDAC. However, the exact role and molecular mechanism of IFIT3 in chemotherapy resistance in PDAC has been unclear. Methods: A group of transcriptome datasets were downloaded and analyzed for the characterization of IFIT3 in PDAC. Highly metastatic PDAC cell line L3.6pl and patient-derived primary cell TBO368 were used and IFIT3 knockdown and the corresponding knockin cells were established for in vitro studies. Chemotherapy-induced apoptosis, ROS production, confocal immunofluorescence, subcellular fractionation, chromatin-immunoprecipitation, co-immunoprecipitation and mass spectrometry analysis were determined to further explore the biological role of IFIT3 in chemotherapy resistance of PDAC. Results: Based on PDAC transcriptome data, we show that IFIT3 expression is associated with the squamous molecular subtype of PDAC and an increase in inflammatory response and apoptosis pathways. We further identify a crucial role for IFIT3 in the regulation of mitochondria-associated apoptosis during chemotherapy. Knockdown of IFIT3 attenuates the chemotherapy resistance of PDAC cells to gemcitabine, paclitaxel, and FOLFIRINOX regimen treatments, independent of individual chemotherapy regimens. While IFIT3 overexpression was found to promote drug resistance. Co-immunoprecipitation identified a direct interaction between IFIT3 and the mitochondrial channel protein VDAC2, an important regulator of mitochondria-associated apoptosis. It was subsequently found that IFIT3 regulates the post-translational modification-O-GlcNAcylation of VDAC2 by stabilizing the interaction of VDAC2 with O-GlcNAc transferase. Increased O-GlcNAcylation of VDAC2 protected PDAC cells from chemotherapy induced apoptosis. Conclusions: These results effectively demonstrate a central mechanism by which IFIT3 expression can affect chemotherapy resistance in PDAC. Targeting IFIT3/VDAC2 may represent a novel strategy to sensitize aggressive forms of pancreatic cancer to conventional chemotherapy regimens.

Recent insights into peroxisome biogenesis and associated diseases.

Peroxisomes are single-membrane organelles present in eukaryotes. The functional importance of peroxisomes in humans is represented by peroxisome-deficient peroxisome biogenesis disorders (PBDs), including Zellweger syndrome. Defects in the genes that encode the 14 peroxins that are required for peroxisomal membrane assembly, matrix protein import and division have been identified in PBDs. A number of recent findings have advanced our understanding of the biology, physiology and consequences of functional defects in peroxisomes. In this Review, we discuss a cooperative cell defense mechanisms against oxidative stress that involves the localization of BAK (also known as BAK1) to peroxisomes, which alters peroxisomal membrane permeability, resulting in the export of catalase, a peroxisomal enzyme. Another important recent finding is the discovery of a nucleoside diphosphate kinase-like protein that has been shown to be essential for how the energy GTP is generated and provided for the fission of peroxisomes. With regard to PBDs, we newly identified a mild mutation, Pex26-F51L that causes only hearing loss. We will also discuss findings from a new PBD model mouse defective in Pex14, which manifested dysregulation of the BDNF-TrkB pathway, an essential signaling pathway in cerebellar morphogenesis. Here, we thus aim to provide a current view of peroxisome biogenesis and the molecular pathogenesis of PBDs.

Downregulation of vdac2 inhibits spermatogenesis via JNK and P53 signalling in mice exposed to cadmium.

Previous studies have reported the reproductive toxicity of cadmium (Cd); however, the effect of Cd on spermatogenesis and the underlying mechanism remain to be elucidated. In this study, mouse Leydig TM3 cells were treated with CdCl2 (0, 5, 10 and 50 µM) for 24 h to evaluate cytotoxicity, and C57BL/6 mice were treated intragastrically with 0.4 mL CdCl2 (0, 0.01, 0.05 and 0.1 g/L) for 2 months to investigate changes in spermatogenesis. The results showed that Cd aggravated apoptosis and proliferation in a dose-dependent manner, concomitant with deteriorated spermatogenesis and testosterone synthesis. For mechanism exploration, RNA-seq was used to profile alterations in gene expression in response to Cd, and the results indicated focus on P53/JNK signalling pathways and membrane proteins. We found that P53/JNK signalling pathways were activated upon Cd treatment, with the Cd-triggered downregulation of the vdac2 gene. P53/JNK pathway blockade ameliorated the Cd-induced inhibition of steroidogenic acute regulatory protein (STAR) expression and testosterone synthesis. Additionally, vdac2 knockdown in TM3 cells contributed to the phosphorylation of JNK/P53 and reduced the testosterone content. Vdac2 overexpression rescued the aforementioned Cd-induced events. Collectively, our study identified an innovative biomarker of Cd exposure in mice. The results demonstrated that vdac2 downregulation inhibits spermatogenesis via the JNK/P53 cascade. This finding may contribute to our understanding of the regulatory mechanism of Cd reproductive toxicity and provide a candidate list for sperm abnormality factors and pathways.