Trafficking of mitochondrial double-stranded RNA from mitochondria to the cytosol.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.

mtDNA release promotes cGAS-STING activation and accelerated aging of postmitotic muscle cells.

The mechanism regulating cellular senescence of postmitotic muscle cells is still unknown. cGAS-STING innate immune signaling was found to mediate cellular senescence in various types of cells, including postmitotic neuron cells, which however has not been explored in postmitotic muscle cells. Here by studying the myofibers from Zmpste24-/- progeria aged mice [an established mice model for Hutchinson-Gilford progeria syndrome (HGPS)], we observed senescence-associated phenotypes in Zmpste24-/- myofibers, which is coupled with increased oxidative damage to mitochondrial DNA (mtDNA) and secretion of senescence-associated secretory phenotype (SASP) factors. Also, Zmpste24-/- myofibers feature increased release of mtDNA from damaged mitochondria, mitophagy dysfunction, and activation of cGAS-STING. Meanwhile, increased mtDNA release in Zmpste24-/- myofibers appeared to be related with increased VDAC1 oligomerization. Further, the inhibition of VDAC1 oligomerization in Zmpste24-/- myofibers with VBIT4 reduced mtDNA release, cGAS-STING activation, and the expression of SASP factors. Our results reveal a novel mechanism of innate immune activation-associated cellular senescence in postmitotic muscle cells in aged muscle, which may help identify novel sets of diagnostic markers and therapeutic targets for progeria aging and aging-associated muscle diseases.

Augmented microglial endoplasmic reticulum-mitochondria contacts mediate depression-like behavior in mice induced by chronic social defeat stress.

Extracellular ATP (eATP) signaling through the P2X7 receptor pathway is widely believed to trigger NLRP3 inflammasome assembly in microglia, potentially contributing to depression. However, the cellular stress responses of microglia to both eATP and stress itself remain largely unexplored. Mitochondria-associated membranes (MAMs) is a platform facilitating calcium transport between the endoplasmic reticulum (ER) and mitochondria, regulating ER stress responses and mitochondrial homeostasis. This study aims to investigate how MAMs influence microglial reaction and their involvement in the development of depression-like symptoms in response to chronic social defeat stress (CSDS). CSDS induced ER stress, MAMs' modifications, mitochondrial damage, and the formation of the IP3R3-GRP75-VDAC1 complex at the ER-mitochondria interface in hippocampal microglia, all concomitant with depression-like behaviors. Additionally, exposing microglia to eATP to mimic CSDS conditions resulted in analogous outcomes. Furthermore, knocking down GRP75 in BV2 cells impeded ER-mitochondria contact, calcium transfer, ER stress, mitochondrial damage, mitochondrial superoxide production, and NLRP3 inflammasome aggregation induced by eATP. In addition, reduced GRP75 expression in microglia of Cx3cr1CreER/+Hspa9f/+ mice lead to reduce depressive behaviors, decreased NLRP3 inflammasome aggregation, and fewer ER-mitochondria contacts in hippocampal microglia during CSDS. Here, we show the role of MAMs, particularly the formation of a tripartite complex involving IP3R3, GRP75, and VDAC1 within MAMs, in facilitating communication between the ER and mitochondria in microglia, thereby contributing to the development of depression-like phenotypes in male mice.

The VDAC1 oligomerization regulated by ATP5B leads to the NLRP3 inflammasome activation in the liver cells under PFOS exposure.

As a persistent organic pollutant, perfluorooctane sulfonate (PFOS) has a serious detrimental impact on human health. It has been suggested that PFOS is associated with liver inflammation. However, the underlying mechanisms are still unclear. Here, PFOS was found to elevate the oligomerization tendency of voltage-dependent anion channel 1 (VDAC1) in the mice liver and human normal liver cells L-02. Inhibition of VDAC1 oligomerization alleviated PFOS-induced nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome activation. Cytoplasmic membrane VDAC1 translocated to mitochondria was also observed in response to PFOS. Therefore, the oligomerization of VDAC1 occurred mainly in the mitochondria. VDAC1 was found to interact with the ATP synthase beta subunit (ATP5B) under PFOS treatment. Knockdown of ATP5B or immobilization of ATP5B to the cytoplasmic membrane alleviated the increased VDAC1 oligomerization and NLRP3 inflammasome activation. Therefore, our results suggested that PFOS induced NLRP3 inflammasome activation through VDAC1 oligomerization, a process dependent on ATP5B to transfer VDAC1 from the plasma membrane to the mitochondria. The findings offer novel perspectives on the activation of the NLRP3 inflammasome, the regulatory mode on VDAC1 oligomerization, and the mechanism of PFOS toxicity.

Gene signatures of endoplasmic reticulum stress and mitophagy for prognostic risk prediction in lung adenocarcinoma.

Genes associated with endoplasmic reticulum stress (ERS) and mitophagy can be conducive to predicting solid tumour prognosis. The authors aimed to develop a prognosis prediction model for these genes in lung adenocarcinoma (LUAD). Relevant gene expression and clinical information were collected from public databases including Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). A total of 265 differentially expressed genes was finally selected (71 up-regulated and 194 downregulated) in the LUAD dataset. Among these, 15 candidate ERS and mitophagy genes (ATG12, CSNK2A1, MAP1LC3A, MAP1LC3B, MFN2, PGAM5, PINK1, RPS27A, SQSTM1, SRC, UBA52, UBB, UBC, ULK1, and VDAC1) might be critical to LUAD based on the expression analysis after crossing with the ERS and mitochondrial autophagy genes. The prediction model demonstrated the ability to effectively predict the 5-, 3-, and 1-year prognoses of LUAD patients in both GEO and TCGA databases. Moreover, high VDAC1 expression was associated with poor overall survival in LUAD (p < 0.001), suggesting it might be a critical gene for LUAD prognosis prediction. Overall, the prognosis model based on ERS and mitophagy genes in LUAD can be useful for evaluating the prognosis of patients with LUAD, and VDAC1 may serve as a promising biomarker for LUAD prognosis.

KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These 'licensed' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

The Hexokinase 1 5'-UTR Mutation in Charcot-Marie-Tooth 4G Disease Alters Hexokinase 1 Binding to Voltage-Dependent Anion Channel-1 and Leads to Dysfunctional Mitochondrial Calcium Buffering.

Demyelinating Charcot-Marie-Tooth 4G (CMT4G) results from a recessive mutation in the 5'UTR region of the Hexokinase 1 (HK1) gene. HK participates in mitochondrial calcium homeostasis by binding to the Voltage-Dependent Anion Channel (VDAC), through its N-terminal porin-binding domain. Our hypothesis is that CMT4G mutation results in a broken interaction between mutant HK1 and VDAC, disturbing mitochondrial calcium homeostasis. We studied a cohort of 25 CMT4G patients recruited in the French gypsy population. The disease was characterized by a childhood onset, an intermediate demyelinating pattern, and a significant phenotype leading to becoming wheelchair-bound by the fifth decade of life. Co-IP and PLA studies indicated a strong decreased interaction between VDAC and HK1 in the patients' PBMCs and sural nerve. We observed that either wild-type HK1 expression or a peptide comprising the 15 aa of the N-terminal wild-type HK1 administration decreased mitochondrial calcium release in HEK293 cells. However, mutated CMT4G HK1 or the 15 aa of the mutated HK1 was unable to block mitochondrial calcium release. Taken together, these data show that the CMT4G-induced modification of the HK1 N-terminus disrupts HK1-VDAC interaction. This alters mitochondrial calcium buffering that has been shown to be critical for myelin sheath maintenance.

Phosphoglycerate mutase 5 aggravates alcoholic liver disease through disrupting VDAC-1-dependent mitochondrial integrity.

Alcoholic liver disease (ALD) poses a substantial global health challenge, with its pathogenesis deeply rooted in mitochondrial dysfunction. Our study explores the pivotal roles of Phosphoglycerate mutase family member 5 (Pgam5) and Voltage-Dependent Anion Channel 1 (VDAC1) in the progression of ALD, providing novel insights into their interplay and impact on mitochondrial integrity. We demonstrate that Pgam5 silencing preserves hepatocyte viability and attenuates ethanol-induced apoptosis, underscoring its detrimental role in exacerbating hepatocyte dysfunction. Pgam5's influence extends to the regulation of VDAC1 oligomerization, a key process in mitochondrial permeability transition pore (mPTP) opening, mitochondrial swelling, and apoptosis initiation. Notably, the inhibition of VDAC1 oligomerization through Pgam5 silencing or pharmacological intervention (VBIT-12) significantly preserves mitochondrial function, evident in the maintenance of mitochondrial membrane potential and reduced reactive oxygen species (ROS) production. In vivo experiments using hepatocyte-specific Pgam5 knockout (Pgam5hKO) and control mice reveal that Pgam5 deficiency mitigates ethanol-induced liver histopathology, inflammation, lipid peroxidation, and metabolic disorder, further supporting its role in ALD progression. Our findings highlight the critical involvement of Pgam5 and VDAC1 in mitochondrial dysfunction in ALD, suggesting potential therapeutic targets. While promising, these findings necessitate further research, including human studies, to validate their clinical applicability and explore broader implications in liver diseases. Overall, our study provides a significant advancement in understanding ALD pathophysiology, paving the way for novel therapeutic strategies targeting mitochondrial pathways in ALD.

AMPK/PGC-1α and p53 modulate VDAC1 expression mediated by reduced ATP level and metabolic oxidative stress in neuronal cells.

Voltage-dependent anion channel 1 (VDAC1) is a pore protein located in the outer mitochondrial membrane. Its channel gating mediates mitochondrial respiration and cell metabolism, and it has been identified as a critical modulator of mitochondria-mediated apoptosis. In many diseases characterized by mitochondrial dysfunction, such as cancer and neurodegenerative diseases, VDAC1 is considered a promising potential therapeutic target. However, there is limited research on the regulatory factors involved in VDAC1 protein expression in both normal and pathological states. In this study, we find that VDAC1 protein expression is up-regulated in various neuronal cell lines in response to intracellular metabolic and oxidative stress. We further demonstrate that VDAC1 expression is modulated by intracellular ATP level. Through the use of pharmacological agonists and inhibitors and small interfering RNA (siRNA), we reveal that the AMPK/PGC-1α signaling pathway is involved in regulating VDAC1 expression. Additionally, based on bioinformatics predictions and biochemical verification, we identify p53 as a potential transcription factor that regulates VDAC1 promoter activity during metabolic oxidative stress. Our findings suggest that VDAC1 expression is regulated by the AMPK/PGC-1α and p53 pathways, which contributes to the maintenance of stress adaptation and apoptotic homeostasis in neuronal cells.

Interleukin 21 Drives a Hypermetabolic State and CD4+ T-Cell-Associated Pathogenicity in Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Incapacitated regulatory T cells (Tregs) contribute to immune-mediated diseases. Inflammatory Tregs are evident during human inflammatory bowel disease; however, mechanisms driving the development of these cells and their function are not well understood. Therefore, we investigated the role of cellular metabolism in Tregs relevant to gut homeostasis. METHODS: Using human Tregs, we performed mitochondrial ultrastructural studies via electron microscopy and confocal imaging, biochemical and protein analyses using proximity ligation assay, immunoblotting, mass cytometry and fluorescence-activated cell sorting, metabolomics, gene expression analysis, and real-time metabolic profiling utilizing the Seahorse XF analyzer. We used a Crohn's disease single-cell RNA sequencing dataset to infer the therapeutic relevance of targeting metabolic pathways in inflammatory Tregs. We examined the superior functionality of genetically modified Tregs in CD4+ T-cell-induced murine colitis models. RESULTS: Mitochondria-endoplasmic reticulum appositions, known to mediate pyruvate entry into mitochondria via voltage-dependent anion channel 1 (VDAC1), are abundant in Tregs. VDAC1 inhibition perturbed pyruvate metabolism, eliciting sensitization to other inflammatory signals reversible by membrane-permeable methyl pyruvate supplementation. Notably, interleukin (IL) 21 diminished mitochondria-endoplasmic reticulum appositions, resulting in enhanced enzymatic function of glycogen synthase kinase 3 ß, a putative negative regulator of VDAC1, and a hypermetabolic state that amplified Treg inflammatory response. Methyl pyruvate and glycogen synthase kinase 3 ß pharmacologic inhibitor (LY2090314) reversed IL21-induced metabolic rewiring and inflammatory state. Moreover, IL21-induced metabolic genes in Tregs in vitro were enriched in human Crohn's disease intestinal Tregs. Adoptively transferred Il21r-/- Tregs efficiently rescued murine colitis in contrast to wild-type Tregs. CONCLUSIONS: IL21 triggers metabolic dysfunction associated with Treg inflammatory response. Inhibiting IL21-induced metabolism in Tregs may mitigate CD4+ T-cell-driven chronic intestinal inflammation.

By regulating the IP3R/GRP75/VDAC1 complex to restore mitochondrial dynamic balance, selenomethionine reduces lipopolysaccharide-induced neuronal apoptosis.

Selenium (Se), as one of the essential trace elements, plays an anti-inflammatory, antioxidation, and immune-enhancing effect in the body. In addition, Se can also improve nervous system damage induced by various factors. Earlier studies have described the important role of mitochondrial dynamic imbalance in lipopolysaccharide (LPS)-induced nerve injury. The inositol 1,4,5-triphosphate receptor (IP3R)/glucose-regulated protein 75 (GRP75)/voltage-dependent anion channel 1 (VDAC1) complex is considered to be the key to regulating mitochondrial dynamics. However, it is not clear whether Selenomethionine (SeMet) has any influence on the IP3R/GRP75/VDAC1 complex. Therefore, the aim of this investigation was to determine whether SeMet can alleviate LPS-induced brain damage and to elucidate the function of the IP3R/GRP75/VDAC1 complex in it. We established SeMet and/or LPS exposure models in vivo and in vitro using laying hens and primary chicken nerve cells. We noticed that SeMet reversed endoplasmic reticulum stress (ERS) and the imbalance in mitochondrial dynamics and significantly prevented the occurrence of neuronal apoptosis. We made this finding by morphological observation of the brain tissue of laying hens and the detection of related genes such as ERS, the IP3R/GRP75/VDAC1 complex, calcium signal (Ca2+), mitochondrial dynamics, and apoptosis. Other than that, we also discovered that the IP3R/GRP75/VDAC1 complex was crucial in controlling Ca2+ transport between the endoplasmic reticulum and the mitochondrion when SeMet functions as a neuroprotective agent. In summary, our results revealed the specific mechanism by which SeMet alleviated LPS-induced neuronal apoptosis for the first time. As a consequence, SeMet has great potential in the treatment and prevention of neurological illnesses (like neurodegenerative diseases).

The inhibition of VDAC1 oligomerization promotes pigmentation through the CaMK-CRTCs/CREB-MITF pathway.

The voltage-dependent anion channel 1 (VDAC1) forms an oligomeric structure on the mitochondrial outer membrane, which plays critical roles in many physiological processes. Research studies have demonstrated that the knockout of VDAC1 increases pigment content and up-regulates the expression of melanogenic genes. Due to its involvement in various physiological processes, the depletion of VDAC1 has significant detrimental effects on cellular functions and the inhibition of VDAC1 oligomerization has recently emerged as a promising strategy for the treatment of several diseases. In this study, we found that VDAC1 oligomerization inhibitors, VBIT-12 and NSC-15364, promote melanogenesis, dendrite formation and melanosome transport in human epidermal melanocytes (HEMCs). Mechanistically, treatment of HEMCs with an oligomerization inhibitor increased the level of cytoplasmic calcium ions, which activated calcium-calmodulin dependent protein kinase (CaMK) and led to the phosphorylation of CREB and the nuclear translocation of CREB-regulated transcription coactivators (CRTCs). Subsequently, CRTCs, p-CREB and CREB-binding protein (CBP) in the nucleus cooperatively recruit the transcription machinery to initiate the transcription of MITF thus promoting pigmentation. Importantly, our study also demonstrates that VDAC1 oligomerization inhibitors increase pigmentation in zebrafish and in human skin explants, highlighting their potential as a therapeutic strategy for skin pigmentation disorders.

Chemoproteomics Reveals Glaucocalyxin A Induces Mitochondria-Dependent Apoptosis of Leukemia Cells via Covalently Binding to VDAC1.

Chronic myelogenous leukemia (CML) that is resistant to tyrosine kinase inhibitors is one of the deadliest hematologic malignancies, and the T315I mutation in the breakpoint cluster region-Abelson (BCR-ABL) kinase domain is the most prominent point mutation responsible for imatinib resistance in CML. Glaucocalyxin A (GLA), a natural bioactive product derived from the Rabdosia rubescens plant, has strong anticancer activity. In this study, the effect and molecular mechanism of GLA on imatinib-sensitive and imatinib-resistant CML cells harboring T315I mutation via a combined deconvolution strategy of chemoproteomics and label-free proteomics is investigated. The data demonstrated that GLA restrains proliferation and induces mitochondria-dependent apoptosis in both imatinib-sensitive and resistant CML cells. GLA covalently binds to the cysteine residues of mitochondrial voltage-dependent anion channels (VDACs), resulting in mitochondrial damage and overflow of intracellular apoptotic factors, eventually leading to apoptosis. In addition, the combination of GLA with elastin, a mitochondrial channel VDAC2/3 inhibitor, enhances mitochondria-dependent apoptosis in imatinib-sensitive and -resistant CML cells, representing a promising therapeutic approach for leukemia treatment. Taken together, the results show that GLA induces mitochondria-dependent apoptosis via covalently targeting VDACs in CML cells. GLA may thus be a candidate compound for the treatment of leukemia.

Voltage-dependent anion channel 1 (VDAC1) overexpression alleviates cardiac fibroblast activation in cardiac fibrosis via regulating fatty acid metabolism.

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix in the myocardium with cardiac fibroblast activation, leading to chronic cardiac remodeling and dysfunction. However, little is known about metabolic alterations in fibroblasts during cardiac fibrosis, and there is a lack of pharmaceutical treatments that target metabolic dysregulation. Here, we provided evidence that fatty acid ß-oxidation (FAO) dysregulation contributes to fibroblast activation and cardiac fibrosis. With transcriptome, metabolome, and functional assays, we demonstrated that FAO was downregulated during fibroblast activation and cardiac fibrosis, and that perturbation of FAO reversely affected the fibroblast-to-myofibroblast transition. The decrease in FAO may be attributed to reduced long-chain fatty acid (LCFA) uptake. Voltage-dependent anion channel 1 (VDAC1), the main gatekeeper of the outer mitochondrial membrane (OMM), serves as the transporter of LCFA into the mitochondria for further utilization and has been shown to be decreased in myofibroblasts. In vitro, the addition of exogenous VDAC1 was shown to ameliorate cardiac fibroblast activation initiated by transforming growth factor beta 1 (TGF-ß1) stimuli, and silencing of VDAC1 displayed the opposite effect. A mechanistic study revealed that VDAC1 exerts a protective effect by regulating LCFA uptake into the mitochondria, which is impaired by an inhibitor of carnitine palmitoyltransferase 1A. In vivo, AAV9-mediated overexpression of VDAC1 in myofibroblasts significantly alleviated transverse aortic constriction (TAC)-induced cardiac fibrosis and rescued cardiac function in mice. Finally, we treated mice with the VDAC1-derived R-Tf-D-LP4 peptide, and the results showed that R-Tf-D-LP4 prevented TAC-induced cardiac fibrosis and dysfunction in mice. In conclusion, this study provides evidence that VDAC1 maintains FAO metabolism in cardiac fibroblasts to repress fibroblast activation and cardiac fibrosis and suggests that the VDAC1 peptide is a promising drug for rescuing fibroblast metabolism and repressing cardiac fibrosis.

HSP90 C-terminal domain inhibition promotes VDAC1 oligomerization via decreasing K274 mono-ubiquitination in Hepatocellular Carcinoma.

Voltage-dependent anion-selective channel protein 1 (VDAC1) is the most abundant protein in the mitochondrial outer membrane and plays a crucial role in the control of hepatocellular carcinoma (HCC) progress. Our previous research found that cytosolic molecular chaperone heat shock protein 90 (Hsp90) interacted with VDAC1, but the effect of the C-terminal and N-terminal domains of Hsp90 on the formation of VDAC1 oligomers is unclear. In this study, we focused on the effect of the C-terminal domain of Hsp90 on VDAC1 oligomerization, ubiquitination, and VDAC1 channel activity. We found that Hsp90 C-terminal domain inhibitor Novobiocin promoted VDAC1 oligomerization, release of cytochrome c, and activated mitochondrial apoptosis pathway. Atomic coarse particle modeling simulation revealed C-terminal domain of Hsp90α stabilized VDAC1 monomers. The purified VDAC1 was reconstituted into a planar lipid bilayer, and electrophysiology experiments of patch clamp showed that the Hsp90 C-terminal inhibitor Novobiocin increased VDAC1 channel conductance via promoting VDAC1 oligomerization. The mitochondrial ubiquitination proteomics results showed that VDAC1 K274 mono-ubiquitination was significantly decreased upon Novobiocin treatment. Site-directed mutation of VDAC1 (K274R) weakened Hsp90α-VDAC1 interaction and increased VDAC1 oligomerization. Taken together, our results reveal that Hsp90 C-terminal domain inhibition promotes VDAC1 oligomerization and VDAC1 channel conductance by decreasing VDAC1 K274 mono- ubiquitination, which provides a new perspective for mitochondria-targeted therapy of HCC.

MIRO-1 interacts with VDAC-1 to regulate mitochondrial membrane potential in Caenorhabditis elegans.

Precise regulation of mitochondrial fusion and fission is essential for cellular activity and animal development. Imbalances between these processes can lead to fragmentation and loss of normal membrane potential in individual mitochondria. In this study, we show that MIRO-1 is stochastically elevated in individual fragmented mitochondria and is required for maintaining mitochondrial membrane potential. We further observe a higher level of membrane potential in fragmented mitochondria in fzo-1 mutants and wounded animals. Moreover, MIRO-1 interacts with VDAC-1, a crucial mitochondrial ion channel located in the outer mitochondrial membrane, and this interaction depends on the residues E473 of MIRO-1 and K163 of VDAC-1. The E473G point mutation disrupts their interaction, resulting in a reduction of the mitochondrial membrane potential. Our findings suggest that MIRO-1 regulates membrane potential and maintains mitochondrial activity and animal health by interacting with VDAC-1. This study provides insight into the mechanisms underlying the stochastic maintenance of membrane potential in fragmented mitochondria.

DYNLT1 promotes mitochondrial metabolism to fuel breast cancer development by inhibiting ubiquitination degradation of VDAC1.

BACKGROUND: Mitochondrial metabolism has been proposed as an attractive target for breast cancer therapy. The discovery of new mechanisms underlying mitochondrial dysfunction will facilitate the development of new metabolic inhibitors to improve the clinical treatment of breast cancer patients. DYNLT1 (Dynein Light Chain Tctex-Type 1) is a key component of the motor complex that transports cellular cargo along microtubules in the cell, but whether and how DYNLT1 affects mitochondrial metabolism and breast cancer has not been reported. METHODS: The expression levels of DYNLT1 were analyzed in clinical samples and a panel of cell lines. The role of DYNLT1 in breast cancer development was investigated using in vivo mouse models and in vitro cell assays, including CCK-8, plate cloning and transwell assay. The role of DYNLT1 in regulating mitochondrial metabolism in breast cancer development is examined by measuring mitochondrial membrane potential and ATP levels. To investigate the underlying molecular mechanism, many methods, including but not limited to Co-IP and ubiquitination assay were used. RESULTS: First, we found that DYNLT1 was upregulated in breast tumors, especially in ER + and TNBC subtypes. DYNLT1 promotes the proliferation, migration, invasion and mitochondrial metabolism in breast cancer cells in vitro and breast tumor development in vivo. DYNLT1 colocalizes with voltage-dependent anion channel 1 (VDAC1) on mitochondria to regulate key metabolic and energy functions. Mechanistically, DYNLT1 stabilizes the voltage-dependent anion channel 1 (VDAC1) by hindering E3 ligase Parkin-mediated VDAC1 ubiquitination and degradation. CONCLUSION: Our data demonstrate that DYNLT1 promotes mitochondrial metabolism to fuel breast cancer development by inhibiting Parkin-mediated ubiquitination degradation of VDAC1. This study suggests that mitochondrial metabolism can be exploited by targeting the DYNLT1-Parkin-VDAC1 axis to improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC).

Increase in primary cilia number and length upon VDAC1 depletion contributes to attenuated proliferation of cancer cells.

Primary cilia (PCs) that are present in most human cells and perform sensory function or signal transduction are lost in many solid tumors. Previously, we identified VDAC1, best known to regulate mitochondrial bioenergetics, to negatively regulate ciliogenesis. Here, we show that downregulation of VDAC1 in pancreatic cancer-derived Panc1 and glioblastoma-derived U-87MG cells significantly increased ciliation. Those PCs were significantly longer than the control cells. Such increased ciliation possibly inhibited cell cycle, which contributed to reduced proliferation of these cells. VDAC1-depletion also led to longer PCs in quiescent RPE1 cells. Therefore, serum-induced PC disassembly was slower in VDAC1-depleted RPE1 cells. Overall, this study reiterates the importance of VDAC1 in modulating tumorigenesis, due to its novel role in regulating PC disassembly and cilia length.

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.

VDAC1 Knockout Affects Mitochondrial Oxygen Consumption Triggering a Rearrangement of ETC by Impacting on Complex I Activity.

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.