Mitochondrial Voltage-dependent Anion Channel Research Articles

Multi-Allelic Mitochondrial DNA Deletions in an Adult Dog with Chronic Weakness, Exercise Intolerance and Lactic Acidemia.

(1) Background: An adult dog was presented to a board-certified veterinary neurologist for evaluation of chronic weakness, exercise intolerance and lactic acidemia. (2) Methods: A mitochondrial myopathy was diagnosed based on the histological and histochemical phenotype of numerous COX-negative muscle fibers. Whole-genome sequencing established the presence of multiple extended deletions in the mitochondrial DNA (mtDNA), with the highest prevalence between the 1-11 kb positions of the approximately 16 kb mitochondrial chromosome. Such findings are typically suggestive of an underlying nuclear genome variant affecting mitochondrial replication, repair, or metabolism. (3) Results: Numerous variants in the nuclear genome unique to the case were identified in the whole-genome sequence data, and one, the insertion of a DYNLT1 retrogene, whose parent gene is a regulator of the mitochondrial voltage-dependent anion channel (VDAC), was considered a plausible causal variant. (4) Conclusions: Here, we add mitochondrial deletion disorders to the spectrum of myopathies affecting adult dogs.

Quantum Sensing of Free Radical Generation in Mitochondria of Human Keratinocytes during UVB Exposure.

Ultraviolet (UV) radiation is known to cause skin issues, such as dryness, aging, and even cancer. Among UV rays, UVB stands out for its ability to trigger problems within cells, including mitochondrial dysfunction, oxidative stress, and DNA damage. Free radicals are implicated in these cellular responses, but they are challenging to measure due to their short lifetime and limited diffusion range. In our study, we used a quantum sensing technique (T1 relaxometry) involving fluorescent nanodiamonds (FNDs) that change their optical properties in response to magnetic noise. This allowed us to monitor the free radical presence in real time. To measure radicals near mitochondria, we coated FNDs with antibodies, targeting mitochondrial protein voltage-dependent anion channel 2 (anti-VDAC2). Our findings revealed a dynamic rise in radical levels on the mitochondrial membrane as cells were exposed to UVB (3 J/cm2), with a significant increase observed after 17 min.

Abstract PO5-23-09: Metabolic alterations in Estrogen Receptor-positive breast cancer contributing to CDK4/6 resistance

Abstract Background CDK4/6 inhibitors (CDK4/6i) are potent FDA-approved agents for the treatment of metastatic and for high-risk early estrogen receptor positive (ER+) breast cancer. However, significant proportion of patients continue to develop disease progression. Response does not correlate well with expression of CDK4/6 or key cell cycle proteins. Ki67, a proliferation marker, is prognostic, but NOT predictive of response. Resistance to CDK4/6i is mediated by multiple mechanisms including metabolic alterations. Herein, we explore the metabolic alterations associated with development of CDK4/6i resistance. Methods To understand the key metabolic alterations associated with resistance, we first developed abemaciclib-resistant ER+ cell lines and compared their metabolic profile with their parental/sensitive counterparts. Seahorse metabolic profiles were performed on parental cells (MCF-7, LCC2, LCC9, T47D and ZR-75.1) and their resistant derivatives. In addition, spatial proteomics analysis was performed using multiplex cyclic immunofluorescence (CycIF) assay for 27 markers including 14 metabolic regulators (MCT1, GLUT1, G6PD, VDAC1/3, LDHA, GAPDH, PGC1A, HK2, ATP5A, GLUD12, CPT1A, CS, C6 Ceramide, COXIV), 9 signaling (CD36, CCND1, CCNE1, pRB, MKI67, pERK, CDH1, GPNMB and MET) and 4 segmentation markers (pan-cytokeratin for epithelial cells, concanavalin A for endoplasmic reticulum, phalloidin for actin, and wheat germ agglutinin (WGA) for Golgi and plasma membrane) in sensitive and resistant cells. Following the image registration (hiPlex) and cell segmentation (CellProfiler), multiplex protein images were clustered for cell phenotypes (HistoCAT). Results Cells resistant to abemaciclib showed G1 phase arrest, but a dramatic increase in invasive capacity. The Seahorse XFp Cell Energy Phenotype Assay demonstrated altered metabolic profiles including decreased extracellular acidification rate (ECAR) and oxygen consumption rate (OCAR) indicative of glycolysis, and mitochondrial respiration, respectively, in resistant cells. Multiplex CycIF utilized a panel of 11 cycles resulting in a total of 27 markers. We demonstrated that three metabolic enzymes, mitochondrial voltage-dependent anion channel protein (VDAC1/3), lactate Dehydrogenase A (LDHA) and PPARγ coactivator 1α (PGC1α) are significantly overexpressed in CDK4/6 resistant cells compared to the control parental sensitive cells. Further analyses are ongoing to better characterize these changes in human samples treated with CDK4/6i. Conclusion Our findings highlight the importance of metabolic pathways in development of resistance to CDK4/6i treatment. Understanding the mechanisms by which these pathways contribute to therapeutic failure will further provide with novel intervention strategies and new avenues for prevent recurrence/resistance in ER+ breast cancer. Citation Format: Mayar Allam, Ahmet Coskun, Thomas Hu, Yuan Gu, Sunil Badve, Yesim Gokmen-Polar. Metabolic alterations in Estrogen Receptor-positive breast cancer contributing to CDK4/6 resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-23-09.

Glutathionylation of a glycolytic enzyme promotes cell death and vigor loss during aging of elm seeds.

Seed deterioration during storage is a major problem in agricultural and forestry production and for germplasm conservation. Our previous studies have shown that a mitochondrial outer membrane protein VOLTAGE-DEPENDENT ANION CHANNEL (VDAC) is involved in programmed cell death (PCD)-like viability loss during the controlled deterioration treatment (CDT) of elm (Ulmus pumila L.) seeds, but its underlying mechanism remains unclear. In this study, we demonstrate that the oxidative modification of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) is functioned in the gate regulation of VDAC during the CDT of elm seeds. Through biochemical and cytological methods and observations of transgenic material [Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and yeast (Saccharomyces cerevisiae)], we demonstrate that cysteine S-glutathionylated UpGAPDH1 interacts with UpVDAC3 during seed aging, which leads to a mitochondrial permeability transition and aggravation of cell death, as indicated by the leakage of the mitochondrial pro-apoptotic factor cytochrome c and the emergence of apoptotic nucleus. Physiological assays and inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that GAPDH glutathionylation is mediated by increased glutathione, which might be caused by increases in the concentrations of free metals, especially Zn. Introduction of the Zn-specific chelator TPEN [(N, N, N', N'-Tetrakis (2-pyridylmethyl)ethylenediamine)] significantly delayed seed aging. We conclude that glutathionylated UpGAPDH1 interacts with UpVDAC3 and serves as a pro-apoptotic protein for VDAC-gating regulation and cell death initiation during seed aging.

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

We previously reported that bakuchiol, a phenolic isoprenoid anti-cancer 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 (MDCK) cell lysates and purified the specific bakuchiol-binding proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using nano-liquid chromatography with tandem mass spectrometry 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 co-localized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein (NP) expression levels and viral titers in the conditioned medium of virus-infected MDCK 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.

Integrating Proteomics and Functional Genomics to Identify Targets for Improving Glucocorticoid-Based Treatment of B-Lymphoblastic Leukemia

Glucocorticoids are a critical component of successful combination chemotherapy regimens for B-lymphoblastic leukemia (B-ALL). Despite advances in immunotherapies, glucocorticoid-based chemotherapy continues to be the essential frontline treatment of patients with B-ALL. This approach has led to high cure rates but at the risk of both acute, potentially life-threatening complications and chronic toxicities. Additionally, glucocorticoids remain ineffective for some patients with B-ALL, which predicts poor outcomes. To achieve better outcomes and reduce the toxicity of treatment, we take a functional genomics approach to understand how glucocorticoids induce cell death and identify strategies to make them more effective. Glucocorticoids work through the glucocorticoid receptor (GR), a ligand-activated transcription factor, to induce cell death in B-ALL. By integrating gene expression and RNA interference screening, we have identified both effector genes (genes whose regulation by glucocorticoids contributes to B-ALL cell death) and sources of resistance that can be targeted to improve treatment (Kruth et al Blood 2017). This identified the network of BCL-2 family proteins (e.g., BCL2, BCL2L11, BMF), whose regulation contributes to apoptosis in B-ALL. Surprisingly, it also showed that suppression of B-cell development genes (e.g., EBF1, PAX5, IL7R) contributes to B-ALL cell death. However, only a fraction of the genes that affect the glucocorticoid sensitivity of B-ALL are effector genes. We therefore sought to identify functional interaction partners of GR, defined as proteins that physically interact with GR and influence glucocorticoid sensitivity. To identify proteins that physically interact with GR, we used proximity labeling. We fused an ascorbate peroxidase domain (APEX2) to the N-terminus of GR and expressed it REH cells, a B-ALL cell line that does not express GR protein. After verifying that APEX2-GR restored glucocorticoid sensitivity in the REH cells, we performed proximity labeling in the presence and absence of dexamethasone. After optimizing these reactions, cells were lysed and labeled proteins were isolated using magnetic streptavidin beads and identified by mass spectrometry. We were able to detect more than 4,000 proteins that interact with GR, including hundreds that are enriched upon addition of dexamethasone. To identify functional interaction, we intersected the proteins enriched upon addition of dexamethasone with proteins shown by us to modulate glucocorticoid sensitivity in B-ALL (Kruth et al Blood 2017). Supporting our working model, we found functional interaction partners that are involved in B-cell development and apoptosis. We found that GR interacts with EBF1 and MEF2C, B-cell development transcription factors that restrain glucocorticoid-induced cell death. This indicates that GR activity and glucocorticoid sensitivity is directly blunted by B-cell development, and further supports the model that glucocorticoids restrain B-cell development. The mechanism of how these interactions influence glucocorticoid sensitivity will guide rational targeting to improve treatment of B-ALL both in general and in different B-ALL subtypes. Additionally, the mitochondrial voltage dependent anion channel 2 (VDAC2) also interacts with GR in a dexamethasone-dependent manner and is integral to glucocorticoid-induced cell death. VDAC2 is important for nucleating and perhaps triggering pore formation through BAK and BAX. How GR influences this activity is not known but suggests a non-genomic role of GR in glucocorticoid-induced cell death. In summary, integration of functional genomics and proteomics provides rich data sets that will help understand the mechanism of glucocorticoid-induced B-ALL cell death. Targeting of non-canonical (B-cell development) and non-genomic (VDAC2) functions of GR will allow for optimization of glucocorticoid-based chemotherapy regimens.

Abstract 17808: Neuroprotective Effects of 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid Inhibition on Voltage-Dependent Anion Channel 1 or Ruthenium Red Inhibition on Mitochondrial Calcium Uniporter After Cardiac Arrest

Introduction: Mitochondrial dysfunction is associated with poor neurological outcomes after resuscitation. Over the past decade, identification of the mitochondrial outer membrane voltage-dependent anion channel 1 (VDAC1) and inner membrane mitochondrial calcium uniporter (MCU) has provided insight into the roles that mitochondrial Ca2+ regulation plays in neuronal function after cardiac arrest (CA). Hypothesis: The neuroprotective effects of 4,4'-Diisothiocyanostilbene-2,2'-disulfonic Acid (DIDS) inhibition on VDAC1 or Ruthenium red (RUR) inhibition on MCU after asphyxial CA in rats were investigated. Methods: Male SD rats, 240-340 g, were subjected to 8 min asphyxial CA and followed by manual cardiopulmonary resuscitation. Sham, Control, DIDS, RUR, DIDS+RUR groups were included. The rats in the DIDS, RUR, DIDS+RUR groups received 7 mg/kg DIDS, 3 mg/kg RUR or 7 mg/kg DIDS combined with 3 mg/kg RUR immediately after return of spontaneous circulation (ROSC). Survival and Neurologic Deficit Score were assessed at 24 h after ROSC. Neurological outcome was assessed. Hippocampal mitochondrial apoptosis was measured. Results: Survival was significantly higher in the RUR group compared to the Control group (85% vs. 40%, P < 0.05). NDS in the DIDS and RUR groups were significantly better than in the Control group (P < 0.05). Neurons were less damaged in the DIDS, RUR, DIDS+RUR groups than in the Control group (Figure). Cyt.C was lower in the DIDS, RUR, DIDS+RUR groups than in the Control group (P < 0.05). Bax was lower in the RUR, DIDS+RUR groups than in the Control group (P < 0.05). Bcl-2 was highly expressed in the RUR group than in the Control group (P < 0.05). Cleaved caspase-3 was lower in the DIDS+RUR group (P < 0.05). AIF was higher in the DIDS and RUR groups compared to the Control group (P < 0.05). Conclusions: Inhibiting VDAC1 or MCU significantly improved survival, neurological outcome and inhibited hippocampal mitochondrial apoptosis after CA.

Mitochondrial VOLTAGE-DEPENDENT ANION CHANNEL 3 regulates stomatal closure by abscisic acid signaling.

In Arabidopsis (Arabidopsis thaliana), stomatal closure mediated by abscisic acid (ABA) is redundantly controlled by ABA receptor family proteins (PYRABACTIN RESISTANCE 1 [PYR1]/PYR1-LIKE [PYLs]) and subclass III SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2 (SnRK2s). Among these proteins, the roles of PYR1, PYL2, and SnRK2.6 are more dominant. A recent discovery showed that ABA-induced accumulation of reactive oxygen species (ROS) in mitochondria promotes stomatal closure. By analyzing stomatal movements in an array of single and higher order mutants, we revealed that the mitochondrial protein VOLTAGE-DEPENDENT ANION CHANNEL 3 (VDAC3) jointly regulates ABA-mediated stomatal closure with a specialized set of PYLs and SnRK2s by affecting cellular and mitochondrial ROS accumulation. VDAC3 interacted with 9 PYLs and all 3 subclass III SnRK2s. Single mutation in VDAC3, PYLs (except PYR1 and PYL2), or SnRK2.2/2.3 had little effect on ABA-mediated stomatal closure. However, knocking out PYR1, PYL1/2/4/8, or SnRK2.2/2.3 in vdac3 mutants resulted in significantly delayed or attenuated ABA-mediated stomatal closure, despite the presence of other PYLs or SnRK2s conferring redundant functions. We found that cellular and mitochondrial accumulation of ROS induced by ABA was altered in vdac3pyl1 mutants. Moreover, H2O2 treatment restored ABA-induced stomatal closure in mutants with decreased stomatal sensitivity to ABA. Our work reveals that VDAC3 ensures redundant control of ABA-mediated stomatal closure by canonical ABA signaling components.

Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial immunometabolic modulation.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs' intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.

Abstract PR006: STING is a vulnerability in RCC independent of its innate immunity function

Abstract 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. Recently, we find 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. We identify mitochondrial voltage dependent anion channel VDAC2 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, our 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. Citation Format: Pengda Liu, Zhichuan Zhu. STING is a vulnerability in RCC independent of its innate immunity function [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR006.

Effect of the metal ion-induced carbonylation modification of mitochondrial membrane channel protein VDAC on cell vitality, seedling growth and seed aging.

Seeds are the most important carrier of germplasm preservation. However, an irreversible decrease in vigor can occur after the maturation of seeds, denoted as seed aging. Mitochondrion is a crucial organelle in initiation programmed cell death during seed aging. However, the underlying mechanism remains unclear. Our previous proteome study found that 13 mitochondria proteins underwent carbonylation modification during the aging of Ulmus pumila L. (Up) seeds. This study detected metal binding proteins through immobilized metal affinity chromatography (IMAC), indicating that metal binding proteins in mitochondria are the main targets of carbonization during seed aging. Biochemistry, molecular and cellular biology methods were adopted to detect metal-protein binding, protein modification and subcellular localization. Yeast and Arabidopsis were used to investigate the biological functions in vivo. In IMAC assay, 12 proteins were identified as Fe2+/Cu2+/Zn2+ binding proteins, including mitochondrial voltage dependent anion channels (VDAC). UpVDAC showed binding abilities to all the three metal ions. His204Ala (H204A) and H219A mutated UpVDAC proteins lost their metal binding ability, and became insensitive to metal-catalyzed oxidation (MCO) induced carbonylation. The overexpression of wild-type UpVDAC made yeast cells more sensitive to oxidative stress, retarded the growth of Arabidopsis seedlings and accelerated the seed aging, while overexpression of mutated UpVDAC weakened these effects of VDAC. These results reveal the relationship between the metal binding ability and carbonylation modification, as well as the probable function of VDAC in regulating cell vitality, seedling growth and seed aging.

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.

PO-01-124 THE CALCIUM HANDLING MACHINERY IS REMODELED IN FRIEDREICH’S ATAXIA

Friedreich's ataxia (FA) is an inherited neurodegenerative disorder that causes progressive nervous system damage. FA is caused by a deficiency in the frataxin protein due to an expansion of GAA repeats in the first intron of the frataxin gene. Reduced frataxin protein expression is thought to negatively affect mitochondrial function, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, it is not completely understood why most patients present with left ventricular systolic dysfunction and die from cardiac events. The objective of our study is to determine whether the abnormal calcium handling machinery is a molecular mechanism that perpetuates cardiac dysfunction in FA. We used the frataxin knock-out (KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. The expression of calcium handling machinery proteins was assessed with proteomics and western blot. In left ventricular myocytes from KO and WT mice, the IonOptix system was used for calcium imaging, the seahorse assay was utilized to measure oxygen consumption rate (OCR) and confocal imaging was used to quantify the mitochondrial membrane potential (Δψm), and reactive oxygen species (ROS). ECG and echocardiography were used to assess cardiac function in the mice. In the proteomic analysis and western blot, SERCA2, Ryr2 and CaMKII were significantly downregulated, and the mitochondrial calcium handling proteins voltage-dependent anion channel and mitochondrial calcium uniporter were significantly upregulated in left ventricular tissue from humans with FA and from KO mice. On the ECG, the RR, PR, QRS, and QTc intervals were significantly longer in the KO mice. Echocardiography showed that the ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the KO mice. Moreover, the calcium transient decay was significantly prolonged in ventricular myocytes from KO mice compared to WT. Additionally, the Δψm was significantly depolarized, the OCR was significantly decreased, and ROS levels were significantly elevated in left ventricular myocytes from KO compared to WT. The development of left ventricular contractile dysfunction in FA is associated with reduced expression of calcium handling proteins, abnormal calcium cycling, and mitochondrial dysfunction.

Alteration of metabolite landscape in hypoxia replicates immune cell programs indicative of dysfunctional immunity in severe COVID-19

Abstract Our prior work has demonstrated that altered metabolic programs in T cells define dysregulated immunity in acute SARS-CoV-2 infection. Apoptotic T cells were defined by high expression of the mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1). These cells were markedly increased in elderly patients and correlated with lymphopenia. We devised in vitro systems in healthy T cells to investigate the mechanism driving this unique phenotype. While SARS-CoV-2 did not directly induce this T cell phenotype, factors that enhanced the development of VDAC1+ T cells, including hypoxia, advanced age of healthy T cell donor, and COVID-19 patient-derived plasma. Analysis of patient-derived plasma revealed robust differences in cytokine and metabolomic profiles between people who did or did not exhibit apoptotic T cells. Increased flux through the tryptophan:kynurenin pathway has been suggested as a biomarker of severe COVID-19 and was further enhanced in individuals with apoptotic T cells. Kynurenine directly induced VDAC1+ T cells in a dose dependent manner in vitro. Together, our work indicates metabolic alterations combined with cellular age and hypoxia may induce apoptosis of T cells following SARS-CoV-2 infection. This work highlights mechanisms leading to cellular alterations following infection and highlights key aspects of the host environment that could be targeted therapeutically. U54CA260492

Prevotella histicola suppresses ferroptosis to mitigate ethanol-induced gastric mucosal lesions in mice

BackgroundEthanol-induced gastric mucosal lesions (EGML) is one of the most common digestive disorders for which current therapies have limited outcomes in clinical practice. Prevotella histicola (P. histicola) has shown probiotic efficacy against arthritis, multiple sclerosis and oestrogen deficiency-induced depression in mice; however, its role in EGML remains unclear in spite of its extensive colonisation of the stomach. Ferroptosis, which is characterised by lipid peroxidation, may be involved in EGML. Herein, we aimed to investigate the effects and underlying mechanism of action of P. histicola on EGML in the ferroptosis-dependent pathway.MethodsP. histicola was intragastrically administered for a week, and deferoxamine (DFO), a ferroptosis inhibitor, was intraperitoneally injected prior to oral ethanol administration. The gastric mucosal lesions and ferroptosis were assessed via histopathological examinations, quantitative real-time PCR, Western blot, immunohistochemistry and immunofluorescence.ResultsP. histicola was originally found to attenuate EGML by reducing histopathological changes and lipid reactive oxygen species (ROS) accumulation. The pro-ferroptotic genes of Transferrin Receptor (TFR1), Solute Carrier Family 39 Member 14 (SLC39A14), Haem Oxygenase-1 (HMOX-1), Acyl-CoA Synthetase Long-chain Family Member 4 (ACSL4), Cyclooxygenase 2 (COX-2) and mitochondrial Voltage-dependent Anion Channels (VDACs) were up-regulated; the anti-ferroptotic System Xc-/Glutathione Peroxidase 4 (GPX4) axis was inhibited after ethanol administration. However, the changes of histopathology and ferroptosis-related parameters induced by ethanol were reversed by DFO. Furthermore, P. histicola treatment significantly downregulated the expression of ACSL4, HMOX-1 and COX-2, as well as TFR1 and SLC39A14, on mRNA or the protein level, while activating the System Xc-/GPX4 axis.ConclusionsWe found that P. histicola reduces ferroptosis to attenuate EGML by inhibiting the ACSL4- and VDAC-dependent pro-ferroptotic pathways and activating the anti-ferroptotic System Xc-/GPX4 axis.

Ablation of GPR56 Causes β-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 β-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 β-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 βH1 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 β-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 β-cell function.

Pro-Apoptotic and Anti-Cancer Activity of the Vernonanthura Nudiflora Hydroethanolic Extract

The mitochondrial voltage-dependent anion channel 1 (VDAC1) protein is involved in several essential cancer hallmarks, including energy and metabolism reprogramming and apoptotic cell death evasion. In this study, we demonstrated the ability of hydroethanolic extracts from three different plants, Vernonanthura nudiflora (Vern), Baccharis trimera (Bac), and Plantago major (Pla), to induce cell death. We focused on the most active Vern extract. We demonstrated that it activates multiple pathways that lead to impaired cell energy and metabolism homeostasis, elevated ROS production, increased intracellular Ca2+, and mitochondria-mediated apoptosis. The massive cell death generated by this plant extract's active compounds involves the induction of VDAC1 overexpression and oligomerization and, thereby, apoptosis. Gas chromatography of the hydroethanolic plant extract identified dozens of compounds, including phytol and ethyl linoleate, with the former producing similar effects as the Vern hydroethanolic extract but at 10-fold higher concentrations than those found in the extract. In a xenograft glioblastoma mouse model, both the Vern extract and phytol strongly inhibited tumor growth and cell proliferation and induced massive tumor cell death, including of cancer stem cells, inhibiting angiogenesis and modulating the tumor microenvironment. Taken together, the multiple effects of Vern extract make it a promising potential cancer therapeutic.

Defining the roles and regulation of the mitochondrial VDAC isoforms one molecule at a time.

The voltage-dependent anion channel (VDAC) is recognized as the key metabolite conduit in the mitochondrial outer membrane and regulator for fluxes of water-soluble metabolites, ions, and polypeptides. The uniqueness of VDAC arises from its position at the interface between the mitochondria and the cytosol making VDAC an important cellular communication hub. VDAC has three isoforms in mammals: VDAC1, 2, and 3 which share high sequence similarity, indicative of a similar pore-forming structure. All three VDAC isoforms form similar highly conductive, anion-selective, voltage-gated channels when reconstituted into planar lipid membranes implying that they can concurrently facilitate the transport of the negatively charged respiratory metabolites. However, cellular model systems and isoform-specific knock-out mice demonstrate distinctive functional roles for each VDAC isoform. We explored the biophysical basis for these differences by reconstituting human, mouse, and zebrafish VDAC isoforms in planar lipid bilayers and performing single channel electrophysiology. We have uncovered that interactions with cytosolic regulators, such as the Parkinson's disease associated alpha-synuclein, vary in their on-rates by two-orders of magnitude between each isoform. Furthermore, each isoform varies in their ability translocate synuclein across the lipid bilayer. Exploring the calcium transport properties of each isoform, we show that VDAC3 has the highest calcium permeability. We demonstrate that VDAC2 can dynamically switch between channel properties of VDAC1 and VDAC3, and explored the structural basis for its unique plasticity using mutational and structural analysis and molecular dynamics simulations. We find that both VDAC2's N-terminal extension and cysteine residues contribute to, but are not sufficient to explain VDAC2's striking behavior. Our results suggest that tuning of beta-barrel and loop flexibility between the isoforms could define their biophysical properties and help to explain distinct physiological roles.

Inhibition of mitochondrial VDAC1 oligomerization alleviates apoptosis and necroptosis of retinal neurons following OGD/R injury

Ischemia-reperfusion (I/R) injury is a common pathological mechanism in many retinal diseases, which can lead to cell death via mitochondrial dysfunction. Voltage-dependent anion channel 1 (VDAC1), which is mainly located in the outer mitochondrial membrane, is the gatekeeper of mitochondria. The permeability of mitochondrial membrane can be regulated by controlling the oligomerization of VDAC1. However, the functional mechanism of VDAC1 in retinal I/R injury was unclear. Our results demonstrate that oxygen-glucose deprivation and re-oxygenation (OGD/R) injury leads to apoptosis, necroptosis, and mitochondrial dysfunction of R28 cells. The OGD/R injury increases the levels of VDAC1 oligomerization. Inhibition of VDAC1 oligomerization by VBIT-12 rescued mitochondrial dysfunction by OGD/R and also reduced apoptosis/necroptosis of R28 cells. In vivo, the use of VBIT-12 significantly reduced aHIOP-induced neuronal death (apoptosis/necroptosis) in the rat retina. Our findings indicate that VDAC1 oligomers may open and enlarge mitochondrial membrane pores during OGD/R injury, leading to the release of death-related factors in mitochondria, resulting in apoptosis and necroptosis. This study provides a potential therapeutic strategy against ocular diseases caused by I/R injury.

Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules

The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands.