Modulation of Cellular Levels of Adenosine Phosphates and Creatine Phosphate in Cultured Primary Astrocytes.

Adenosine triphosphate (ATP) is the main energy currency of all cells, while creatine phosphate (CrP) is considered as a buffer of high energy-bond phosphate that facilitates rapid regeneration of ATP from adenosine diphosphate (ADP). Astrocyte-rich primary cultures contain ATP, ADP and adenosine monophosphate (AMP) in average specific contents of 36.0 ± 6.4 nmol/mg, 2.9 ± 2.1 nmol/mg and 1.7 ± 2.1 nmol/mg, respectively, which establish an adenylate energy charge of 0.92 ± 0.04. The average specific cellular CrP level was found to be 25.9 ± 10.8 nmol/mg and the CrP/ATP ratio was 0.74 ± 0.28. The specific cellular CrP content, but not the ATP content, declined with the age of the culture. Absence of fetal calf serum for 24 h caused a partial loss in the cellular contents of both CrP and ATP, while application of creatine for 24 h doubled the cellular CrP content and the CrP/ATP ratio, but did not affect ATP levels. In glucose-deprived astrocytes, the high cellular ATP and CrP contents were rapidly depleted within minutes after application of the glycolysis inhibitor 2-deoxyglucose and the respiratory chain inhibitor antimycin A. For those conditions, the decline in CrP levels always preceded that of ATP contents. In contrast, incubation of glucose-fed astrocytes for up to 30 min with antimycin A had little effect on the high cellular ATP content, while the CrP level was significantly lowered. These data demonstrate the importance of cellular CrP for maintaining a high cellular ATP content in astrocytes during episodes of impaired ATP regeneration.

[Inactivation of Ras1 in Fission Yeast Aggravates the Oxidative Stress Response Induced by Tert Butyl Hydroperoxide (tBHP)].

Ras proteins are small GTPases and function as molecular switches to regulate cellular homeostasis. Ras-dependent signalling pathways regulate several essential processes such as cell cycle progression, growth, migration, apoptosis, and senescence. The dysregulation of Ras signaling pathway has been linked to several pathological outcomes. A potential role of RAS in regulating the redox signalling pathway has been established that includes the manipulation of ROS levels to provide a redox milieu that might be conducive to carcinogenesis. Reactive oxygen species (ROS) and mitochondrial impairment have been proposed as major factors affecting the physiology of cells and implicated in several pathologies. The present study was conducted to evaluate the role of Ras1, tert Butyl hydroperoxide (tBHP), and antimycin A in oxidative stress response in Schizosaccharomyces pombe cells. We observed decreased cell survival, higher levels of ROS, and mitochondrial dysfunctionality in ras1Δ cells and tBHP as well as respiratory inhibitor, antimycin A treated wild type cells. Furthermore, these defects were more profound in ras1Δ cells treated with tBHP or antimycin A. Additionally, Ras1 also has been shown to regulate the expression and activity of several antioxidant enzymes like glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), and catalase. Together, these results suggest the potential role of S. pombe Ras1 in mitigating oxidative stress response.

Identification of antimycin A as a c-Myc degradation accelerator via high-throughput screening.

c-Myc is a critical regulator of cell proliferation and growth. Elevated levels of c-Myc cause transcriptional amplification, leading to various types of cancers. Small molecules that specifically inhibit c-Myc-dependent regulation are potentially invaluable for anticancer therapy. Because c-Myc does not have enzymatic activity or targetable pockets, researchers have attempted to obtain small molecules that inhibit c-Myc cofactors, activate c-Myc repressors, or target epigenetic modifications to regulate the chromatin of c-Myc-addicted cancer without any clinical success. In this study, we screened for c-Myc inhibitors using a cell-dependent assay system in which the expression of c-Myc and its transcriptional activity can be inferred from monomeric Keima and enhanced GFP fluorescence, respectively. We identified one mitochondrial inhibitor, antimycin A, as a hit compound. The compound enhanced the c-Myc phosphorylation of threonine-58, consequently increasing the proteasome-mediated c-Myc degradation. The mechanistic analysis of antimycin A revealed that it enhanced the degradation of c-Myc protein through the activation of glycogen synthetic kinase 3 by reactive oxygen species (ROS) from damaged mitochondria. Furthermore, we found that the inhibition of cell growth by antimycin A was caused by both ROS-dependent and ROS-independent pathways. Interestingly, ROS-dependent growth inhibition occurred only in the presence of c-Myc, which may reflect the representative features of cancer cells. Consistently, the antimycin A sensitivity of cells was correlated to the endogenous c-Myc levels in various cancer cells. Overall, our study provides an effective strategy for identifying c-Myc inhibitors and proposes a novel concept for utilizing ROS inducers for cancer therapy.

Identification of In Vitro Inhibitors of Monkeypox Replication.

Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity in vitro against two orthopoxviruses. IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.

Antimycin A induced apoptosis in HCT-116 colorectal cancer cells through the up- and downregulation of multiple signaling pathways.

Colorectal cancer is the third most life-threatening cancer in the western countries. For the treatment, several chemotherapeutic drugs are using those that have severe side effects on the patient. So, finding alternative drugs is important. In the present research antimycin A was selected to evaluate the anticancer properties on the HCT-116 colorectal cancer cells. Antimycin A inhibited HCT-116 cells proliferation with the IC50 value of 29 µg/mL concentration. As a long-term effect, HCT-116 cells were incubated with 10-40 µg/mL concentration of antimycin A for 7 days. No colony was observed in the treated wells. Apoptotic features in HCT-116 cells were observed in antimycin A treated cells after being stained with Hoechst 33342 dye. Apoptosis was further confirmed by FITC-annexin V/PI. Role of caspase-3 protein in the apoptosis process was also confirmed by the caspase-3 inhibitor. After treatment of HCT-116 cells with antimycin A, apoptotic related gene expression was checked by reverse transcription polymerase chain reaction. p53 and caspase-9 genes were upregulated consequently mitogen-activated protein kinases (MAPK), poly(ADP-Ribose) polymerase (PARP), and nuclear factor kappa B (NF-κB) genes were downregulated. Molecular docking simulation indicated significant binding affinity of antimycin A with the five proteins. The results indicated antimycin A would be a promising anticancer agent for further anticancer research.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

How to Use Respiratory Chain Inhibitors in Toxicology Studies-Whole-Cell Measurements.

Mitochondrial electron transport chain (ETC) inhibition is a phenomenon interesting in itself and serves as a tool for studying various cellular processes. Despite the fact that searching the term "rotenone" in PubMed returns more than 6900 results, there are many discrepancies regarding the directions of changes reported to be caused by this RTC inhibitor in the delicate redox balance of the cell. Here, we performed a multifaceted study of the popular ETC inhibitors rotenone and antimycin A, involving assessment of mitochondrial membrane potential and the production of hydrogen peroxide and superoxide anions at cellular and mitochondrial levels over a wide range of inhibitor concentrations (1 nmol/dm3-100 µmol/dm3). All measurements were performed with whole cells, with accompanying control of ATP levels. Antimycin A was more potent in hindering HepG2 cells' abilities to produce ATP, decreasing ATP levels even at a 1 nmol/dm3 concentration, while in the case of rotenone, a 10,000-times greater concentration was needed to produce a statistically significant decrease. The amount of hydrogen peroxide produced in the course of antimycin A biological activity increased rapidly at low concentrations and decreased below control level at a high concentration of 100 µmol/dm3. While both inhibitors influenced cellular superoxide anion production in a comparable manner, rotenone caused a greater increase in mitochondrial superoxide anions compared to a modest impact for antimycin A. IC50 values for rotenone and antimycin A with respect to HepG2 cell survival were of the same order of magnitude, but the survival curve of cells treated with rotenone was clearly biphasic, suggesting a concentration-dependent mode of biological action. We propose a clear experimental setup allowing for complete and credible analysis of the redox state of cells under stress conditions which allows for better understanding of the effects of ETC inhibition.

Reprint of: Ubisemiquinone Is the Electron Donor for Superoxide Formation by Complex III of Heart Mitochondria.

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.

An ancient function of PGR5 in iron delivery?

In all phototrophic organisms, the photosynthetic apparatus must be protected from light-induced damage. One important mechanism that mitigates photodamage in plants is antimycin A (AA)-sensitive cyclic electron flow (CEF), the evolution of which remains largely obscure. Here we show that proton gradient regulation 5 (PGR5), a key protein involved in AA-sensitive CEF, displays intriguing commonalities - including sequence and structural features - with a group of ferritin-like proteins. We therefore propose that PGR5 may originally have been involved in prokaryotic iron mobilization and delivery, which facilitated a primordial type of CEF as a side effect. The abandonment of the bacterioferritin system during the transformation of cyanobacterial endosymbionts into chloroplasts might have allowed PGR5 to functionally specialize in CEF.

The protective effect of N-acetylcysteine on antimycin A-induced respiratory chain deficiency in mesenchymal stem cells.

Transplantation of mesenchymal stem cells (MSCs) is an effective treatment in tissue injuries though it is limited due to the early death of stem cells within the first few days. The main reason could be a deficiency in the respiratory chain of injured tissues which is linked to the oxidative stress (OS) and disruption of energy metabolism. The disruption in energy metabolism and OS both inhibit the homing of stem cells in the hypoxic micro-environment, however on other hand, the key functions of stem cells are mainly regulated by their cellular redox status and energy metabolism. Because of that, strategies are being developed to improve the bio-functional properties of MSCs, including preconditioning of the stem cells in hypoxic conditions and pretreatment of antioxidants. To achieve this purpose, in this study N-acetylcysteine (NAC) was used for the protection of cells from oxidative stress and the disruption in energy metabolism was induced by Antimycin A (AMA) via blocking the cytochrome C complex. Then several parameters were analyzed, including cell viability/apoptosis, mitochondrial membrane potential, and redox molecular homeostasis. Based on our findings, upon the exposure of the MSCs to the conditions of deficient respiratory chain, the cells failed to scavenge the free radicals, and energy metabolism was disrupted. The use of NAC was found to alleviate the DNA damage, cell apoptosis, and oxidative stress via Nrf2/Sirt3 pathway though without any effect on the mitochondrial membrane potential. It means that antioxidants protect the cells from OS but the problem of ATP metabolism yet remains unresolved in the hypoxic conditions.

Interaction of picolinamide fungicide primary metabolites UK-2A and CAS-649 with the cytochrome bc1 complex Qi site: mutation effects and modelling in Saccharomyces cerevisiae.

BACKGROUND: Fenpicoxamid and florylpicoxamid are picolinamide fungicides targeting the Qi site of the cytochrome bc1 complex, via their primary metabolites UK-2A and CAS-649, respectively. We explore binding interactions and resistance mechanisms for picolinamides, antimycin A and ilicicolin H in yeast by testing effects of cytochrome b amino acid changes on fungicide sensitivity and interpreting results using molecular docking. RESULTS: Effects of amino acid changes on sensitivity to UK-2A and CAS-649 were similar, with highest resistance associated with exchanges involving G37 and substitutions N31K and L198F. These changes, as well as K228M, also affected antimycin A, while ilicicolin H was affected by changes at G37 and L198, as well as Q22E. N31 substitution patterns suggest that a lysine at position 31 introduces an electrostatic interaction with neighbouring D229, causing disruption of a key salt-bridge interaction with picolinamides. Changes involving G37 and L198 imply resistance primarily through steric interference. G37 changes also showed differences between CAS-649 and UK-2A or antimycin A with respect to branched versus unbranched amino acids. N31K and substitution of G37 by large amino acids reduced growth rate substantially while L198 substitutions showed little effect on growth. CONCLUSION: Binding of UK-2A and CAS-649 at the Qi site involves similar interactions such that general cross-resistance between fenpicoxamid and florylpicoxamid is anticipated in target pathogens. Some resistance mutations reduced growth rate and could carry a fitness penalty in pathogens. However, certain changes involving G37 and L198 carry little or no growth penalty and may pose the greatest risk for resistance development in the field. © 2022 Society of Chemical Industry.

Contributing role of mitochondrial energy metabolism on platelet adhesion, activation and thrombus formation under blood flow conditions.

Platelets have an active energy metabolism mediated by mitochondria. However, the role of mitochondria in platelet adhesion, activation, and thrombus formation under blood flow conditions remains to be elucidated. Blood specimens were obtained from healthy adult volunteers. The consumption of glucose molecules by platelets was measured after 24 hours. Platelet adhesion, activation, and thrombus formation on collagen fibrils and immobilized von Willebrand factor (VWF) at a wall shear rate of 1,500 s-1 were detected by fluorescence microscopy with an ultrafast laser confocal unit in the presence or absence of mitochondrial functional inhibitors of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, and oligomycin. Consumption of glucose molecules within the first 24 h of 4.21 × 10-15 ± 4.46 x 10-15 (n = 6) increased to 13.82 × 10-15 ± 3.46 x 10-15 (n = 4) in the presence of FCCP, 12.11 × 10-15 ± 2.33 x 10-15 (n = 4) in the presence of antimycin A, and 11.87 × 10-15 ± 3.56 x 10-15 (n = 4) in the presence of oligomycin (p < .05). These mitochondrial functional blockers did not influence both surface area coverage by platelets and the 3-dimensional size of platelet thrombi formed on the collagen fibrils. However, a rapid increase in the intracellular calcium ion concentration ([Ca2+]i) upon adhering on immobilized VWF decreased significantly from 405.5 ± 86.2 nM in control to 198.0 ± 79.2 nM in the presence of FCCP (p < .005). A similar decrease in the rapid increase in ([Ca2+]i) was observed in the presence of antimycin A and oligomycin. Mitochondrial function is necessary for platelet activation represented by a rapid increase in [Ca2+]i after platelet adhesion on VWF. However, the influence could not be detected as changes in platelet adhesion or 3-dimensional growth of platelet thrombi on collagen fibrils.

Electron transfer via cytochrome b6f complex displays sensitivity to antimycin A upon STT7 kinase activation.

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.

Zhaoshumycins A and B, Two Unprecedented Antimycin-Type Depsipeptides Produced by the Marine-Derived Streptomyces sp. ITBB-ZKa6.

Marine actinomycetes are prolific chemical sources of complex and novel natural products, providing an excellent chance for new drug discovery. The chemical investigation of the marine-derived Streptomyces sp. ITBB-ZKa6, from Zhaoshu island, Hainan, led to the discovery of two unique antimycin-type depsipeptides, zhaoshumycins A (1) and B (2), along with the isolation of the four known neoantimycins A (3), F (4), D (5), and E (6). The structures of the new compounds 1 and 2 were elucidated on the basis of the analysis of diverse spectroscopic data and biogenetic consideration. Zhaoshumycins A (1) and B (2) represent a new class of depsipeptides, featuring two neoantimycin monomers (only neoantimycin D or neoantimycins D and E) linked to a 1,4-disubstituted benzene ring via an imino group. Initial toxicity tests of 1-6 in MCF7 human breast cancer cells revealed that compounds 5 and 6 possess weak cytotoxic activity. Further structure-activity relationship analysis suggested the importance of the NH2 group at C-34 in 5 and 6 for cytotoxicity in MCF7 cells.

Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model.

Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter's transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate's attenuation of oxaloacetate's inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells.

Identifying the Effects of Reactive Oxygen Species on Mitochondrial Dynamics and Cytoskeleton Stability in Dictyostelium discoideum.

Defects in mitochondrial dynamics, fission, fusion, and motility have been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and Charcot-Marie-Tooth disease. Another key feature of neurodegeneration is the increase in reactive oxygen species (ROS). Previous work has shown that the cytoskeleton, in particular the microtubules, and ROS generated by rotenone significantly regulate mitochondrial dynamics in Dictyostelium discoideum. The goal of this project is to study the effects of ROS on mitochondrial dynamics within our model organism D. discoideum to further understand the underlying issues that are the root of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. We chose three likely ROS inducers, cumene hydroperoxide, hydroxylamine hydrochloride, and Antimycin A. Our work demonstrates that alteration of the microtubule cytoskeleton is not required to alter dynamics in response to ROS and there is no easy way to predict how mitochondrial dynamics will be altered based on which ROS generator is used. This research contributes to the better understanding of the cellular mechanisms that induce the pathogenesis of incurable neurodegenerative diseases with the hope that it will translate into developing new and more effective treatments for patients afflicted by them.

Antimycin A-induced mitochondrial dysfunction regulates inflammasome signaling in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a severe retinal eye disease where dysfunctional mitochondria and damaged mitochondrial DNA in retinal pigment epithelium (RPE) have been demonstrated to underlie the pathogenesis of this devastating disease. In the present study, we aimed to examine whether damaged mitochondria induce inflammasome activation in human RPE cells. Therefore, ARPE-19 cells were primed with IL-1α and exposed to the mitochondrial electron transport chain complex III inhibitor, antimycin A. We found that antimycin A-induced mitochondrial dysfunction caused caspase-1-dependent inflammasome activation and subsequent production of mature IL-1ß and IL-18 in human RPE cells. AIM2 and NLRP3 appeared to be the responsible inflammasome receptors upon antimycin A-induced mitochondrial damage. We aimed at verifying our findings using hESC-RPE cells but antimycin A was absorbed by melanin. Therefore, results were repeated on D407 RPE cell cultures. Antimycin A-induced mitochondrial and NADPH oxidase-dependent ROS production occurred upstream of inflammasome activation, whereas K+ efflux was not required for inflammasome activation in antimycin A-treated human RPE cells. Collectively, our data emphasize that dysfunctional mitochondria regulate the assembly of inflammasome multiprotein complexes in the human RPE cells. The present study associates AIM2 with the pathogenesis of AMD.

PGRL2 triggers degradation of PGR5 in the absence of PGRL1.

In plants, inactivation of either of the thylakoid proteins PGR5 and PGRL1 impairs cyclic electron flow (CEF) around photosystem I. Because PGR5 is unstable in the absence of the redox-active PGRL1, but not vice versa, PGRL1 is thought to be essential for CEF. However, we show here that inactivation of PGRL2, a distant homolog of PGRL1, relieves the need for PGRL1 itself. Conversely, high levels of PGRL2 destabilize PGR5 even when PGRL1 is present. In the absence of both PGRL1 and PGRL2, PGR5 alters thylakoid electron flow and impairs plant growth. Consequently, PGR5 can operate in CEF on its own, and is the target of the CEF inhibitor antimycin A, but its activity must be modulated by PGRL1. We conclude that PGRL1 channels PGR5 activity, and that PGRL2 triggers the degradation of PGR5 when the latter cannot productively interact with PGRL1.

Bioenergetic Profiling of Human Pluripotent Stem Cells.

Cellular metabolism contributes to cell fate decisions. Bioenergetic profiling can therefore provide considerable insights into cellular identity and specification. Given the current importance of human pluripotent stem cells (hPSCs) for biomedical applications, assessing the bioenergetic properties of hPSCs and derivatives can unveil relevant mechanisms in the context of development biology and molecular disease modeling. Here, we describe a method to facilitate bioenergetic profiling of hPSCs in a reproducible and scalable manner. After simultaneous assessment of mitochondrial respiration and glycolytic capacity using Seahorse XFe96 Analyzer, we measure lactate concentration in the cellular media. Finally, we normalize the values based on DNA amount. We describe the procedures with specific requirements related to hPSCs . However, the same protocol can be easily adapted to other cell types, including differentiated progenies from hPSCs .

Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells.

Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.