bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒05‒08
33 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Biomed Opt Express. 2022 Apr 01. 13(4): 2103-2116
      Mitochondrial redox is an important indicator of cell metabolism and health, with implications in cancer, diabetes, aging, neurodegenerative diseases, and mitochondrial disease. The most common method to observe redox of individual cells and mitochondria is through fluorescence of NADH and FAD+, endogenous cofactors serve as electron transport inputs to the mitochondrial respiratory chain. Yet this leaves out redox within the respiratory chain itself. To a degree, the missing information can be filled in by exogenous fluorophores, but at the risk of disturbed mitochondrial permeability and respiration. Here we show that variations in respiratory chain redox can be detected up by visible-wavelength transient absorption microscopy (TAM). In TAM, the selection of pump and probe wavelengths can provide multiphoton imaging contrast between non-fluorescent molecules. Here, we applied TAM with a pump at 520nm and probe at 450nm, 490nm, and 620nm to elicit redox contrast from mitochondrial respiratory chain hemeproteins. Experiments were performed with reduced and oxidized preparations of isolated mitochondria and whole muscle fibers, using mitochondrial fuels (malate, pyruvate, and succinate) to set up physiologically relevant oxidation levels. TAM images of muscle fibers were analyzed with multivariate curve resolution (MCR), revealing that the response at 620nm probe provides the best redox contrast and the most consistent response between whole cells and isolated mitochondria.
  2. Cell Biol Toxicol. 2022 May 04.
      Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals.
    Keywords:  ATP; Machine learning; Membrane potential; Mitochondria; Morphology
  3. Blood Adv. 2022 May 02. pii: bloodadvances.2021005776. [Epub ahead of print]
      IFNγ is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher than normal OCR. Whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh Syndrome patient monocytes) or NADPH oxidase (DPI treatment or chronic granulomatous disease (CGD) patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes.
  4. Cell Metab. 2022 May 03. pii: S1550-4131(22)00133-4. [Epub ahead of print]34(5): 775-782.e9
      The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic donor of folate-mediated 1C units in cancer cells has been thoroughly investigated, a potential role of glycine oxidation remains unclear. We developed an approach for quantifying mitochondrial glycine cleavage system (GCS) flux by combining stable and radioactive isotope tracing with computational flux decomposition. We find high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis. Surprisingly, other than supplying 1C units, we found that GCS is important for maintaining protein lipoylation and mitochondrial activity. Genetic silencing of glycine decarboxylase inhibits the lipoylation and activity of pyruvate dehydrogenase and impairs tumor growth, suggesting a novel drug target for HCC. Considering the physiological role of liver glycine cleavage, our results support the notion that tissue of origin plays an important role in tumor-specific metabolic rewiring.
    Keywords:  GCS; GLDC; PDH; glycine cleavage system; glycine decarboxylase; hepatocellular carcinoma; one-carbon metabolism; protein P; protein lipoylation; pyruvate dehydrogenase
  5. Dev Cell. 2022 Apr 22. pii: S1534-5807(22)00254-4. [Epub ahead of print]
      Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically.
    Keywords:  CRISPR; ISL2; pancreatic cancer; tumor suppressor
  6. Mitochondrion. 2022 Apr 30. pii: S1567-7249(22)00038-1. [Epub ahead of print]
      Mitochondrial dysfunction plays a vital role in growth and malignancy of tumors. In recent scenarios, mitochondrial transplantation therapy is considered as an effective method to remodel mitochondrial function in mitochondria-related diseases. However, the mechanism by which mitochondrial transplantation blocks tumor cell proliferation is still not determined. In addition, mitochondria are maternal inheritance in evolution, and mitochondria obtained from genders exhibit differences in mitochondrial activity. Therefore, the study indicates the inhibitory effect of mitochondria from different genders on hepatocellular carcinoma and explores the molecular mechanism. The results reveal that the healthy mitochondria can retard the proliferation of the hepatocellular carcinoma cells in vitro and in vivo through arresting cell cycle and inducing apoptosis. The molecular mechanism suggests that mitochondrial transplantation therapy can decrease aerobic glycolysis, and down-regulate the expression of cycle-related proteins while up-regulate apoptosis-related proteins in tumor cells. In addition, the antitumor activity of mitochondria from female mice (F-Mito) is relatively higher than that of mitochondria from male mice (M-Mito), which would be related to the evidence that the F-Mito process higher activity than the M-Mito. This study clarifies the mechanism of exogenous mitochondria inhibiting the proliferation of hepatocellular carcinoma and contributes a new biotechnology for therapy of mitochondria-related diseases from different genders.
    Keywords:  apoptosis; cell cycle arrest; hepatocellular carcinoma; mitochondrial therapy
  7. Nat Commun. 2022 May 05. 13(1): 2483
      The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states - glucose, galactose, OXPHOS inhibition, and absence of pyruvate - designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments.
  8. Am J Physiol Renal Physiol. 2022 May 02.
      Caloric restriction (CR) prevents obesity and increases resilience against pathological stimuli in laboratory rodents. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and capacities, avoiding Ca2+-induced mitochondrial permeability transition. Dietary restriction has also been shown to increase kidney resistance against damaging stimuli, but if these effects are related to similar mitochondrial adaptations has not been uncovered. Here, we characterized changes in mitochondrial function in response to six months CR in rats, measuring bioenergetic parameters, redox balance and calcium homeostasis. CR promoted an increase in succinate-supported mitochondrial oxygen consumption rates. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2O2 release was enhanced in a succinate-dependent manner. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+-induced mitochondrial permeability transition, in a manner reversed by antioxidant dithiothreitol. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials or amounts of the mitochondrial calcium uniporter (MCU). Instead, increased mitochondrial calcium uptake rates in CR kidneys correlate with a loss of MICU2, an MCU modulator. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet-sensitive regulatory role controlling mitochondrial calcium homeostasis. Together, our results highlight the organ-specific bioenergetic, redox, and ionic transport effects of CR, with some unexpected deleterious effects in kidney.
    Keywords:  Calorie restriction; Mitochondria; Reactive oxygen species; calcium; kidney
  9. Mitochondrion. 2022 Apr 30. pii: S1567-7249(22)00040-X. [Epub ahead of print]
      Mitochondria, known as the powerhouse of the cell, are at the center of healthy physiology and provide cells with energy in the form of ATP. These unique organelles are also implicated in many pathological conditions affecting a variety of organs in various systems. Recently, mitochondrial transplantation, inspired by mitochondria's endosymbiotic origin, has been attempted as a potential biotherapy in mitigating a variety of pathological conditions. Mitochondrial transplantation consists of the process of isolation, transfer, and uptake of exogenous, intact mitochondria into damaged cells. Here, we discuss mitochondrial transplantation in the context of clinical medicine practiced in neurology, cardiology, pulmonary medicine, and oncology, among others. We outline the role of mitochondria in various pathologies and discuss the state-of-the-art research that potentially form the basis of new therapeutics for the treatment of a variety of diseases due to mitochondrial dysfunction. Lastly, we explore some of the challenges associated with mitochondrial transplantation that must be addressed before mitochondrial transplantation becomes a viable therapeutic option in clinical settings.
    Keywords:  Mitochondrial transplantation; clinical medicine; mitochondrial transfer; mitochondrial transplantation in medicine
  10. EMBO Mol Med. 2022 May 06. e15203
      The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins, and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anticancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein, we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) and has potent and selective activity against multiple AML subtypes. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.
    Keywords:  DHODH; acute myeloid leukemia; leukemic stem cells; protein translation
  11. RSC Adv. 2020 Nov 27. 10(71): 43383-43388
      Controlled intracellular chemical reactions to regulate cellular functions remain a challenge in biology mimetic systems. Herein, we developed an intra-mitochondrial bio-orthogonal reaction to induce aggregation induced emission. In situ carbonyl ligation inside mitochondria drives the molecules to form nano-aggregates with green fluorescence, which leads to depolarization of the mitochondrial membrane, generation of ROS, and subsequently mitochondrial dysfunction. This intra-mitochondrial carbonyl ligation shows great potential for anticancer treatment in various cancer cell lines.
  12. Cancer Res. 2022 May 02. pii: canres.3868.2021. [Epub ahead of print]
      Branched-chain amino acid transaminase 1 (BCAT1) is upregulated selectively in human isocitrate dehydrogenase (IDH) wildtype (WT) but not mutant glioblastoma multiforme (GBM) and promotes IDHWT GBM growth. Through a metabolic synthetic lethal screen, we report here that α-ketoglutarate (AKG) kills IDHWT GBM cells when BCAT1 protein is lost, which is reversed by re-expression of BCAT1 or supplementation with branched-chain α-ketoacids (BCKAs), downstream metabolic products of BCAT1. In patient-derived IDHWT GBM tumors in vitro and in vivo, co-treatment of BCAT1 inhibitor gabapentin and AKG resulted in synthetic lethality. However, AKG failed to evoke a synthetic lethal effect with loss of BCAT2, BCKDHA, or GPT2 in IDHWT GBM cells. Mechanistically, loss of BCAT1 increased the NAD+/NADH ratio but impaired oxidative phosphorylation, mTORC1 activity, and nucleotide biosynthesis. These metabolic alterations were synergistically augmented by AKG treatment, thereby causing mitochondrial dysfunction and depletion of cellular building blocks, including ATP, nucleotides, and proteins. Partial restoration of ATP, nucleotides, proteins, and mTORC1 activity by BCKA supplementation prevented IDHWT GBM cell death conferred by the combination of BCAT1 loss and AKG. These findings define a targetable metabolic vulnerability in the most common subset of GBM that is currently incurable.
  13. Nat Commun. 2022 May 03. 13(1): 2403
      C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.
  14. J Biol Chem. 2022 May 02. pii: S0021-9258(22)00448-3. [Epub ahead of print] 102008
      Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and non-alcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced non-alcoholic steatohepatitis and in human livers from patients diagnosed with NAFLD. We used CRISPR-Cas9 to delete ATAD3A in Huh7 human hepatocellular carcinoma cells, and used RNAi to silence ATAD3A expression in human hepatocytes isolated from humanized liver-chimeric mice to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor of acetyl-CoA acetyltransferase. Our results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells, and also that triglyceride (TG) levels were significantly increased in ATAD3A KO Huh7 cells following inhibition of lipolysis mediated by upregulation of lipid droplet-binding protein perilipin-2. Moreover, loss of ATAD3A upregulated autophagosome-associated light chain 3-II protein and p62 in Huh7 cells and fresh human hepatocytes through blockage of autophagosome degradation. Finally, we show the mitophagy mediator, PTEN-induced kinase 1, was downregulated in ATAD3A KO Huh7 cells, suggesting that ATAD3A KO inhibits mitophagy. These results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. Taken together, we conclude that loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, TG, and damaged mitochondria in hepatocytes.
    Keywords:  ATAD3A; NAFLD; autophagy; cholesterol; fatty acid oxidation; free fatty acid; mitochondrial respiration; mitophagy; triglyceride
  15. Biochem Biophys Res Commun. 2022 Apr 15. pii: S0006-291X(22)00582-4. [Epub ahead of print]612 30-36
      Human embryonic stem cells (hESCs) can self-renew infinitely and differentiate into the cell types of all lineages of our body, holding great promise for investigating early human embryo development and providing functional cells for disease treatment. For the full application of hESCs, it is necessary to elucidate how hESCs maintain their identity. Recent studies have shown that glycolysis and mitochondrial respiration are linked to pluripotency states. However, the function of mitochondrial respiration in hESCs has not been fully understood. Herein, we report that the adenosine triphosphate (ATP) production rate is comparable between mitochondrial respiration and glycolysis, suggesting an important contribution of mitochondrial respiration to ATP production in conventionally cultured hESCs. To investigate the function of mitochondrial respiration, we silence OGDH expression in hESCs by the inducible CRISPRi method, and find that OGDH knockdown (KD) results in disrupted TCA (tricarboxylic acid) cycle, and diminished mitochondrial respiration activity and total ATP level. Moreover, OGDH KD leads to hESC death and aberrant transcriptional program. Interestingly, blockage of the electron transport chain (ETC) by small molecule inhibitors gives rise to the phenotype similar to that observed in OGDH deficient hESCs. Therefore, genetic and pharmacological perturbations of the mitochondrial respiration impair identity of hESCs. Collectively, our study highlights the pivotal role of the mitochondrial respiration activity for the stemness maintenance of primed hESCs, and unveils OGDH as a key regulator for the proper production of ATP and TCA cycle metabolites in primed hESCs.
    Keywords:  ATP production; Human embryonic stem cells; Mitochondrial respiration; OGDH; Primed pluripotency
  16. Cell. 2022 Apr 28. pii: S0092-8674(22)00462-7. [Epub ahead of print]
      Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.
    Keywords:  fitness; genetically engineered mouse model; lineage tracing; lung cancer; phylogenetics; plasticity; single cell; transcriptome heterogeneity; tumor evolution
  17. RSC Adv. 2020 Jul 15. 10(45): 26874-26879
      A lower pH level and high hydrogen peroxide (H2O2) concentration in mitochondria is closely associated with a variety of diseases including cancer and inflammation. Thus, determination of changes in the level of acidic pH and H2O2 is of great importance and could provide new insights into the key functions under both physiological and pathological conditions. Herein, we present a novel mitochondria-targetable probe NIR-pH-H2O2, as the first near infrared (NIR) fluorescent small molecule, to monitor changes of endogenous pH (pk a = 6.17) and H2O2 with high sensitivity, good compatibility and low cytotoxicity. Futhermore, it was successfully employed to monitor pH and H2O2 in a mouse acute inflammation model. These results demonstrate that NIR-pH-H2O2 is a novel bifunctional mitochondrial-targeted NIR probe to sense acidic pH and H2O2 in vitro and in vivo, indicating its huge potential for the diagnosis of pH and H2O2-related diseases.
  18. Bioengineered. 2022 Apr;13(4): 11187-11207
      Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinomas, with high mortality and poor prognoses worldwide. Succinate dehydrogenase (SDH) consists of four nuclear-encoded subunits and it is the only complex involved in both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Previous studies have shown decreased SDH activity in ccRCC. However, the role and underlying molecular mechanisms of SDH in ccRCC initiation and development remain unclear. In the present study, pan-cancer analysis of SDH gene expression was analyzed and the relationship between SDH gene expression and clinicopathological parameters was assessed using different databases. cBioPortal, UACLAN, and Tumor Immune Estimation Resource (TIMER) were subsequently utilized to analyze genetic alterations, methylation, and immune cell infiltration of SDH genes in ccRCC patients. We found SDHs were significantly downregulated in ccRCC tissues and correlated with ccRCC progression. Increased methylation and high SDH promoter mutation rates may be the cause of reduced expression of SDHs in ccRCC. Moreover, the interaction network showed that SDH genes were correlated with ferroptosis-related genes. We further demonstrated that SDH inhibition dampened oxidative phosphorylation, reduced ferroptotic events, and restored ferroptotic cell death, characterized by eliminated mitochondrial ROS levels, decreased cellular ROS and diminished peroxide accumulation. Collectively, this study provides new insights into the regulatory role of SDH in the carcinogenesis and progression of ccRCC, introducing a potential target for advanced antitumor therapy through ferroptosis.
    Keywords:  Succinate dehydrogenase; ferroptosis; oxidative phosphorylation; prognosis; renal cell carcinoma
  19. Mol Cell. 2022 May 05. pii: S1097-2765(22)00375-6. [Epub ahead of print]82(9): 1613-1615
      Jouandin et al. (2022) show that lysosomal-derived cysteine serves as a signal to promote the tricarboxylic acid (TCA) cycle and suppress TORC1 signaling for Drosophila to endure starvation periods.
  20. Biochemistry (Mosc). 2022 Feb;87(2): 179-190
      In this review, we discuss the mechanisms of generation of membrane-bound protons using different energy sources in model and natural systems. Analysis of these mechanisms revealed that all three types of reactions include the same principal stage, which is dissociation of electrically neutral Brønsted acids at the interface during transition from the hydrophobic phase to water with a low dielectric constant. Special attention is paid to the fact that in one of the analyzed model systems, membrane-bound protons provide energy for the reaction of ATP synthesis. Similar mechanism for the generation of membrane-bound protons has been found in natural membranes involved in oxidative phosphorylation, in particular, on the membranes of mitoplasts and mitochondria. The energy of oxidative reactions required for ATP synthesis, is stored at the intermediate stage not only in the form of transmembrane electrochemical potential of protons, but also and perhaps mostly, as protons attached to the inner mitochondrial membrane. The process of energy storage in mitochondria is linked to the transfer of protons that simultaneously perform two functions. Protons on the membrane surface carry free energy and, at the same time, act as substrates facilitating the movement of F1F0-ATP-synthase biological machine.
    Keywords:  hydrogen ion; membrane; membrane bound proton; mitochondria; oxidative phosphorylation; supercapacitor
  21. Cell Death Discov. 2022 May 06. 8(1): 253
      Ferroptosis is a caspase-independent form of regulated cell death strongly linked to the accumulation of reactive lipid hydroperoxides. Lipid hydroperoxides are neutralized in cells by glutathione peroxidase 4 (GPX4) and inhibitors of GPX4 are potent ferroptosis inducers with therapeutic potential in cancer. Here we report that siRNA-mediated silencing of the AMPK-related kinase NUAK2 suppresses cell death by small-molecule inducers of ferroptosis but not apoptosis. Mechanistically we find that NUAK2 suppresses the expression of GPX4 at the RNA level and enhances ferroptosis triggered by GPX4 inhibitors in a manner independent of its kinase activity. NUAK2 is amplified along with MDM4 in a subset of breast cancers, particularly the claudin-low subset, suggesting that this may predict vulnerability to GPX4 inhibitors. These findings identify a novel pathway regulating GPX4 expression as well as ferroptotic sensitivity with potential as a biomarker of breast cancer patients that might respond to GPX4 inhibition as a therapeutic strategy.
  22. Nucleic Acids Res. 2022 May 07. pii: gkac306. [Epub ahead of print]
      Mitochondria are subcellular organelles present in almost all eukaryotic cells, which play a central role in cellular metabolism. Different tissues, health and age conditions are characterized by a difference in mitochondrial structure and composition. The visual data mining platform mitoXplorer 1.0 was developed to explore the expression dynamics of genes associated with mitochondrial functions that could help explain these differences. It, however, lacked functions aimed at integrating mitochondria in the cellular context and thus identifying regulators that help mitochondria adapt to cellular needs. To fill this gap, we upgraded the mitoXplorer platform to version 2.0 (mitoXplorer 2.0). In this upgrade, we implemented two novel integrative functions, network analysis and transcription factor enrichment, to specifically help identify signalling or transcriptional regulators of mitochondrial processes. In addition, we implemented several other novel functions to allow the platform to go beyond simple data visualization, such as an enrichment function for mitochondrial processes, a function to explore time-series data, the possibility to compare datasets across species and an IDconverter to help facilitate data upload. We demonstrate the usefulness of these functions in three specific use cases. mitoXplorer 2.0 is freely available without login at
  23. J Mol Biol. 2022 Apr 29. pii: S0022-2836(22)00198-X. [Epub ahead of print] 167618
      The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.
    Keywords:  Mitochondrial Unfolded Protein Response; Molecular Chaperone; Protein misfolding; Proteostasis; Proteotoxic stress; Ribosome Quality Control; Stress Response; TOM complex; Vms1
  24. Cell Rep Methods. 2022 Apr 25. 2(4): 100192
      Macrophages are dynamic immune cells that can adopt several activation states. Fundamental to these functional activation states is the regulation of cellular metabolic processes. Especially in mice, metabolic alterations underlying pro-inflammatory or homeostatic phenotypes have been assessed using various techniques. However, researchers new to the field may encounter ambiguity in choosing which combination of techniques is best suited to profile immunometabolism. To address this need, we have developed a toolbox to assess cellular metabolism in a semi-high-throughput 96-well-plate-based format. Application of the toolbox to activated mouse and human macrophages enables fast metabolic pre-screening and robust measurement of extracellular fluxes, mitochondrial mass and membrane potential, and glucose and lipid uptake. Moreover, we propose an application of SCENITH technology for ex vivo metabolic profiling. We validate established activation-induced metabolic rewiring in mouse macrophages and report new insights into human macrophage metabolism. By thoroughly discussing each technique, we hope to guide readers with practical workflows for investigating immunometabolism.
    Keywords:  immunometabolism; macrophages; metabolism; semi-high throughput screening; toolbox
  25. Proc Natl Acad Sci U S A. 2022 May 10. 119(19): e2120595119
      SignificanceGlutamine is the most abundant amino acid in human plasma, although it is challenging to determine glutamine's metabolic fate noninvasively. In this work, we utilize established chemical methods to develop a platform for imaging glutamine metabolism using hyperpolarized magnetic resonance imaging. Using this strategy, we are able to spatially measure glutaminolysis in vivo as well as develop a biomarker for the inhibition of glutaminase. Combining this biomarker with isotope tracing metabolomics connects this inhibition to reduced glutamine contribution to the tricarboxylic acid cycle. This provides an approach for future imaging of glutamine metabolism in humans.
    Keywords:  cancer metabolism; magnetic resonance imaging; metabolic imaging; pancreatic cancer
  26. Comput Struct Biotechnol J. 2022 ;20 1829-1840
      The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters.
    Keywords:  AAC, ADP/ADP carrier; ATP translocases; CATR, carboxyatractyloside; CoA, coenzyme A; GDC, Graves disease carrier protein, or SLC25A16; MCF, mitochondrial carrier family; MD simulation, molecular dynamics simulation PCA, Principal component analysis; Mitochondrial ADP; OXPHOS, oxidative phosphorylation; SCaMCs, short Ca2+-binding mitochondrial carrier, or Mg-ATP/Pi carrier; Solute carrier family 25, molecular dynamics simulation; Substrate recognition; Transporter; c-state, cytosol-open state; m-state, matrix-open state
  27. Nat Commun. 2022 May 03. 13(1): 2412
      Human neurodegenerative disorders often exhibit similar pathologies, suggesting a shared aetiology. Key pathological features of Parkinson's disease (PD) are also observed in other neurodegenerative diseases. Pantothenate Kinase-Associated Neurodegeneration (PKAN) is caused by mutations in the human PANK2 gene, which catalyzes the initial step of de novo CoA synthesis. Here, we show that fumble (fbl), the human PANK2 homolog in Drosophila, interacts with PINK1 genetically. fbl and PINK1 mutants display similar mitochondrial abnormalities, and overexpression of mitochondrial Fbl rescues PINK1 loss-of-function (LOF) defects. Dietary vitamin B5 derivatives effectively rescue CoA/acetyl-CoA levels and mitochondrial function, reversing the PINK1 deficiency phenotype. Mechanistically, Fbl regulates Ref(2)P (p62/SQSTM1 homolog) by acetylation to promote mitophagy, whereas PINK1 regulates fbl translation by anchoring mRNA molecules to the outer mitochondrial membrane. In conclusion, Fbl (or PANK2) acts downstream of PINK1, regulating CoA/acetyl-CoA metabolism to promote mitophagy, uncovering a potential therapeutic intervention strategy in PD treatment.
  28. Elife. 2022 May 04. pii: e75492. [Epub ahead of print]11
      Cancer survivors suffer from progressive frailty, multimorbidity and premature morbidity. We hypothesize that therapy-induced senescence and senescence progression via bystander effects is a significant cause of this premature ageing phenotype. Accordingly, the study addresses the question whether a short anti-senescence intervention is able to block progression of radiation-induced frailty and disability in a pre-clinical setting. Male mice were sub-lethally irradiated at 5 months of age and treated (or not) with either a senolytic drug (Navitoclax or dasatinib + quercetin) for 10 days or with the senostatic metformin for 10 weeks. Follow up was for one year. Treatments commencing within a month after irradiation effectively reduced frailty progression (p<0.05) and improved muscle (p<0.01) and liver (p<0.05) function as well as short-term memory (p<0.05) until advanced age with no need for repeated interventions. Senolytic interventions that started late, after radiation-induced premature frailty was manifest, still had beneficial effects on frailty (p<0.05) and short-term memory (p<0.05). Metformin was similarly effective as senolytics. At therapeutically achievable concentrations metformin acted as a senostatic neither via inhibition of mitochondrial complex I, nor via improvement of mitophagy or mitochondrial function, but by reducing non-mitochondrial ROS production via NOX4 inhibition in senescent cells. Our study suggests that the progression of adverse long-term health and quality-of-life effects of radiation exposure, as experienced by cancer survivors, might be rescued by short-term adjuvant anti-senescence interventions.
    Keywords:  cancer biology; cell biology; mouse
  29. Cell Stem Cell. 2022 May 05. pii: S1934-5909(22)00160-6. [Epub ahead of print]29(5): 760-775.e10
      Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4+ T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4+ T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool.
    Keywords:  CD4(+) T cells; acute myeloid leukemia; antigen presentation; hematopoietic stem cells; immune regulation; immunosurveillance; leukemia
  30. Sci Rep. 2022 May 06. 12(1): 7453
      Intestinal homeostasis is highly dependent on optimal epithelial barrier function and permeability. Intestinal epithelial cells (IEC) regulate these properties acting as cellular gatekeepers by selectively absorbing nutrients and controlling the passage of luminal bacteria. These functions are energy demanding processes that are presumably met through mitochondrial-based processes. Routine methods for examining IEC mitochondrial function remain sparse, hence, our objective is to present standardized methods for quantifying mitochondrial energetics in an immortalized IEC line. Employing the murine IEC4.1 cell line, we present adapted methods and protocols to examine mitochondrial function using two well-known platforms: the Seahorse Extracellular Flux Analyzer and Oxygraph-2 k. To demonstrate the applicability of these protocols and instruments, IEC were treated with and without the murine colitogenic agent, dextran sulfate sodium (DSS, 2% w/v). Profound impairments with DSS treatment were found with both platforms, however, the Oxygraph-2 k allowed greater resolution of affected pathways including short-chain fatty acid metabolism. Mitochondrial functional analysis is a novel tool to explore the relationship between IEC energetics and functional consequences within the contexts of health and disease. The outlined methods offer an introductory starting point for such assessment and provide the investigator with insights into platform-specific capabilities.
  31. Theory Biosci. 2022 May 02.
      Mechanisms coupling the chemical reactions of oxidation and ATP synthesis in cellular metabolism by the fundamental biological process of oxidative phosphorylation (OX PHOS) in mitochondria provide > 90% of the energy requirements in living organisms. Mathematical graph theory methods have been extensively used to characterize various metabolic, regulatory, and disease networks in biology. However, networks of energy coupling mechanisms in OX PHOS have not been represented and analyzed previously by these approaches. Here, the problem of biological energy coupling is translated into a graph-theoretical framework, and all possible coupling schemes between oxidation and ATP synthesis are represented as graphs connecting these processes by various intermediates or states. The problem is shown to be transformed into the hard problem of finding a Hamiltonian tour in the networks of possible constituent mechanisms, given the constraints of a cyclical nature of operation of enzymes and biological molecular machines. Accessible mathematical proofs of three theorems that guarantee sufficient conditions for the existence of a Hamiltonian cycle in simple graphs are provided. The results of the general theorems are applied to the set of possible coupling mechanisms in OX PHOS and shown to (1) unequivocally differentiate between the major theories and mechanisms of energy coupling, (2) greatly reduce the possibilities for detailed consideration, and (3) deduce the biologically selected mechanism using additional constraints from the cumulative experimental record. Finally, an algorithm is constructed to implement the graph-theoretical procedure. In summary, the enormous power and generality of mathematical theorems and approaches in graph theory are shown to help solve a fundamental problem in biology.
    Keywords:  ATP synthesis; Algorithms; Bioenergetics; Bondy–Chvátal theorem; Cell metabolism; Closure; Coupling mechanisms; Dirac’s theorem; Energy coupling; F O F 1-ATP synthase; Graph theory; Graphs; Hamiltonian cycles; Mathematical biology; Metabolic regulation and control; Mitchell’s chemiosmotic theory; Molecular motors; Nath’s torsional mechanism of energy transduction and ATP synthesis; Nath’s two-ion theory of energy coupling and ATP synthesis; Networks; Ore’s theorem; Oxidative phosphorylation (OX PHOS); Sufficient conditions for Hamiltonian circuits; Traveling Salesman Problem
  32. Cell Discov. 2022 May 03. 8(1): 40
      Ferroptosis is a regulated iron-dependent cell death characterized by the accumulation of lipid peroxidation. A myriad of facets linking amino acid, lipid, redox, and iron metabolisms were found to drive or to suppress the execution of ferroptosis. However, how the cells decipher the diverse pro-ferroptotic stress to activate ferroptosis remains elusive. Here, we report that protein O-GlcNAcylation, the primary nutrient sensor of glucose flux, orchestrates both ferritinophagy and mitophagy for ferroptosis. Following the treatment of ferroptosis stimuli such as RSL3, a commonly used ferroptosis inducer, there exists a biphasic change of protein O-GlcNAcylation to modulate ferroptosis. Pharmacological or genetic inhibition of O-GlcNAcylation promoted ferritinophagy, resulting in the accumulation of labile iron towards mitochondria. Inhibition of O-GlcNAcylation resulted in mitochondria fragmentation and enhanced mitophagy, providing an additional source of labile iron and rendering the cell more sensitive to ferroptosis. Mechanistically, we found that de-O-GlcNAcylation of the ferritin heavy chain at S179 promoted its interaction with NCOA4, the ferritinophagy receptor, thereby accumulating labile iron for ferroptosis. Our findings reveal a previously uncharacterized link of dynamic O-GlcNAcylation with iron metabolism and decision-making for ferroptosis, thus offering potential therapeutic intervention for fighting disease.
  33. Proc Natl Acad Sci U S A. 2022 May 10. 119(19): e2201921119
      SignificanceThe ATP synthases of many anaerobic archaea have an unusual motor subunit c that otherwise is only found in eukaryotic V1VO ATPases. The evolutionary switch from synthase to hydrolase is thought to be caused by a doubling of the rotor subunit c, followed by a loss of the ion binding site. By purification and reconstitution of an ATP synthase with a V-type c subunit, we have unequivocally demonstrated, against expectations, the capability of such an enzyme to synthesize ATP at physiological relevant driving forces of 90 to 150 mV. This is the long-awaited answer to an eminent question in microbial energetics and physiology, especially for life near the thermodynamic limit of ATP synthesis.
    Keywords:  ATP synthesis; acetogenic bacteria; archaea; bioenergetics; driving forces