bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2022‒01‒02
thirty papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit

  1. Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01671-5. [Epub ahead of print]37(13): 110171
      Macrophages are often prominently present in the tumor microenvironment, where distinct macrophage populations can differentially affect tumor progression. Although metabolism influences macrophage function, studies on the metabolic characteristics of ex vivo tumor-associated macrophage (TAM) subsets are rather limited. Using transcriptomic and metabolic analyses, we now reveal that pro-inflammatory major histocompatibility complex (MHC)-IIhi TAMs display a hampered tricarboxylic acid (TCA) cycle, while reparative MHC-IIlo TAMs show higher oxidative and glycolytic metabolism. Although both TAM subsets rapidly exchange lactate in high-lactate conditions, only MHC-IIlo TAMs use lactate as an additional carbon source. Accordingly, lactate supports the oxidative metabolism in MHC-IIlo TAMs, while it decreases the metabolic activity of MHC-IIhi TAMs. Lactate subtly affects the transcriptome of MHC-IIlo TAMs, increases L-arginine metabolism, and enhances the T cell suppressive capacity of these TAMs. Overall, our data uncover the metabolic intricacies of distinct TAM subsets and identify lactate as a carbon source and metabolic and functional regulator of TAMs.
    Keywords:  TCA cycle break; immunometabolism; immunosuppression; lactate; macrophage metabolism; metabolomics; non-small-cell lung carcinoma; single-cell metabolic profiling; tumor microenvironment; tumor-associated macrophages
  2. Methods Mol Biol. 2022 ;2445 227-239
      Mitophagy, a process of selective elimination of mitochondria by autophagy, is a mechanism of mitochondrial quality control that maintains mitochondrial network functionality. The elimination of damaged mitochondria through autophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition. Mitophagy impairment is linked to various pathologies; thus, removal of malfunctioning or even harmful mitochondria is vital to cellular physiology. Here, we describe methods that can be applied to the investigation of mitophagy.
    Keywords:  Autophagy; Confocal microscopy; Flow cytometry; Mitochondria; Mitophagy; Respiration
  3. Front Plant Sci. 2021 ;12 804468
      A fundamental principle shared by all organisms is the metabolic conversion of nutrients into energy for cellular processes and structural building blocks. A highly precise spatiotemporal programming is required to couple metabolic capacity with energy allocation. Cellular metabolism is also able to adapt to the external time, and the mechanisms governing such an adaptation rely on the circadian clock. Virtually all photosensitive organisms have evolved a self-sustained timekeeping mechanism or circadian clock that anticipates and responds to the 24-h environmental changes that occur during the day and night cycle. This endogenous timing mechanism works in resonance with the environment to control growth, development, responses to stress, and also metabolism. Here, we briefly describe the prevalent role for the circadian clock controlling the timing of mitochondrial activity and cellular energy in Arabidopsis thaliana. Evidence that metabolic signals can in turn feedback to the clock place the spotlight onto the molecular mechanisms and components linking the circadian function with metabolic homeostasis and energy.
    Keywords:  ATP; Arabidopsis thaliana; circadian clock; metabolism; mitochondria
  4. Free Radic Biol Med. 2021 Dec 27. pii: S0891-5849(21)01169-2. [Epub ahead of print]
      Mitochondria are central to the metabolic circuitry that generates superoxide radicals/anions (O2•-) as a by-product of oxygen metabolism. By regulating superoxide levels, manganese superoxide dismutase plays important roles in numerous biochemical and molecular events essential for the survival of aerobic life. In this study, we used MitoParaquat (mPQ) to generate mitochondria-specific O2•- and stable isotope-resolved metabolomics tracing in primary human epidermal keratinocytes to investigate how O2•- generated in mitochondria regulates gene expression. The results reveal that isocitrate is blocked from conversion to α-ketoglutarate and that acetyl-coenzyme A (CoA) accumulates, which is consistent with a reduction in oxygen consumption rate and inactivation of isocitrate dehydrogenase (IDH) activity. Since acetyl-CoA is linked to histone acetylation and gene regulation, we determined the effect of mPQ on histone acetylation. The results demonstrate an increase in histone H3 acetylation at lysines 9 and 14. Suppression of IDH increased histone acetylation, providing a direct link between metabolism and epigenetic alterations. The activity of histone acetyltransferase p300 increased after mPQ treatment, which is consistent with histone acetylation. Importantly, mPQ selectively increased the nuclear levels and activity of the oxidative stress-sensitive nuclear factor erythroid 2-related factor 2. Together, the results establish a new paradigm that recognizes O2•- as an initiator of metabolic reprogramming that activates epigenetic regulation of gene transcription in response to mitochondrial dysfunction.
    Keywords:  Epigenetics; Metabolism; Mitochondria; Superoxide; TCA cycle; Transcription
  5. Cancer Metastasis Rev. 2021 Dec 28.
      Branched-chain amino acids (BCAA) are essential amino acids utilized in anabolic and catabolic metabolism. While extensively studied in obesity and diabetes, recent evidence suggests an important role for BCAA metabolism in cancer. Elevated plasma levels of BCAA are associated with an increased risk of developing pancreatic cancer, namely pancreatic ductal adenocarcinoma (PDAC), a tumor with one of the highest 1-year mortality rates. The dreadful prognosis for PDAC patients could be attributable also to the early and frequent development of cancer cachexia, a fatal host metabolic reprogramming leading to muscle and adipose wasting. We propose that BCAA dysmetabolism is a unifying component of several pathological conditions, i.e., obesity, insulin resistance, and PDAC. These conditions are mutually dependent since PDAC ranks among cancers tightly associated with obesity and insulin resistance. It is also well-established that PDAC itself can trigger insulin resistance and new-onset diabetes. However, the exact link between BCAA metabolism, development of PDAC, and tissue wasting is still unclear. Although tissue-specific intracellular and systemic metabolism of BCAA is being intensively studied, unresolved questions related to PDAC and cancer cachexia remain, namely, whether elevated circulating BCAA contribute to PDAC etiology, what is the biological background of BCAA elevation, and what is the role of adipose tissue relative to BCAA metabolism during cancer cachexia. To cover those issues, we provide our view on BCAA metabolism at the intracellular, tissue, and whole-body level, with special emphasis on different metabolic links to BCAA intermediates and the role of insulin in substrate handling.
    Keywords:  Adipose tissue; BCAA metabolism; Cancer cachexia; Insulin resistance; PDAC
  6. Elife. 2021 Dec 31. pii: e68213. [Epub ahead of print]10
      Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.
    Keywords:  assembly; biochemistry; cell biology; chemical biology; mitochondria; oxidative phosphorylation; ribosome; translation
  7. Methods Mol Biol. 2022 ;2445 329-335
      Cancer cells possess an elevated demand for nutrients and metabolites due to their uncontrolled proliferation and need to survive in unfavorable conditions. Autophagy is a conservative degradation pathway that counters lack of nutrients and provides organelle and protein quality control, beyond maintenance of cellular metabolism.Mass spectrometry-based metabolomics is a powerful tool to study the metabolome of a cell. Such analysis requires proper sample preparation including the extraction of metabolites. Here, we provide a protocol for the extraction of metabolites from adherent cancer cells suitable for global metabolome profiling by mass spectrometry.
    Keywords:  Autophagy; CE-MS; Cancer metabolism; Chaperone-mediated autophagy; GC-MS; LC-MS; Mass spectrometry; Metabolism; Methanol extraction
  8. Cancer Metastasis Rev. 2021 Dec 27.
      Pheochromocytoma (PHEO) and paraganglioma (PGL) (together PPGL) are tumors with poor outcomes that arise from neuroendocrine cells in the adrenal gland, and sympathetic and parasympathetic ganglia outside the adrenal gland, respectively. Many follow germline mutations in genes coding for subunits of succinate dehydrogenase (SDH), a tetrameric enzyme in the tricarboxylic acid (TCA) cycle that both converts succinate to fumarate and participates in electron transport. Germline SDH subunit B (SDHB) mutations have a high metastatic potential. Herein, we review the spectrum of model organisms that have contributed hugely to our understanding of SDH dysfunction. In Saccharomyces cerevisiae (yeast), succinate accumulation inhibits alpha-ketoglutarate-dependent dioxygenase enzymes leading to DNA demethylation. In the worm Caenorhabditis elegans, mutated SDH creates developmental abnormalities, metabolic rewiring, an energy deficit and oxygen hypersensitivity (the latter is also found in Drosophila melanogaster). In the zebrafish Danio rerio, sdhb mutants display a shorter lifespan with defective energy metabolism. Recently, SDHB-deficient pheochromocytoma has been cultivated in xenografts and has generated cell lines, which can be traced back to a heterozygous SDHB-deficient rat. We propose that a combination of such models can be efficiently and effectively used in both pathophysiological studies and drug-screening projects in order to find novel strategies in PPGL treatment.
    Keywords:  Model organisms; Paraganglioma; Pheochromocytoma; SDH; Succinate dehydrogenase
  9. Semin Immunol. 2021 Dec 25. pii: S1044-5323(21)00114-7. [Epub ahead of print] 101583
      Neutrophils are critical innate immune cells for the host anti-bacterial defense. Throughout their lifecycle, neutrophils are exposed to different microenvironments and modulate their metabolism to survive and sustain their functions. Although tumor cell metabolism has been intensively investigated, how neutrophil metabolism is affected in cancer remains largely to be discovered. Neutrophils are described as mainly glycolytic cells. However, distinct tumor-associated neutrophil (TAN) states may co-exist in tumors and adapt their metabolism to exert different or even opposing activities ranging from tumor cell killing to tumor support. In this review, we gather evidence about the metabolic mechanisms that underly TANs' pro- or anti-tumoral functions in cancer. We first discuss how tumor-secreted factors and the heterogenous tumor microenvironment can have a strong impact on TAN metabolism. We then describe alternative metabolic pathways used by TANs to exert their functions in cancer, from basic glycolysis to more recently-recognized but less understood metabolic shifts toward mitochondrial oxidative metabolism, lipid and amino acid metabolism and even autophagy. Last, we discuss promising strategies targeting neutrophil metabolism to combat cancer.
    Keywords:  Cancer metabolism; Neutrophil metabolism; Tumor-associated neutrophils
  10. Front Oncol. 2021 ;11 783856
      Mitochondrial dysfunction and aberrant glycolysis are hallmarks of human clear cell renal cell carcinoma (ccRCC). Whereas glycolysis is thoroughly studied, little is known about the mitochondrial contribution to the pathology of ccRCC. Mitochondrial Ndufa4l2 is predictive of poor survival of ccRCC patients, and in kidney cancer cell lines the protein supports proliferation and colony formation. Its role in ccRCC, however, remains enigmatic. We utilized our established ccRCC model, termed Transgenic Cancer of the Kidney (TRACK), to generate a novel genetically engineered mouse model in which dox-regulated expression of an shRNA decreases Ndufa4l2 levels specifically in the renal proximal tubules (PT). This targeted knockdown of Ndufa4l2 reduced the accumulation of neutral renal lipid and was associated with decreased levels of the ccRCC markers carbonic anhydrase 9 (CA9) and Enolase 1 (ENO1). These findings suggest a link between mitochondrial dysregulation (i.e. high levels of Ndufa4l2), lipid accumulation, and the expression of ccRCC markers ENO1 and CA9, and demonstrate that lipid accumulation and ccRCC development can potentially be attenuated by inhibiting Ndufa4l2.
    Keywords:  HIF1; clear cell renal cell carcinoma; hypoxia; lipids; mitochondria; proximal tubules; von Hippel-Lindau
  11. Metab Eng. 2021 Dec 22. pii: S1096-7176(21)00193-2. [Epub ahead of print]
      Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary 13C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Metabolic flux analysis results show that resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.
    Keywords:  Blood platelets; Metabolic flux analysis; Metabolomics; Thrombin
  12. J Biol Chem. 2021 Dec 24. pii: S0021-9258(21)01350-8. [Epub ahead of print] 101540
      Persistent inactivity promotes skeletal muscle atrophy, marked by mitochondrial aberrations that affect strength, mobility, and metabolic health leading to the advancement of disease. Mitochondrial quality control (MQC) pathways include biogenesis (synthesis), mitophagy/lysosomal turnover, and the mitochondrial unfolded protein response (UPRmt) which serve to maintain an optimal organelle network. Tumor suppressor p53 has been implicated in regulating muscle mitochondria in response to cellular stress; however, its role in the context of muscle disuse has yet to be explored, and whether p53 is necessary for MQC remains unclear. To address this, we subjected p53 muscle-specific knockout (mKO) and wild-type (WT) mice to unilateral denervation. Transcriptomic and pathway analyses revealed dysregulation of pathways pertaining to mitochondrial function, and especially turnover, in mKO muscle following denervation. Protein and mRNA data of the MQC pathways indicated activation of the UPRmt and mitophagy-lysosome systems along with reductions in mitochondrial biogenesis and content in WT and mKO tissue following chronic denervation. However, p53 ablation also attenuated the expression of autophagy/mitophagy machinery, reduced autophagic flux, and enhanced lysosomal dysfunction. While similar reductions in mitochondrial biogenesis and content were observed between genotypes, MQC dysregulation exacerbated mitochondrial dysfunction in mKO fibers, evidenced by elevated reactive oxygen species (ROS). Moreover, acute experiments indicate that p53 mediates the expression of transcriptional regulators of MQC pathways as early as 1 day following denervation. Together, our data illustrate exacerbated mitochondrial dysregulation with denervation stress in p53 mKO tissue, thus indicating that p53 contributes to organellar maintenance via regulation of MQC pathways during muscle atrophy.
    Keywords:  lysosome; mitochondria; mitochondrial biogenesis; mitochondrial quality control; mitophagy; muscle atrophy; p53; skeletal muscle; transcriptomics; unfolded protein response (UPR)
  13. Mol Carcinog. 2021 Dec 27.
      Tumor metabolism has emerged as a hallmark of cancer and is involved in carcinogenesis and tumor growth. Reprogramming of tumor metabolism is necessary for cancer cells to sustain high proliferation rates and enhanced demands for nutrients. Recent studies suggest that metabolic plasticity in cancer cells can decrease the efficacy of anticancer therapies by enhancing antioxidant defenses and DNA repair mechanisms. Studying radiation-induced metabolic changes will lead to a better understanding of radiation response mechanisms as well as the identification of new therapeutic targets, but there are few robust studies characterizing the metabolic changes induced by radiation therapy in cancer. In this review, we will highlight studies that provide information on the metabolic changes induced by radiation and oxidative stress in cancer cells and the associated underlying mechanisms.
    Keywords:  cancer; metabolism; oxidative stress; radiation therapy; radiosensitivity
  14. Front Microbiol. 2021 ;12 793664
      Though all theories for the origin of life require a source of energy to promote primordial chemical reactions, the nature of energy that drove the emergence of metabolism at origins is still debated. We reasoned that evidence for the nature of energy at origins should be preserved in the biochemical reactions of life itself, whereby changes in free energy, ΔG, which determine whether a reaction can go forward or not, should help specify the source. By calculating values of ΔG across the conserved and universal core of 402 individual reactions that synthesize amino acids, nucleotides and cofactors from H2, CO2, NH3, H2S and phosphate in modern cells, we find that 95-97% of these reactions are exergonic (ΔG ≤ 0 kJ⋅mol-1) at pH 7-10 and 80-100°C under nonequilibrium conditions with H2 replacing biochemical reductants. While 23% of the core's reactions involve ATP hydrolysis, 77% are ATP-independent, thermodynamically driven by ΔG of reactions involving carbon bonds. We identified 174 reactions that are exergonic by -20 to -300 kJ⋅mol-1 at pH 9 and 80°C and that fall into ten reaction types: six pterin dependent alkyl or acyl transfers, ten S-adenosylmethionine dependent alkyl transfers, four acyl phosphate hydrolyses, 14 thioester hydrolyses, 30 decarboxylations, 35 ring closure reactions, 31 aromatic ring formations, and 44 carbon reductions by reduced nicotinamide, flavins, ferredoxin, or formate. The 402 reactions of the biosynthetic core trace to the last universal common ancestor (LUCA), and reveal that synthesis of LUCA's chemical constituents required no external energy inputs such as electric discharge, UV-light or phosphide minerals. The biosynthetic reactions of LUCA uncover a natural thermodynamic tendency of metabolism to unfold from energy released by reactions of H2, CO2, NH3, H2S, and phosphate.
    Keywords:  bioenergetics; biosynthesis; early evolution; energetics; last universal common ancestor; metabolism; origin of life; thermodynamics
  15. Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01653-3. [Epub ahead of print]37(13): 110157
      Lipid storage in fat tissue is important for energy homeostasis and cellular functions. Through RNAi screening in Drosophila fat body, we found that knockdown of a Drosophila NAD kinase (NADK), which phosphorylates NAD to synthesize NADP de novo, causes lipid storage defects. NADK sustains lipogenesis by maintaining the pool of NADPH. Promoting NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals. Furthermore, NADK and fatty acid synthase 1 (FASN1) regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids. Reducing the level of acetyl-CoA or increasing the synthesis of cardiolipin (CL), a mitochondrion-specific phospholipid, partially rescues the mitochondrial defects of NADK RNAi. Therefore, NADK- and FASN1-mediated fatty acid synthesis coordinates lipid storage and mitochondrial function.
    Keywords:  Drosophila; FASN; NADK; lipogenesis; mitochondrial metabolism
  16. Mol Metab. 2021 Dec 22. pii: S2212-8778(21)00282-9. [Epub ahead of print] 101424
      Glucocorticoids (GCs) are one of the most widely prescribed anti-inflammatory drugs. By acting through their cognate receptor, the glucocorticoid receptor (GR), GCs down-regulate the expression of pro-inflammatory genes, and up-regulate anti-inflammatory genes. Metabolic pathways have recently been identified as key parts of both the inflammatory activation and anti-inflammatory polarization of macrophages, immune cells responsible for acute inflammation and tissue repair. It is currently unknown whether GCs control macrophage metabolism, and if so, to what extent metabolic regulation by GCs confers anti-inflammatory activity. Using transcriptomic and metabolomic profiling of macrophages, we identified GC controlled pathways involved in metabolism, especially in mitochondrial function. Metabolic analyses revealed that GCs repress glycolysis in inflammatory myeloid cells and promote tricarboxylic acid (TCA) cycle flux, promoting succinate metabolism and preventing intracellular accumulation of succinate. Inhibition of ATP synthase attenuated GC induced transcriptional changes, likely through stalling of TCA cycle anaplerosis. We further identified a glycolytic regulatory transcription factor, HIF1α, as regulated by GCs, and as a key regulator of GC responsiveness during inflammatory challenge, further linking metabolism to GC action in macrophages.
    Keywords:  Glucocorticoids; Succinate; TCA Cycle; immunometabolism; macrophage
  17. Eur J Immunol. 2021 Dec 27.
      Alterations in cell metabolism can shift the differentiation of immune cells towards a regulatory or inflammatory phenotype, thus opening up new therapeutic opportunities for immune-related diseases. Indeed, growing knowledge on T cell metabolism has revealed differences in the metabolic programs of suppressive regulatory T cells (Tregs) as compared to inflammatory Th1 and Th17 cells. In addition to Tregs, IL-10-producing regulatory B cells are crucial for maintaining tolerance, inhibiting inflammation and autoimmunity. Yet, the metabolic networks regulating diverse B lymphocyte responses are not well known. Here, we show that glutaminase blockade decreased downstream mTOR activation and attenuated IL-10 secretion. Direct suppression of mTOR activity by rapamycin selectively impaired IL-10 production by B cells whereas secretion was restored upon GSK3 inhibition. Mechanistically, we found mTORC1 activation leads to GSK3 inhibition, identifying a key signalling pathway regulating IL-10 secretion by B lymphocytes. Thus, our results identify glutaminolysis and the mTOR/GSK3 signalling axis, as critical regulators of the generation of IL-10 producing B cells with regulatory functions. This article is protected by copyright. All rights reserved.
    Keywords:  B10; GSK3; IL-10; glutamine; immunotherapy; mTOR; metabolism; regulatory B cells
  18. Compr Physiol. 2021 Dec 29. 12(1): 2995-3044
      The proximal tubule of the kidney is programmed to reabsorb all filtered glucose and fructose. Glucose is taken up by apical sodium-glucose cotransporters SGLT2 and SGLT1 whereas SGLT5 and potentially SGLT4 and GLUT5 have been implicated in apical fructose uptake. The glucose taken up by the proximal tubule is typically not metabolized but leaves via the basolateral facilitative glucose transporter GLUT2 and is returned to the systemic circulation or used as an energy source by distal tubular segments after basolateral uptake via GLUT1. The proximal tubule generates new glucose in metabolic acidosis and the postabsorptive phase, and fructose serves as an important substrate. In fact, under physiological conditions and intake, fructose taken up by proximal tubules is primarily utilized for gluconeogenesis. In the diabetic kidney, glucose is retained and gluconeogenesis enhanced, the latter in part driven by fructose. This is maladaptive as it sustains hyperglycemia. Moreover, renal glucose retention is coupled to sodium retention through SGLT2 and SGLT1, which induces secondary deleterious effects. SGLT2 inhibitors are new anti-hyperglycemic drugs that can protect the kidneys and heart from failing independent of kidney function and diabetes. Dietary excess of fructose also induces tubular injury. This can be magnified by kidney formation of fructose under pathological conditions. Fructose metabolism is linked to urate formation, which partially accounts for fructose-induced tubular injury, inflammation, and hemodynamic alterations. Fructose metabolism favors glycolysis over mitochondrial respiration as urate suppresses aconitase in the tricarboxylic acid cycle, and has been linked to potentially detrimental aerobic glycolysis (Warburg effect). © 2022 American Physiological Society. Compr Physiol 12:2995-3044, 2022.
  19. Am J Physiol Cell Physiol. 2021 12 29.
      Selective autophagy of the mitochondria, known as mitophagy, is a major mitochondrial quality control pathway in the heart that is involved in removing unwanted or dysfunctional mitochondria from the cell. Baseline mitophagy is critical for maintaining the fitness of the mitochondrial population by continuous turnover of aged and less functional mitochondria. Mitophagy is also critical in adapting to stress associated with mitochondrial damage or dysfunction. The removal of damaged mitochondria prevents ROS-mediated damaged to proteins and DNA and suppresses activation of inflammation and cell death. Impairments in mitophagy are associated with the pathogenesis of many diseases, including cancers, inflammatory diseases, neurodegeneration, and cardiovascular disease. Mitophagy is a highly regulated and complex process that requires the coordination of labeling dysfunctional mitochondria for degradation while simultaneously promoting de novo autophagosome biogenesis adjacent to the cargo. In this review, we provide an update on our current understanding of these steps in mitophagy induction and discuss the physiological and pathophysiological consequences of altered mitophagy in the heart.
    Keywords:  Parkin; autophagy; heart; mitochondria; mitophagy
  20. J Biol Chem. 2021 Dec 28. pii: S0021-9258(21)01357-0. [Epub ahead of print] 101547
      Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as tricarboxylic cycle metabolite). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research.
    Keywords:  Gas trap; adipocyte; bicarbonate; carbon dioxide; cell metabolism; glucose; oxidation
  21. Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01651-X. [Epub ahead of print]37(13): 110155
      During somatic reprogramming, cellular energy metabolism fundamentally switches from predominantly mitochondrial oxidative phosphorylation toward glycolysis. This metabolic reprogramming, also called the Warburg effect, is critical for the induction of pluripotency, but its molecular mechanisms remain poorly defined. Notably, SIRT2 is consistently downregulated during the reprogramming process and regulates glycolytic switch. Here, we report that downregulation of SIRT2 increases acetylation of mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) at Lys175, resulting in activation of extracellular signal-regulated kinases (ERKs) and subsequent activation of the pro-fission factor dynamin-related protein 1 (DRP1). In parallel, downregulation of SIRT2 hyperacetylates the serine/threonine protein kinase AKT1 at Lys20 in a non-canonical way, activating DRP1 and metabolic reprogramming. Together, our study identified two axes, SIRT2-MEK1-ERK-DRP1 and SIRT2-AKT1-DRP1, that critically link mitochondrial dynamics and oxidative phosphorylation to the somatic reprogramming process. These upstream signals, together with SIRT2's role in glycolytic switching, may underlie the Warburg effect observed in human somatic cell reprogramming.
    Keywords:  AKT1; DRP1; MEK1-ERK axis; OXPHOS; SIRT2; Warburg-like effect; human somatic cell reprogramming; induced pluripotent stem cells; metabolic reprogramming; mitochondrial remodeling
  22. Front Nutr. 2021 ;8 783164
      Anaerobic glycolysis is the process by which glucose is broken down into pyruvate and lactate and is the primary metabolic pathway in sepsis. The pyruvate dehydrogenase complex (PDHC) is a multienzyme complex that serves as a critical hub in energy metabolism. Under aerobic conditions, pyruvate translocates to mitochondria, where it is oxidized into acetyl-CoA through the activation of PDHC, thereby accelerating aerobic oxidation. Both phosphorylation and acetylation affect PDHC activity and, consequently, the regulation of energy metabolism. The mechanisms underlying the protective effects of PDHC in sepsis involve the regulation on the balance of lactate, the release of inflammatory mediators, the remodeling of tricarboxylic acid (TCA) cycle, as well as on the improvement of lipid and energy metabolism. Therapeutic drugs that target PDHC activation for sepsis treatment include dichloroacetate, thiamine, amrinone, TNF-binding protein, and ciprofloxacin. In this review, we summarize the recent findings regarding the metabolic regulation of PDHC in sepsis and the therapies targeting PDHC for the treatment of this condition.
    Keywords:  glycolysis; metabolism; pyruvate dehydrogenase complex; sepsis; therapy
  23. Methods Mol Biol. 2022 ;2445 99-115
      Autophagy and autophagy-associated genes are implicated in a growing list of cellular, physiological, and pathophysiological processes and conditions. Therefore, it is ever more important to be able to reliably monitor and quantify autophagic activity. Whereas autophagic markers, such as LC3 can provide general indications about autophagy, specific and accurate detection of autophagic activity requires assessment of autophagic cargo flux. Here, we provide protocols on how to monitor bulk and selective autophagy by the use of inducible expression of exogenous probes based on the fluorescent coral protein Keima. To exemplify and demonstrate the power of this system, we provide data obtained by analyses of cytosolic and mitochondrially targeted Keima probes in human retinal epithelial cells treated with the mTOR-inhibitor Torin1 or with the iron chelator deferiprone (DFP). Our data indicate that Torin1 induces autophagic flux of cytosol and mitochondria to a similar degree, that is, compatible with induction of bulk autophagy, whereas DFP induces a highly selective form of mitophagy that efficiently excludes cytosol.
    Keywords:  Autophagic cargo flux; Autophagy; Bulk autophagy; Deferiprone; LDHB-mKeima; Mito-mKeima; Mitophagy; Selective autophagy; Torin1; mKeima
  24. J Mol Cell Cardiol. 2021 Dec 23. pii: S0022-2828(21)00232-7. [Epub ahead of print]165 9-18
      ATP synthase (F1Fo) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F1Fo transports exclusively H+, our laboratory has demonstrated that, in addition to H+, F1Fo ATP synthase transports a significant fraction of ΔΨm-driven charge as K+ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the 'core' mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K+/H+ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F1Fo ATP synthase confers mitochondria with a significant energetic advantage compared to K+ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K+-cycling mechanism requires a KHEmito that exchanges matrix K+ for intermembrane space H+, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4➔3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the 'core' mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨm, mitochondrial K+ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K+ concentration. The model also explores the role played by KHEmito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K+ and H+ transport through the F1Fo ATP synthase.
    Keywords:  Energy supply-demand matching; F(1)F(o) ATP synthase; Mitochondrial K(+) uptake; Mitochondrial K(+)/H(+) exchanger
  25. Curr Opin Syst Biol. 2021 Dec;28 None
      Metabolites are prone to damage, either via enzymatic side reactions, which collectively form the underground metabolism, or via spontaneous chemical reactions. The resulting non-canonical metabolites that can be toxic, are mended by dedicated "metabolite repair enzymes." Deficiencies in the latter can cause severe disease in humans, whereas inclusion of repair enzymes in metabolically engineered systems can improve the production yield of value-added chemicals. The metabolite damage and repair loops are typically not yet included in metabolic reconstructions and it is likely that many remain to be discovered. Here, we review strategies and associated challenges for unveiling non-canonical metabolites and metabolite repair enzymes, including systematic approaches based on high-resolution mass spectrometry, metabolome-wide side-activity prediction, as well as high-throughput substrate and phenotypic screens.
    Keywords:  Metabolite repair enzymes; Non-canonical metabolites; Underground metabolism; Untargeted metabolomics
  26. Proc Natl Acad Sci U S A. 2022 Jan 04. pii: e2023328119. [Epub ahead of print]119(1):
      Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2 •-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2 •- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2 •- toxicity, which only requires <1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.
    Keywords:  glycolysis; oxygen sensing; pentose phosphate pathway; redox signaling; superoxide dismutase
  27. Mol Cell. 2021 Dec 20. pii: S1097-2765(21)01038-8. [Epub ahead of print]
      Ferroptosis is a unique type of non-apoptotic cell death resulting from the unrestrained occurrence of peroxidized phospholipids, which are subject to iron-mediated production of lethal oxygen radicals. This cell death modality has been detected across many organisms, including in mammals, where it can be used as a defense mechanism against pathogens or even harnessed by T cells to sensitize tumor cells toward effective killing. Conversely, ferroptosis is considered one of the main cell death mechanisms promoting degenerative diseases. Emerging evidence suggests that ferroptosis represents a vulnerability in certain cancers. Here, we critically review recent advances linking ferroptosis vulnerabilities of dedifferentiating and persister cancer cells to the dependency of these cells on iron, a potential Achilles heel for small-molecule intervention. We provide a perspective on the mechanisms reliant on iron that contribute to the persister cancer cell state and how this dependency may be exploited for therapeutic benefits.
  28. Compr Physiol. 2021 Dec 29. 12(1): 2769-2798
      Nearly every system within the body contains an intrinsic cellular circadian clock. The circadian clock contributes to the regulation of a variety of homeostatic processes in mammals through the regulation of gene expression. Circadian disruption of physiological systems is associated with pathophysiological disorders. Here, we review the current understanding of the molecular mechanisms contributing to the known circadian rhythms in physiological function. This article focuses on what is known in humans, along with discoveries made with cell and rodent models. In particular, the impact of circadian clock components in metabolic, cardiovascular, endocrine, musculoskeletal, immune, and central nervous systems are discussed. © 2021 American Physiological Society. Compr Physiol 11:1-30, 2021.
  29. Cell. 2021 Dec 22. pii: S0092-8674(21)01426-4. [Epub ahead of print]
    Immunoprofiler Consortium
      Cancers display significant heterogeneity with respect to tissue of origin, driver mutations, and other features of the surrounding tissue. It is likely that individual tumors engage common patterns of the immune system-here "archetypes"-creating prototypical non-destructive tumor immune microenvironments (TMEs) and modulating tumor-targeting. To discover the dominant immune system archetypes, the University of California, San Francisco (UCSF) Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub-compartments uncovered dominant patterns of immune composition across cancers. These archetypes were profound insofar as they also differentiated tumors based upon unique immune and tumor gene-expression patterns. They also partitioned well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune organization and provides a rational path forward to learn how to modulate these to improve therapy.
    Keywords:  Pan Cancer analysis; immune profiling; solid tumor microenvironement; system immunology; tumor immunology; unsupervised clustering
  30. Elife. 2021 Dec 31. pii: e70016. [Epub ahead of print]10
      Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.
    Keywords:  E. coli; molecular biophysics; structural biology