bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒10‒02
38 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. FEBS Lett. 2022 Sep 30.
      Complex I converts oxidoreduction energy into a proton electrochemical gradient across the inner mitochondrial or bacterial cell membrane. This gradient is the primary source of energy for aerobic synthesis of ATP. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) by ubiquinone (Q) yields NAD+ and ubiquinol (QH2 ), which is tightly coupled to translocation of four protons from the negatively to the positively charged side of the membrane. Electrons from NADH oxidation reach the iron-sulfur centre N2 positioned near the bottom of a tunnel that extends ca. 30Å from the membrane domain into the hydrophilic domain of the complex. The tunnel is occupied by ubiquinone, which can take a distal position near the N2 centre, or proximal positions closer to the membrane. Here, we review important structural, kinetic and thermodynamic properties of ubiquinone that define its role in complex I function. We suggest that this function exceeds that of a mere substrate or electron acceptor, and propose that ubiquinone may be the redox element of complex I coupling electron transfer to proton translocation.
    Keywords:  energy conservation; mitochondria; oxidative phosphorylation; proton pumping
  2. Sci Adv. 2022 Sep 30. 8(39): eabq0117
      The fate of pyruvate is a defining feature in many cell types. One major fate is mitochondrial entry via the mitochondrial pyruvate carrier (MPC). We found that diffuse large B cell lymphomas (DLBCLs) consume mitochondrial pyruvate via glutamate-pyruvate transaminase 2 to enable α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid cycle in DLBCLs. As a result, MPC inhibition led to decreased glutaminolysis in DLBCLs, opposite to previous observations in other cell types. We also found that MPC inhibition or genetic depletion decreased DLBCL proliferation in an extracellular matrix (ECM)-like environment and xenografts, but not in a suspension environment. Moreover, the metabolic profile of DLBCL cells in ECM is markedly different from cells in a suspension environment. Thus, we conclude that the synergistic consumption and assimilation of glutamine and pyruvate enables DLBCL proliferation in an extracellular environment-dependent manner.
  3. Nature. 2022 Sep 28.
      CD4+ T cell differentiation requires metabolic reprogramming to fulfil the bioenergetic demands of proliferation and effector function, and enforce specific transcriptional programmes1-3. Mitochondrial membrane dynamics sustains mitochondrial processes4, including respiration and tricarboxylic acid (TCA) cycle metabolism5, but whether mitochondrial membrane remodelling orchestrates CD4+ T cell differentiation remains unclear. Here we show that unlike other CD4+ T cell subsets, T helper 17 (TH17) cells have fused mitochondria with tight cristae. T cell-specific deletion of optic atrophy 1 (OPA1), which regulates inner mitochondrial membrane fusion and cristae morphology6, revealed that TH17 cells require OPA1 for its control of the TCA cycle, rather than respiration. OPA1 deletion amplifies glutamine oxidation, leading to impaired NADH/NAD+ balance and accumulation of TCA cycle metabolites and 2-hydroxyglutarate-a metabolite that influences the epigenetic landscape5,7. Our multi-omics approach revealed that the serine/threonine kinase liver-associated kinase B1 (LKB1) couples mitochondrial function to cytokine expression in TH17 cells by regulating TCA cycle metabolism and transcriptional remodelling. Mitochondrial membrane disruption activates LKB1, which restrains IL-17 expression. LKB1 deletion restores IL-17 expression in TH17 cells with disrupted mitochondrial membranes, rectifying aberrant TCA cycle glutamine flux, balancing NADH/NAD+ and preventing 2-hydroxyglutarate production from the promiscuous activity of the serine biosynthesis enzyme phosphoglycerate dehydrogenase (PHGDH). These findings identify OPA1 as a major determinant of TH17 cell function, and uncover LKB1 as a sensor linking mitochondrial cues to effector programmes in TH17 cells.
  4. Clin Lymphoma Myeloma Leuk. 2022 Oct;pii: S2152-2650(22)01253-8. [Epub ahead of print]22 Suppl 2 S229
      CONTEXT: Ferroptosis, a form of non-apoptotic cell death regulated by iron-dependent lipid peroxidation, has drawn extensive attention as a potential anti-cancer strategy. However, it remains to be explored in hematologic malignancies. We here investigate the molecular mechanisms of ferroptosis in acute myeloid leukemia (AML) and its therapeutic potential of co-targeting mitochondrial respiration.RESULTS: Analyses of publicly available databases (TCGA, DepMap) showed that AML patients with higher mRNA expression of major anti-ferroptotic genes had significantly shorter survival and that leukemia cells are one of the cell types that are highly dependent on GPX4 among other cancers. This suggests the potential prognostic impact and relevance in therapeutics of this pathway in AML. Indeed, pharmacological inhibition or genetic knockdown of a well-established anti-ferroptosis regulator GPX4 induced profound ferroptosis, evidenced by dependency on iron and lipid peroxidation. Importantly, the induced cell death was agonistic of TP53 mutational status. As a potentially AML-specific mechanism that is unusual in solid tumor models, we found that GPX4 inhibition-induced ferroptosis was efficiently blocked by the mitochondria-targeted ubiquinone MitoQ in AML cells. Ubiquinone is an endogenous antioxidant protecting cells from lipid peroxidation, and in the mitochondria, the respiratory chain is essential for the recycling of ubiquinone. Thus, we hypothesized that mitochondria protect AML cells from ferroptosis by activating the ubiquinone cycle. Consistently, mitochondrial DNA-depleted HL60 Rho0 cells, which lack mitochondrial respiration, were more sensitive to ML210 compared to the parental cells. To translate this finding to a pharmacological approach, we utilized the imipridone ONC201, which hyperactivates mitochondrial protease ClpP to degrade mitochondrial proteins, including the respiratory chain complex, and exerts cancer-selective lethality (Ishizawa et al. Cancer Cell 2019). Indeed, the combination of ONC201 and ML210 resulted in synergistic anti-leukemia effects in primary AML cells. The combinatorial effect was also validated by utilizing genetically engineered AML cells (genetic knockdown of GPX4 or overexpression of hyperactivated mutant ClpP).
    CONCLUSIONS: GPX4 inhibition induces ferroptosis involving mitochondrial redox machinery in AML. Combinatorial targeting of mitochondrial respiration with GPX4 inhibition exerts synergistic anti-leukemia effects. Further studies are in progress to assess the molecular mechanisms and the in-vivo efficacy of the combinatorial treatments.
    Keywords:  AML; ClpP; GPX4; ferroptosis; mitochondrial respiration
  5. Elife. 2022 Sep 26. pii: e80919. [Epub ahead of print]11
      Mitochondrial electron transport chain (ETC) dysfunction due to mutations in the nuclear or mitochondrial genome is a common cause of metabolic disease in humans and displays striking tissue specificity depending on the affected gene. The mechanisms underlying tissue specific phenotypes are not understood. Complex I (cI) is classically considered the entry point for electrons into the ETC, and in vitro experiments indicate that cI is required for basal respiration and maintenance of the NAD+/NADH ratio, an indicator of cellular redox status. This finding has largely not been tested in vivo. Here, we report that mitochondrial complex I is dispensable for homeostasis of the adult mouse liver; animals with hepatocyte-specific loss of cI function display no overt phenotypes or signs of liver damage, and maintain liver function, redox and oxygen status. Further analysis of cI-deficient livers did not reveal significant proteomic or metabolic changes, indicating little to no compensation is required in the setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult hepatocytes results in decreased liver function, impaired oxygen handling, steatosis, and liver damage, accompanied by significant metabolomic and proteomic perturbations. Our results support a model whereby complex I loss is tolerated in the mouse liver because hepatocytes use alternative electron donors to fuel the mitochondrial ETC.
    Keywords:  cell biology; genetics; genomics; mouse
  6. Biochemistry (Mosc). 2022 Aug;87(8): 683-688
      The conclusions made in the three papers published in Function by Juhaszova et al. [Function, 3, 2022, zqab065, zqac001, zqac018], can be seen as a breakthrough in bioenergetics and mitochondrial medicine. For more than half a century, it has been believed that mitochondrial energetics is solely protonic and is based on the generation of electrochemical potential of hydrogen ions across the inner mitochondrial membrane upon oxidation of respiratory substrates, resulting in the generation of ATP via reverse transport of protons through the ATP synthase complex. Juhaszova et al. demonstrated that ATP synthase transfers not only protons, but also potassium ions, with the generation of ATP. This mechanism seems logical, given the fact that in eukaryotic cells, the concentration of potassium ions is several million times higher than the concentration of protons. The transport of K+ through the ATP synthase was enhanced by the activators of mitochondrial ATP-dependent K+ channel (mK/ATP), leading to the conclusion that ATP synthase is the material essence of mK/ATP. Beside ATP generation, the transport of osmotically active K+ to the mitochondrial matrix is accompanied by water entry to the matrix, leading to an increase in the matrix volume and activation of mitochondrial respiration with the corresponding increase in the ATP synthesis, which suggests an advantage of such transport for energy production. The driving force for K+ transport into the mitochondria is the membrane potential; an excess of K+ is exported from the matrix by the hypothetical K+/H+ exchangers. Inhibitory factor 1 (IF1) plays an important role in the activation of mK/ATP by increasing the chemo-mechanical efficiency of ATP synthase, which may be a positive factor in the protective anti-ischemic signaling.
    Keywords:  ATP synthase; bioenergetics; ischemia; membrane potential; mitochondria; mitochondrial ATP-dependent potassium channel; potassium ions; protons; rotation; transport
  7. Biochemistry (Mosc). 2022 Aug;87(8): 812-822
      Pyrrolomycins C (Pyr_C) and D (Pyr_D) are antibiotics produced by Actinosporangium and Streptomyces. The mechanism of their antimicrobial activity consists in depolarization of bacterial membrane, leading to the suppression of bacterial bioenergetics through the uncoupling of oxidative phosphorylation, which is based on the protonophore action of these antibiotics [Valderrama et al., Antimicrob. Agents Chemother. (2019) 63, e01450]. Here, we studied the effect of pyrrolomycins on the isolated rat liver mitochondria. Pyr_C was found to be more active than Pyr_D and uncoupled mitochondria in the submicromolar concentration range, which was observed as the mitochondrial membrane depolarization and stimulation of mitochondrial respiration. In the case of mitoplasts (isolated mitochondria with impaired outer membrane integrity), the difference in the action of Pyr_C and Pyr_D was significantly less pronounced. By contrast, in inverted submitochondrial particles (SMPs), Pyr_D was more active as an uncoupler, which caused collapse of the membrane potential even at the nanomolar concentrations. The same ratio of the protonophoric activity of Pyr_D and Pyr_C was obtained by us on liposomes loaded with the pH indicator pyranine. The protonophore activity of Pyr_D in the planar bilayer lipid membranes (BLMs) was maximal at ~pH 9, i.e., at pH values close to pKa of this compound. Pyr_D functions as a typical anionic protonophore; its activity in the BLM could be reduced by the addition of the dipole modifier phloretin. The difference between the protonophore activity of Pyr_C and Pyr_D in the mitochondria and BLMs can be attributed to a higher ability of Pyr_C to penetrate the outer mitochondrial membrane.
    Keywords:  membrane potential; mitochondria; protonophore; pyrrolomycin; respiration; uncoupler
  8. Science. 2022 Sep 30. 377(6614): 1519-1529
      Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.
  9. Autophagy. 2022 Sep 28.
      Mitochondria rely on efficient protein import across their membranes for optimal function. We have shown that numerous mitochondrial stressors all converge on a common pathway disrupting this import efficiency. We identified a novel pathway involving NLRX1 and RRBP1 that responds to this import stress, resulting in LC3 lipidation, mitochondrial targeting and ultimate degradation. Furthermore, we demonstrated the relevance of this mitophagy axis in murine skeletal muscle following acute exercise. We propose that mitochondrial protein import stress is an underlying, common trigger for mitophagy, offering a novel avenue for therapeutic exploration and mechanistic insight.
    Keywords:  Autophagy; NLR; exercise; import; mitochondria; mitophagy; proteostasis
  10. J Biol Chem. 2022 Sep 23. pii: S0021-9258(22)00976-0. [Epub ahead of print] 102533
      Mitochondrial morphology and dynamics maintain mitochondrial integrity by regulating its size, shape, distribution, and connectivity, thereby modulating various cellular processes. Several studies have established a functional link between mitochondrial dynamics, mitophagy, and cell death, but further investigation is needed to identify specific proteins involved in mitochondrial dynamics. Any alteration in the integrity of the mitochondria has severe ramifications that include disorders like cancer and neurodegeneration. In this study, we used budding yeast as a model organism and found that Pil1, the major component of the eisosome complex, also localizes to the periphery of mitochondria. Interestingly, the absence of Pil1 causes the branched tubular morphology of mitochondria to be abnormally fused or aggregated, whereas its overexpression leads to mitochondrial fragmentation. Most importantly, pil1Δ cells are defective in mitophagy and bulk autophagy, resulting in elevated levels of ROS and protein aggregates. In addition, we show that pil1Δ cells are more prone to cell death. Yeast two-hybrid analysis and co-immunoprecipitations show the interaction of Pil1 with two major proteins in mitochondrial fission, Fis1 and Dnm1. Additionally, our data suggest that the role of Pil1 in maintaining mitochondrial shape is dependent on Fis1 and Dnm1, but it functions independently in mitophagy and cell death pathways. Together, our data suggest that Pil1, an eisosome protein, is a novel regulator of mitochondrial morphology, mitophagy, and cell death.
    Keywords:  Saccharomyces cerevisiae; autophagy; cell death; mitochondria; mitophagy; protein aggregation; reactive oxygen species (ROS)
  11. Biochimie. 2022 Sep 26. pii: S0300-9084(22)00234-6. [Epub ahead of print]
      Mitoregulin (Mtln) is a recently identified 56 aminoacid long mitochondrial peptide conserved in vertebrates. Mtln is known to enhance function of respiratory complex I, which is likely mediated by modulation of lipid composition. To address an influence of Mtln gene on the metabolism we created knockout mice deficient in Mtln gene. In line with accumulation of triglycerides observed earlier on a model of Mtln knockout cell lines, we observed Mtln KO mice to develop obesity on a high fat diet. An increased weight gain could be attributed to enhanced fat accumulation according to the magnetic resonance live imaging. In addition, Mtln KO mice demonstrate elevated serum triglycerides and other oxidation substrates accompanied by an exhaustion of tricarboxylic acids cycle intermediates, suggesting suboptimal oxidation of respiration substrates by mitochondria lacking Mtln.
    Keywords:  Metabolism; Mitochondria; Oxidative phosphorylation; Short open reading frame; Small peptide
  12. Front Cell Dev Biol. 2022 ;10 984245
      Mitochondria are the primary sites for cellular energy production and are required for many essential cellular processes. Mitochondrial DNA (mtDNA) is a 16.6 kb circular DNA molecule that encodes only 13 gene products of the approximately 90 different proteins of the respiratory chain complexes and an estimated 1,200 mitochondrial proteins. MtDNA is, however, crucial for organismal development, normal function, and survival. MtDNA maintenance requires mitochondrially targeted nuclear DNA repair enzymes, a mtDNA replisome that is unique to mitochondria, and systems that control mitochondrial morphology and quality control. Here, we provide an overview of the current literature on mtDNA repair and transcription machineries and discuss how dynamic functional interactions between the components of these systems regulate mtDNA maintenance and transcription. A profound understanding of the molecular mechanisms that control mtDNA maintenance and transcription is important as loss of mtDNA integrity is implicated in normal process of aging, inflammation, and the etiology and pathogenesis of a number of diseases.
    Keywords:  DNA repair; base excision repair (BER); base excision repair (BER)glycosylases; mitochdrial damage; mitochondria; transcription
  13. Med Oncol. 2022 Sep 29. 39(12): 227
      Metabolic reprogramming wherein the cancer cells exhibit altered energetics is a hallmark of cancer. Although recent discoveries have enhanced our understanding of tumor metabolism, the therapeutic utility of targeting tumor metabolism is not yet realized. Glutamine, a non-essential amino acid, plays a critical role in regulating tumor metabolism and provides an alternative tumor energy source. In this study, we investigate the molecular mechanism regulated by glutamine and elucidate if targeting glutamine metabolism would enhance the efficacy of cancer chemotherapy. Using clonogenic and cell cycle analysis, we found that deprivation of glutamine suppress the growth of cancer cells. Mechanistically we demonstrate that glutamine stabilizes myc by preventing its ubiquitination through alpha-ketoglutarate. Inhibition of glutamine metabolism enhanced the sensitivity of tumor cells to chemotherapeutic agent paclitaxel. Our results delineate the mechanism behind glutamine-induced myc stabilization, and they provide a viable strategy to target cancer with a glutamine metabolism inhibitor in the clinic.
    Keywords:  Cancer; Glutamine; Metabolism; Myc
  14. Sci Rep. 2022 Sep 27. 12(1): 16066
      Mitochondrial metabolism varies significantly between individuals of the same species and can influence animal performance, such as growth. However, growth rate is usually determined before the mitochondrial assay. The hypothesis that natural variation in mitochondrial metabolic traits is linked to differences in both previous and upcoming growth remains untested. Using biopsies to collect tissue in a non-lethal manner, we tested this hypothesis in a fish model (Dicentrarchus labrax) by monitoring individual growth rate, measuring mitochondrial metabolic traits in the red muscle, and monitoring the growth of the same individuals after the mitochondrial assay. Individual variation in growth rate was consistent before and after the mitochondrial assay; however, the mitochondrial traits that explained growth variation differed between the growth rates determined before and after the mitochondrial assay. While past growth was correlated with the activity of the cytochrome c oxidase, a measure of mitochondrial density, future growth was linked to mitochondrial proton leak respiration. This is the first report of temporal shift in the relationship between growth rate and mitochondrial metabolic traits, suggesting an among-individual variation in temporal changes in mitochondrial traits. Our results emphasize the need to evaluate whether mitochondrial metabolic traits of individuals can change over time.
  15. Cancer Res. 2022 Sep 26. pii: CAN-22-1039. [Epub ahead of print]
      Autophagy is a conserved catabolic process that maintains cellular homeostasis. Autophagy supports lung tumorigenesis and is a potential therapeutic target in lung cancer. A better understanding of the importance of tumor cell-autonomous versus systemic autophagy in lung cancer could facilitate clinical translation of autophagy inhibition. Here, we exploited inducible expression of Atg5 shRNA to temporally control Atg5 levels and generate reversible tumor-specific and systemic autophagy loss mouse models of KrasG12D/+;p53-/- (KP) non-small cell lung cancer (NSCLC). Transient suppression of systemic but not tumor Atg5 expression significantly reduced established KP lung tumor growth without damaging normal tissues. In vivo 13C isotope tracing and metabolic flux analyses demonstrated that systemic Atg5 knockdown specifically led to reduced glucose and lactate uptake. As a result, carbon flux from glucose and lactate to major metabolic pathways, including the tricarboxylic acid cycle, glycolysis, and serine biosynthesis, was significantly reduced in KP NSCLC following systemic autophagy loss. Furthermore, systemic Atg5 knockdown increased tumor T cell infiltration, leading to T cell-mediated tumor killing. Importantly, intermittent transient systemic Atg5 knockdown, which resembles what would occur during autophagy inhibition for cancer therapy, significantly prolonged lifespan of KP lung tumor-bearing mice, resulting in recovery of normal tissues but not tumors. Thus, systemic autophagy supports the growth of established lung tumors by promoting immune evasion and sustaining cancer cell metabolism for energy production and biosynthesis, and the inability of tumors to recover from loss of autophagy provides further proof of concept that inhibition of autophagy is a valid approach to cancer therapy.
  16. Cell Rep. 2022 Sep 27. pii: S2211-1247(22)01256-6. [Epub ahead of print]40(13): 111415
      Sphingolipids play important signaling and structural roles in cells. Here, we find that during de novo sphingolipid biosynthesis, a toxic metabolite is formed with critical implications for cancer cell survival. The enzyme catalyzing the first step in this pathway, serine palmitoyltransferase complex (SPT), is upregulated in breast and other cancers. SPT is dispensable for cancer cell proliferation, as sphingolipids can be salvaged from the environment. However, SPT activity introduces a liability as its product, 3-ketodihydrosphingosine (3KDS), is toxic and requires clearance via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). In cancer cells, but not normal cells, targeting KDSR induces toxic 3KDS accumulation leading to endoplasmic reticulum (ER) dysfunction and loss of proteostasis. Furthermore, the antitumor effect of KDSR disruption can be enhanced by increasing metabolic input (via high-fat diet) to allow greater 3KDS production. Thus, de novo sphingolipid biosynthesis entails a detoxification requirement in cancer cells that can be therapeutically exploited.
    Keywords:  CP: Cancer; CP: Metabolism; cancer metabolism; cancer therapy; endoplasmic reticulum; ketodihydrosphingosine reductase; serine palmitoyltransferase
  17. J Exp Clin Cancer Res. 2022 Sep 24. 41(1): 283
      BACKGROUND: Alternative treatment strategies in melanoma beyond immunotherapy and mutation-targeted therapy are urgently needed. Wild-type isocitrate dehydrogenase 1 (wtIDH1) has recently been implicated as a metabolic dependency in cancer. The enzyme protects cancer cells under metabolic stress, including nutrient limited conditions in the tumor microenvironment. Specifically, IDH1 generates NADPH to maintain redox homeostasis and produces α-ketoglutarate to support mitochondrial function through anaplerosis. Herein, the role of wtIDH1 in melanoma is further explored.METHODS: The expression of wtIDH1 was determined by qRT-PCR, and Western blot in melanoma cell lines and the effect of wtIDH1 on metabolic reprogramming in melanoma was interrogated by LC-MS. The impact of wtIDH1 inhibition alone and in combination with chemotherapy was determined in cell culture and mouse melanoma models.
    RESULTS: Melanoma patients express higher levels of the wtIDH1 enzyme compared to normal skin tissue, and elevated wtIDH1 expression portends poor patient survival. Knockdown of IDH1 by RNA interference inhibited cell proliferation and migration under low nutrient levels. Suppression of IDH1 expression in melanoma also decreased NADPH and glutathione levels, resulting in increased reactive oxygen species. An FDA-approved inhibitor of mutant IDH1, ivosidenib (AG-120), exhibited potent anti-wtIDH1 properties under low magnesium and nutrient levels, reflective of the tumor microenvironment in natura. Thus, similar findings were replicated in murine models of melanoma. In light of the impact of wtIDH1 inhibition on oxidative stress, enzyme blockade was synergistic with conventional anti-melanoma chemotherapy in pre-clinical models.
    CONCLUSIONS: These results demonstrate the clinical potential of wtIDH1 inhibition as a novel and readily available combination treatment strategy for patients with advanced and refractory melanoma. Schematic shows increased wild-type IDH1 expression and activity as an adaptive response to metabolic stress induced by chemotherapy.
    Keywords:  Chemoresistance; Combination therapy; IDH1; Ivosidenib; Melanoma
  18. Toxicol Rep. 2022 ;9 1566-1573
      Previous studies have shown that inhibition or depletion of N-acetyltransferase 1 (NAT1) in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. To understand molecular and cellular processes that NAT1 contributes to and generate novel hypotheses in regard to NAT1's role in breast cancer, we performed an unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines. Among 4890 proteins identified, 737 proteins were found significantly (p < 0.01) upregulated, and 651 proteins were significantly (p < 0.01) downregulated in both NAT1 KO cell lines. We performed enrichment analyses to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set. Among the proteins upregulated in NAT1 KO cells, pathways associated with MHC (major histocompatibility complex) I-mediated antigen presentation were significantly enriched. This raises an interesting and new hypothesis that upregulation of NAT1 in breast cancer cells may aid them evade immune detection. Multiple pathways involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells, consistent with reported observations that NAT1 KO cells exhibit a slower growth rate both in vitro and in vivo. Thus, mitochondrial dysfunction in NAT1 KO cells likely contributes to growth retardation.
    Keywords:  ATP synthase; Antigen presentation; Cell cycle; MHC-I, major histocompatibility complex I; Mitochondria; NAT1, arylamine N-acetyltransferase 1; Proteomics
  19. NPJ Breast Cancer. 2022 Sep 26. 8(1): 111
      Recurrent cancer cells that evade therapy is a leading cause of death in breast cancer patients. This risk is high for women showing an overexpression of human epidermal growth factor receptor 2 (Her2). Cells that persist can rely on different substrates for energy production relative to their primary tumor counterpart. Here, we characterize metabolic reprogramming related to tumor dormancy and recurrence in a doxycycline-induced Her2+/Neu model of breast cancer with varying times to recurrence using longitudinal fluorescence microscopy. Glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE) imaging metabolically phenotype mammary tumors as they transition to regression, dormancy, and recurrence. "Fast-recurrence" tumors (time to recurrence ~55 days), transition from glycolysis to mitochondrial metabolism during regression and this persists upon recurrence. "Slow-recurrence" tumors (time to recurrence ~100 days) rely on both glycolysis and mitochondrial metabolism during recurrence. The increase in mitochondrial activity in fast-recurrence tumors is attributed to a switch from glucose to fatty acids as the primary energy source for mitochondrial metabolism. Consequently, when fast-recurrence tumors receive treatment with a fatty acid inhibitor, Etomoxir, tumors report an increase in glucose uptake and lipid synthesis during regression. Treatment with Etomoxir ultimately prolongs survival. We show that metabolic reprogramming reports on tumor recurrence characteristics, particularly at time points that are essential for actionable targets. The temporal characteristics of metabolic reprogramming will be critical in determining the use of an appropriate timing for potential therapies; namely, the notion that metabolic-targeted inhibition during regression reports long-term therapeutic benefit.
  20. Front Cell Dev Biol. 2022 ;10 918691
      Endoplasmic reticulum (ER) functions critically depend on a suitable ATP supply to fuel ER chaperons and protein trafficking. A disruption of the ability of the ER to traffic and fold proteins leads to ER stress and the unfolded protein response (UPR). Using structured illumination super-resolution microscopy, we revealed increased stability and lifetime of mitochondrial associated ER membranes (MAM) during ER stress. The consequent increase of basal mitochondrial Ca2+ leads to increased TCA cycle activity and enhanced mitochondrial membrane potential, OXPHOS, and ATP generation during ER stress. Subsequently, OXPHOS derived ATP trafficking towards the ER was increased. We found that the increased lifetime and stability of MAMs during ER stress depended on the mitochondrial fusion protein Mitofusin2 (MFN2). Knockdown of MFN2 blunted mitochondrial Ca2+ effect during ER stress, switched mitochondrial F1FO-ATPase activity into reverse mode, and strongly reduced the ATP supply for the ER during ER stress. These findings suggest a critical role of MFN2-dependent MAM stability and lifetime during ER stress to compensate UPR by strengthening ER ATP supply by the mitochondria.
    Keywords:  ER stress; mitochondria; mitochondria-associated membranes (MAM); mitochondrial Ca2+; mitofusin 2
  21. Sci Adv. 2022 Sep 30. 8(39): eabq5575
      The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response-dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11, and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress-induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.
  22. Redox Biol. 2022 Sep 24. pii: S2213-2317(22)00263-4. [Epub ahead of print]57 102491
      Ascorbate is a crucial antioxidant and essential cofactor of biosynthetic and regulatory enzymes. Unlike humans, mice can synthesize ascorbate thanks to the key enzyme gulonolactone oxidase (Gulo). In the present study, we used the Gulo-/- mouse model, which cannot synthesize their own ascorbate to determine the impact of this vitamin on the liver proteome of specific subcellular organelles. We performed label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) global quantitative proteomic profiling to identify and quantify proteins in microsomal enriched liver extracts (MEE) from Gulo-/- mice treated with 0-0.4% (w/v) ascorbate in drinking water until the age of four months. Using a principal component analysis on normalized and imputed data of the label-free protein quantifications, a sex-based difference in MEE proteome profiles was observed for all the different ascorbate treated mice. Suboptimal hepatic ascorbate concentrations affected the levels of more proteins and hence biochemical processes in females than in males. Nevertheless, Pearson correlation analyses revealed that the MS intensities of various proteins involved in complement activation inversely correlated with liver ascorbate concentrations in both Gulo-/- males and females. Moreover, the correlation analyses also indicated that several proteins in the mitochondrial complex III of the electron transport chain positively correlated with liver ascorbate concentrations in both Gulo-/- females and males. Consequently, the mitochondrial complex III activity in Gulo-/- female and male mice treated with suboptimal hepatic concentrations of ascorbate was significantly lower than Gulo-/- mice treated with optimal ascorbate concentration. Finally, the whole liver of ascorbate-deficient Gulo-/- mice exhibited lower ATP levels and increased reactive oxygen species. These findings provide new information on how ascorbate deficiency potentially induces mitochondrial dysfunction in the liver of mice.
    Keywords:  Gulonolactone oxidase; Liver; Mass spectrometry; Mitochondrial complex III; Mouse; Reactive oxygen species; Sex-related differences; Vitamin C
  23. Pharmacol Res. 2022 Sep 26. pii: S1043-6618(22)00412-1. [Epub ahead of print] 106466
      Until recently it was thought that most humans only harbor one type of mitochondrial DNA (mtDNA), however, deep sequencing and single-cell analysis has shown the converse - that mixed populations of mtDNA (heteroplasmy) are the norm. This is important because heteroplasmy levels can change dramatically during transmission in the female germ line, leading to high levels causing severe mitochondrial diseases. There is also emerging evidence that low level mtDNA mutations contribute to common late onset diseases such as neurodegenerative disorders and cardiometabolic diseases because the inherited mutation levels can change within developing organs and non-dividing cells over time. Initial predictions suggested that the segregation of mtDNA heteroplasmy was largely stochastic, with an equal tendency for levels to increase or decrease. However, transgenic animal work and single-cell analysis have shown this not to be the case during germ-line transmission and in somatic tissues during life. Mutation levels in specific mtDNA regions can increase or decrease in different contexts and the underlying molecular mechanisms are starting to be unraveled. In this review we provide a synthesis of recent literature on the mechanisms of selection for and against mtDNA variants. We identify the most pertinent gaps in our understanding and suggest ways these could be addressed using state of the art techniques.
    Keywords:  mitochondria; mitophagy; mtDNA; mutant; selection; selfish
  24. Nat Commun. 2022 Sep 28. 13(1): 5696
      Fatty liver is a highly heterogenous condition driven by various pathogenic factors in addition to the severity of steatosis. Protein insufficiency has been causally linked to fatty liver with incompletely defined mechanisms. Here we report that fatty liver is a sulfur amino acid insufficient state that promotes metabolic inflexibility via limiting coenzyme A availability. We demonstrate that the nutrient-sensing transcriptional factor EB synergistically stimulates lysosome proteolysis and methionine adenosyltransferase to increase cysteine pool that drives the production of coenzyme A and glutathione, which support metabolic adaptation and antioxidant defense during increased lipid influx. Intriguingly, mice consuming an isocaloric protein-deficient Western diet exhibit selective hepatic cysteine, coenzyme A and glutathione deficiency and acylcarnitine accumulation, which are reversed by cystine supplementation without normalizing dietary protein intake. These findings support a pathogenic link of dysregulated sulfur amino acid metabolism to metabolic inflexibility that underlies both overnutrition and protein malnutrition-associated fatty liver development.
  25. Trends Cancer. 2022 Sep 21. pii: S2405-8033(22)00195-9. [Epub ahead of print]
      To thrive in a hypoxic and nutrient-limited tumor microenvironment, pancreatic ductal adenocarcinoma (PDAC) cells rewire their metabolism. Understanding PDAC cell metabolism may uncover vulnerabilities that can be targeted for improved therapy. Three recent studies find that the PDAC tumor microenvironment modulates the functional consequences of depleting the mitochondrially localized aspartate transaminase GOT2, thus providing new insights into the metabolism of this lethal cancer.
  26. Sci Rep. 2022 Sep 26. 12(1): 16028
      Metabolic programming of the innate immune cells known as dendritic cells (DCs) changes in response to different stimuli, influencing their function. While the mechanisms behind increased glycolytic metabolism in response to inflammatory stimuli are well-studied, less is known about the programming of mitochondrial metabolism in DCs. We used lipopolysaccharide (LPS) and interferon-β (IFN-β), which differentially stimulate the use of glycolysis and oxidative phosphorylation (OXPHOS), respectively, to identify factors important for mitochondrial metabolism. We found that the expression of peroxisome proliferator-activated receptor gamma co-activator 1β (PGC-1β), a transcriptional co-activator and known regulator of mitochondrial metabolism, decreases when DCs are activated with LPS, when OXPHOS is diminished, but not with IFN-β, when OXPHOS is maintained. We examined the role of PGC-1β in bioenergetic metabolism of DCs and found that PGC-1β deficiency indeed impairs their mitochondrial respiration. PGC-1β-deficient DCs are more glycolytic compared to controls, likely to compensate for reduced OXPHOS. PGC-1β deficiency also causes decreased capacity for ATP production at steady state and in response to IFN-β treatment. Loss of PGC-1β in DCs leads to increased expression of genes in inflammatory pathways, and reduced expression of genes encoding proteins important for mitochondrial metabolism and function. Collectively, these results demonstrate that PGC-1β is a key regulator of mitochondrial metabolism and negative regulator of inflammatory gene expression in DCs.
  27. Cell Mol Immunol. 2022 Sep 30.
      Serine metabolism is reportedly involved in immune cell functions, but whether and how serine metabolism regulates macrophage polarization remain largely unknown. Here, we show that suppressing serine metabolism, either by inhibiting the activity of the key enzyme phosphoglycerate dehydrogenase in the serine biosynthesis pathway or by exogenous serine and glycine restriction, robustly enhances the polarization of interferon-γ-activated macrophages (M(IFN-γ)) but suppresses that of interleukin-4-activated macrophages (M(IL-4)) both in vitro and in vivo. Mechanistically, serine metabolism deficiency increases the expression of IGF1 by reducing the promoter abundance of S-adenosyl methionine-dependent histone H3 lysine 27 trimethylation. IGF1 then activates the p38-dependent JAK-STAT1 axis to promote M(IFN-γ) polarization and suppress STAT6-mediated M(IL-4) activation. This study reveals a new mechanism by which serine metabolism orchestrates macrophage polarization and suggests the manipulation of serine metabolism as a therapeutic strategy for macrophage-mediated immune diseases.
    Keywords:  IGF1; Macrophage polarization; PHGDH; SAM; Serine metabolism; p38
  28. Front Mol Biosci. 2022 ;9 995421
      MitoNEET is a mitochondrial outer membrane protein that regulates energy metabolism, iron homeostasis, and production of reactive oxygen species in cells. Aberrant expression of mitoNEET in tissues has been linked to type II diabetes, neurodegenerative diseases, and several types of cancer. Structurally, the N-terminal domain of mitoNEET has a single transmembrane alpha helix that anchors the protein to mitochondrial outer membrane. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine and one histidine residues. Here we report that the reduced [2Fe-2S] cluster in the C-terminal cytosolic domain of mitoNEET (mitoNEET45-108) is able to bind nitric oxide (NO) without disruption of the cluster. Importantly, binding of NO at the reduced [2Fe-2S] cluster effectively inhibits the redox transition of the cluster in mitoNEET45-108. While the NO-bound [2Fe-2S] cluster in mitoNEET45-108 is stable, light excitation releases NO from the NO-bound [2Fe-2S] cluster and restores the redox transition activity of the cluster in mitoNEET45-108. The results suggest that NO may regulate the electron transfer activity of mitoNEET in mitochondrial outer membrane via reversible binding to its reduced [2Fe-2S] cluster.
    Keywords:  electron transfer (ET); iron-sulfur; mitochondia; nitric oxide; oxidation reduction
  29. Chem Biol Interact. 2022 Sep 23. pii: S0009-2797(22)00380-5. [Epub ahead of print] 110175
      Several members of the aldehyde dehydrogenase (ALDH) family, especially ALDH1 isoenzymes, have been identified as biomarkers of cancer stem cells (CSCs), a small subpopulation of oncogenic cells with self-renewal and multipotency capability. Consistent with this contention, cell populations with high ALDH enzymatic activity exhibit greater carcinogenic potential. It has been reported that ALDH1, especially ALDH1A1, serves as a valuable biomarker for colon CSCs. However, the functional roles of ALDHs in CSCs and solid tumors of the colon tissue is not fully understood. The aim of the present study was to identify molecular signature associated with high ALDH activity in human colorectal adenocarcinoma (COLO320DM) cells by proteomics profiling. Aldefluor™ assay was performed to sort COLO320DM cells exhibiting high (ALDHhigh) and low (ALDHlow) ALDH activity. Label-free quantitative proteomics analyses were conducted on these two cell populations. Proteomics profiling revealed a total of 229 differentially expressed proteins (DEPs) in ALDHhigh relative to ALDHlow cells, of which 182 were down-regulated and 47 were up-regulated. In agreement with previous studies, ALDH1A1 appeared to be the principal ALDH isozyme contributing to the Aldefluor™ assay activity in COLO320DM cells. Ingenuity pathway analysis of the proteomic datasets indicated that DEPs were associated with mitochondrial dysfunction, sirtuin signaling, oxidative phosphorylation and nucleotide excision repair. Our proteomics study predicts that high ALDH1A1 activity may be involved in these cellular pathways to promote a metabolic switch and cellular survival of CSCs.
  30. Clin Lymphoma Myeloma Leuk. 2022 Oct;pii: S2152-2650(22)01218-6. [Epub ahead of print]22 Suppl 2 S211-S212
      CONTEXT: Acute myeloid leukemia (AML) is an aggressive, heterogenous disease characterized by clonal expansion of immature myeloid blasts. For the last five decades, intensive induction chemotherapy was the primary treatment option for AML. The 5-year survival rate for younger patients following induction chemotherapy is 40%-50%, which is far superior to the dismal 10%-15% 5-year survival seen in older adult patients, who constitute most AML cases. The specific Bcl-2 antagonist venetoclax was approved in combination with Ara-C or azacitidine for newly diagnosed AML or older adult patients unable to tolerate intensive chemotherapy. However, responses are incomplete and relapse rates remain high, necessitating the development of novel treatment strategies to augment current therapies and improve outcomes. To this end, targeting dysregulated sphingolipid metabolism in AML shows promise. Once overlooked as mere structural membrane components, sphingolipids are now appreciated as important regulators of cell signaling and are involved in many hallmarks of cancer that are of critical importance in AML. Acid ceramidase (AC), a ceramide-catabolizing lipid hydrolase, is upregulated in AML and correlates with poorer patient survival. The breakdown of ceramide, a bona fide pro-death lipid, by AC promotes chemotherapeutic resistance and leukemic survival in part by upregulating Mcl-1, a known venetoclax resistance factor.OBJECTIVE: Our ongoing research aims to characterize the efficacy of venetoclax combined with AC inhibition in AML.
    RESULTS: SACLAC, a ceramide analog and irreversible AC inhibitor, augmented venetoclax sensitivity and synergistically decreased cell viability and induced apoptosis in AML cell lines with de novo and acquired venetoclax resistance. Remarkably, combinatorial SACLAC and venetoclax treatment was quantitatively more synergistic than standard venetoclax-containing AML treatment regimens (venetoclax+Ara-C and venetoclax+azacitidine) by Bliss analysis in over 60% of primary patient samples tested in vitro. The SACLAC and venetoclax regimen was less toxic to normal peripheral blood mononuclear cells and bone marrow cells relative to leukemic samples and exhibited toxicity towards these normal cell types comparable to venetoclax+Ara-C and venetoclax+azacitidine. Mechanistically, combination treatment potently increased ceramides, synergistically disrupted mitochondrial membrane potential, and depleted several proteins that inhibit apoptosis, including XIAP and cIAP1.
    CONCLUSIONS: Overall, these data provide the rationale for additional mechanistic and preclinical in-vivo studies targeting AC and Bcl-2 in AML.
    Keywords:  AML; Bcl-2; acid ceramidase; acute myeloid leukemia; ceramide; sphingolipids
  31. Mol Carcinog. 2022 Sep 26.
      Lentivirus-based transduction systems are widely used in biological science and cancer biology, including cancer immunotherapy. However, in in vivo transplanted tumor model, the immunogenicity of these transduced cells was not appropriately addressed. Here, we used empty vector-transduced mouse melanoma (B16) and carcinoma (lewis lung carcinoma) cells transplanted tumor model to study the immune response due to the transduction processes. We showed that the overall in vivo tumor growth rate gets reduced in transduced cells only in immune-competent mice but not in nude mice. This data indicate the involvement of the immune system in the in vivo tumor growth restriction in the transduced group. Further studies showed that specific activation of CD8+ T cells might be responsible for restricted tumor growth. Mechanistically, transduced tumor cells show the higher activity of type I interferon, which might play an essential role in this activation. Overall, our data indicate the modulation of the immune system by lentiviral vector transduced tumor cells, which required further studies to explore the mechanisms and better understand the biological significance. Our data also indicate the importance of considering the immunogenicity of transduced cells when analyzing in vivo results, especially in studies related to immunotherapy.
    Keywords:  CD8+ T cells; lentivirus; type I interferon
  32. J Biol Chem. 2022 Sep 26. pii: S0021-9258(22)00984-X. [Epub ahead of print] 102541
      Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔμH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, i.e., the γ subunit (CF1-γ). When the ΔμH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔμH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔμH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants.
    Keywords:  Chloroplast ATP synthase; oxidation; proton electrochemical gradient; redox regulation; thioredoxin
  33. EMBO Rep. 2022 Sep 26. e54746
      Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD -induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.
    Keywords:  MCU; ROS; calcium; melanoma; mitochondria
  34. Cancer Biol Ther. 2022 Dec 31. 23(1): 1-8
      Defects in tRNA expressions and modifications had been linked to various types of tumorigenesis and progression in recent studies, including colorectal cancer. In the present study, we evaluated transcript levels of mitochondrial tyrosyl-tRNA synthetase YARS2 in both colorectal cancer tissues and normal colorectal tissues using qRT-PCR. The results revealed that the mRNA expression level of YARS2 in colorectal cancer tissues was significantly higher than those in normal intestinal tissues. Knockdown of YARS2 in human colon cancer cell-line SW620 leads to significant inhibition of cell proliferation and migration. The steady-state level of tRNATyr, OCR, and ATP synthesis were decreased in the YARS2 knockdown cells. Moreover, our data indicated that inhibition of YARS2 is associated with increased reactive oxygen species levels which sensitize these cells to 5-FU treatment. In conclusion, our study revealed that targeting YARS2 could inhibit colorectal cancer progression. Thus, YARS2 might be a carcinogenesis candidate gene and can serve as a potential target for clinical therapy.
    Keywords:  YARS2; colorectal cancer; mitochondrial; reactive oxygen; tyrosyl-tRNA
  35. Cell Death Differ. 2022 Sep 28.
      Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.
  36. Clin Cancer Res. 2022 Sep 27. pii: CCR-22-2661. [Epub ahead of print]
      PURPOSE: Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICIs) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient BMI.EXPERIMENTAL DESIGN: Associations between BMI [normal (NL < 25) or OW/OB (BMI ≥ 25] and tumor or microbiome characteristics were examined in specimens from 782 metastatic melanoma patients across 7 cohorts. DNA associations were evaluated in the TCGA cohort. RNASeq from 4 cohorts (n=357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x=36) by LC/MS, and in flow-sorted melanoma tumor cells (x=37) and patient-derived melanoma cell lines (x=17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n=371).
    RESULTS: DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI.
    CONCLUSIONS: These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies.
  37. Biophys J. 2022 Sep 28. pii: S0006-3495(22)00784-6. [Epub ahead of print]
      FOF1 ATP synthase, a ubiquitous enzyme that synthesizes most ATP in living cells, is composed of two rotary motors, a membrane-embedded proton-driven FO motor, and a catalytic F1 motor. These motors share both central and peripheral stalks. Although both FO and F1 have pseudo-symmetric structures, their symmetries do not match. How symmetry mismatch is solved remains elusive because of the missing intermediate structures of the rotational steps. Here, for the case of Bacillus PS3 ATP synthases with 3- and 10-fold symmetries in F1 and FO, respectively, we uncovered the mechanical couplings between FO and F1 at every 36º-rotation step via molecular dynamics simulations and comparative studies of cryo-electron microscopy (EM) structures from three species. We found that the mismatch could be solved using several elements: (1) the F1 head partially rotates relative to the FO a-subunit via elastic distortion of the b-subunits, (2) the rotor is twisted, and (3) comparisons of cryo-EM structures further suggest that the c-ring rotary angles can deviate from the symmetric ones. In addition, the F1 motor may have non-canonical structures, relieving stronger frustration. Thus, we provide new insights for solving the symmetry mismatch problem.
    Keywords:  F(O)F(1) ATP synthase; Molecular dynamics simulation; Molecular motor