The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production

Mammalian cells increase transcription of genes for adaptation to hypoxia through the stabilization of hypoxia-inducible factor 1α (HIF-1α) protein. How cells transduce hypoxic signals to stabilize the HIF-1α protein remains unresolved. We demonstrate that cells deficient in the complex III subunit cytochrome b, which are respiratory incompetent, increase ROS levels and stabilize the HIF-1α protein during hypoxia. RNA interference of the complex III subunit Rieske iron sulfur protein in the cytochrome b–null cells and treatment of wild-type cells with stigmatellin abolished reactive oxygen species (ROS) generation at the Qo site of complex III. These interventions maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Antioxidants maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Exogenous hydrogen peroxide under normoxia prevented hydroxylation of HIF-1α protein and stabilized HIF-1α protein. These results provide genetic and pharmacologic evidence that the Qo site of complex III is required for the transduction of hypoxic signal by releasing ROS to stabilize the HIF-1α protein

Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.

Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing

E2F1 Suppresses Oxidative Metabolism and Endothelial Differentiation of Bone Marrow Progenitor Cells

RATIONALE: The majority of current cardiovascular cell therapy trials use bone marrow progenitor cells (BM PCs) and achieve only modest efficacy; the limited potential of these cells to differentiate into endothelial-lineage cells is one of the major barriers to the success of this promising therapy. We have previously reported that the E2F transcription factor 1 (E2F1) is a repressor of revascularization after ischemic injury. OBJECTIVE: We sought to define the role of E2F1 in the regulation of BM PC function. METHODS AND RESULTS: Ablation of E2F1 (E2F1 deficient) in mouse BM PCs increases oxidative metabolism and reduces lactate production, resulting in enhanced endothelial differentiation. The metabolic switch in E2F1-deficient BM PCs is mediated by a reduction in the expression of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase kinase 2; overexpression of pyruvate dehydrogenase kinase 4 reverses the enhancement of oxidative metabolism and endothelial differentiation. Deletion of E2F1 in the BM increases the amount of PC-derived endothelial cells in the ischemic myocardium, enhances vascular growth, reduces infarct size, and improves cardiac function after myocardial infarction. CONCLUSION: Our results suggest a novel mechanism by which E2F1 mediates the metabolic control of BM PC differentiation, and strategies that inhibit E2F1 or enhance oxidative metabolism in BM PCs may improve the effectiveness of cell therapy

Oxidative phosphorylation selectively orchestrates tissue macrophage homeostasis

We are grateful to N.-G. Larsson, F. Sa´ nchez-Madrid, G. Sabio, R.D. Palmiter, E. Gottlieb, C.T.Moraes, and M.A. del Pozofor sharing essential reagents.We thank S. Iborra, his team, M. Sa´ nchez-A´ lvarez, I. Nikolic, and members of the D.S. laboratory for discussions and critical reading of the manuscript. We thank the staff at the CNIC technical units; foremost the animal, cellomics, histology, metabolomics, genomics,microscopy, and bioinformaticsfacilities; and the SIdI of the Universidad Auto´ noma de Madrid for technical support. This project was supported by the ‘‘la Caixa’’ Foundation (ID 100010434) Postdoctoral Junior Leader Fellowship code LCF/BQ/PR20/11770008 (S.K.W.); ‘‘la Caixa’’ Foundation (ID 100010434) INPhINIT Fellowship code LCF/BQ/IN17/11620074 (I.H.-M.); Spanish Ministry of Education FPU fellowship code FPU20/01418 (M.G.); Ministerio de Ciencia e Innovacio´ n (MCIN) PID2019-104233RB-100/AEI/10.13039/ 501100011033 (S.L.); and NIH grants P01AG049665-08, RO1A148190, and P01HL154998 (N.S.C.). The J.A.E. laboratory is supported by the CNIC and a grant by Ministerio de Ciencia, Innovacio´ n y Universidades (MCNU); Agencia Estatal de Investigacio´ n (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (RTI2018-099357-B-I00); the Biomedical Research Networking Center on Frailty and Healthy Ageing (CIBERFES-ISCiii-CB16/10/00289); and the HFSP agency (RGP0016/2018). Work in the D.S. laboratory is funded by the CNIC; by the European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator grant 725091; by Spanish Ministerio de Ciencia e Innovacio´ n PID2019-108157RB/AEI/ and CPP2021-008310/AEI/10.13039/ 501100011033; by Comunidad de Madrid (P2022/BMD-7333 INMUNOVARCM); and by ‘‘la Caixa’’ Foundation (LCF/PR/HR20/00075 and LCF/PR/HR22/ 00253). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S funded by MCIN/AEI/10.13039/501100011033).S

Oxidation of Alpha-Ketoglutarate Is Required for Reductive Carboxylation in Cancer Cells with Mitochondrial Defects

SummaryMammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA) cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG) via NADPH-dependent isocitrate dehydrogenase (IDH). It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here, we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse

Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.M.Q.F. is a recipient of the following grants: FIS PI13/00430 and FIS PI16/00354 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF) and AECC Scientific Foundation (Beca de Retorno 2010). R.C. is a recipient of the following grants: FIS PI11/00832 and FIS PI14/00726 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF), II14/00009 and PIE15/00068 from the Ministerio de Sanidad, Spain. N.S.C. is a recipient of an NIH grant (5R35CA197532). O.Y.T. is a recipient of the grants BFU2014-57466 from the Ministerio de Economia y Competitividad (MINECO). J.P.B. is funded by MINECO (SAF2016-78114-R), Instituto de Salud Carlos III (RD12/0043/0021), Junta de Castilla y Leon (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigacion Biomedicina 2017 Fundacion BBVA, and Fundacion Ramon Areces. This study was partially supported by the generous donations from Fundacion CRIS Contra el Cancer and AVON Spain. We thank Drs. Erwin Wagner and Nabil Djouder for their critical review of the paper.S

Polyamines Drive Myeloid Cell Survival by Buffering Intracellular pH to Promote Immunosuppression in Glioblastoma

Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution

Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.publishedVersionPeer reviewe