bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2022‒06‒26
fifty-one papers selected by
Christian Frezza
University Hospital Cologne


  1. Nat Metab. 2022 Jun 20.
      Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.
    DOI:  https://doi.org/10.1038/s42255-022-00583-z
  2. Curr Biol. 2022 Jun 20. pii: S0960-9822(22)00765-5. [Epub ahead of print]32(12): R618-R623
      Mitochondria are central to cellular metabolism. They provide intermediate metabolites that are used in biosynthetic pathways and they process diet-derived nutrients into the energy-rich compound ATP. Mitochondrial ATP biosynthesis is a marvel of thermodynamic efficiency. Via the tricarboxylic acid cycle (TCA) and fatty acid β-oxidation, mitochondria extract electrons from dietary carbon compounds and pass them to nucleotides that ultimately deliver them to the respiratory chain complexes located in invaginations in the inner mitochondrial membrane (IMM) known as cristae. The respiratory chain complexes donate electrons in stepwise redox reactions to molecular oxygen and, with the exception of complex II, use the liberated energy to pump protons across the proton-impermeable IMM, generating a proton electrochemical gradient. This gradient is then utilized by the ATP synthase, which, in a rotary mechanism, catalyzes the formation of the high-energy γ-phosphate chemical bond between ADP and inorganic phosphate. The conversion of the chemical energy of carbon compounds into a physical, vectorial form of energy (the electrochemical gradient) maximizes the yield of the ATP biosynthetic process and is perhaps one of the foundations of life as we know it.
    DOI:  https://doi.org/10.1016/j.cub.2022.05.006
  3. Redox Biol. 2022 Jun 17. pii: S2213-2317(22)00140-9. [Epub ahead of print]54 102368
      Cell models of cardiac ischemia-reperfusion (IR) injury are essential to facilitate understanding, but current monolayer cell models poorly replicate the in vivo IR injury that occurs within a three-dimensional tissue. Here we show that this is for two reasons: the residual oxygen present in many cellular hypoxia models sustains mitochondrial oxidative phosphorylation; and the loss of lactate from cells into the incubation medium during ischemia enables cells to sustain glycolysis. To overcome these limitations, we incubated isolated adult mouse cardiomyocytes anoxically while inhibiting lactate efflux. These interventions recapitulated key markers of in vivo ischemia, notably the accumulation of succinate and the loss of adenine nucleotides. Upon reoxygenation after anoxia the succinate that had accumulated during anoxia was rapidly oxidized in association with extensive mitochondrial superoxide/hydrogen peroxide production and cell injury, mimicking reperfusion injury. This cell model will enable key aspects of cardiac IR injury to be assessed in vitro.
    Keywords:  Cardiomyocytes; Hydrogen peroxide; Ischemia-reperfusion injury; Metabolism; Mitochondria
    DOI:  https://doi.org/10.1016/j.redox.2022.102368
  4. Nat Metab. 2022 Jun 20.
      Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue's metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs-elicited by the loss of Rag GTPases-inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.
    DOI:  https://doi.org/10.1038/s42255-022-00584-y
  5. Nat Metab. 2022 Jun 23.
      Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can be a proliferation bottleneck that is influenced by environmental conditions. However, a comprehensive quantitative understanding of metabolic processes that may be affected by NAD+ deficiency is currently missing. Here, we show that de novo lipid biosynthesis can impose a substantial NAD+ consumption cost in proliferating cancer cells. When electron acceptors are limited, environmental lipids become crucial for proliferation because NAD+ is required to generate precursors for fatty acid biosynthesis. We find that both oxidative and even net reductive pathways for lipogenic citrate synthesis are gated by reactions that depend on NAD+ availability. We also show that access to acetate can relieve lipid auxotrophy by bypassing the NAD+ consuming reactions. Gene expression analysis demonstrates that lipid biosynthesis strongly anti-correlates with expression of hypoxia markers across tumor types. Overall, our results define a requirement for oxidative metabolism to support biosynthetic reactions and provide a mechanistic explanation for cancer cell dependence on lipid uptake in electron acceptor-limited conditions, such as hypoxia.
    DOI:  https://doi.org/10.1038/s42255-022-00588-8
  6. Cancers (Basel). 2022 Jun 16. pii: 2983. [Epub ahead of print]14(12):
      Despite the development of metabolism-based therapy for a variety of malignancies, resistance to single-agent treatment is common due to the metabolic plasticity of cancer cells. Improved understanding of how malignant cells rewire metabolic pathways can guide the rational selection of combination therapy to circumvent drug resistance. Here, we show that human T-ALL cells shift their metabolism from oxidative decarboxylation to reductive carboxylation when the TCA cycle is disrupted. The α-ketoglutarate dehydrogenase complex (KGDHC) in the TCA cycle regulates oxidative decarboxylation by converting α-ketoglutarate (α-KG) to succinyl-CoA, while isocitrate dehydrogenase (IDH) 1 and 2 govern reductive carboxylation. Metabolomics flux analysis of T-ALL reveals enhanced reductive carboxylation upon genetic depletion of the E2 subunit of KGDHC, dihydrolipoamide-succinyl transferase (DLST), mimicking pharmacological inhibition of the complex. Mechanistically, KGDHC dysfunction causes increased demethylation of nuclear DNA by α-KG-dependent dioxygenases (e.g., TET demethylases), leading to increased production of both IDH1 and 2. Consequently, dual pharmacologic inhibition of the TCA cycle and TET demethylases demonstrates additive efficacy in reducing the tumor burden in zebrafish xenografts. These findings provide mechanistic insights into how T-ALL develops resistance to drugs targeting the TCA cycle and therapeutic strategies to overcome this resistance.
    Keywords:  DNA demethylation; T-cell acute lymphoblastic leukemia; TCA cycle; oxidative phosphorylation; reductive carboxylation; α-ketoglutarate
    DOI:  https://doi.org/10.3390/cancers14122983
  7. Sci Adv. 2022 Jun 24. 8(25): eabn9699
      Hürthle cell carcinomas (HCCs) display two exceptional genotypes: near-homoplasmic mutation of mitochondrial DNA (mtDNA) and genome-wide loss of heterozygosity (gLOH). To understand the phenotypic consequences of these genetic alterations, we analyzed genomic, metabolomic, and immunophenotypic data of HCC and other thyroid cancers. Both mtDNA mutations and profound depletion of citrate pools are common in HCC and other thyroid malignancies, suggesting that thyroid cancers are broadly equipped to survive tricarboxylic acid cycle impairment, whereas metabolites in the reduced form of NADH-dependent lysine degradation pathway were elevated exclusively in HCC. The presence of gLOH was not associated with metabolic phenotypes but rather with reduced immune infiltration, indicating that gLOH confers a selective advantage partially through immunosuppression. Unsupervised multimodal clustering revealed four clusters of HCC with distinct clinical, metabolomic, and microenvironmental phenotypes but overlapping genotypes. These findings chart the metabolic and microenvironmental landscape of HCC and shed light on the interaction between genotype, metabolism, and the microenvironment in cancer.
    DOI:  https://doi.org/10.1126/sciadv.abn9699
  8. Nat Commun. 2022 Jun 23. 13(1): 3585
      Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular details of substrate binding remain unresolved. Here, we evaluate the role of the solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of asparagine/arginine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are directed through a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.
    DOI:  https://doi.org/10.1038/s41467-022-31366-5
  9. Curr Biol. 2022 Jun 20. pii: S0960-9822(22)00707-2. [Epub ahead of print]32(12): R684-R696
      Maintaining nutrient and energy homeostasis is crucial for the survival and function of cells and organisms in response to environmental stress. Cells have evolved a stress-induced catabolic pathway, termed autophagy, to adapt to stress conditions such as starvation. During autophagy, damaged or non-essential cellular structures are broken down in lysosomes, and the resulting metabolites are reused for core biosynthetic processes or energy production. Recent studies have revealed that autophagy can target and degrade different types of nutrient stores and produce a variety of metabolites and fuels, including amino acids, nucleotides, lipids and carbohydrates. Here, we will focus on how autophagy functions to balance cellular nutrient and energy demand and supply - specifically, how energy deprivation switches on autophagic catabolism, how autophagy halts anabolism by degrading the protein synthesis machinery, and how bulk and selective autophagy-derived metabolites recycle and feed into a variety of bioenergetic and anabolic pathways during stress conditions. Recent new insights and progress in these areas provide a better understanding of how resource mobilization and reallocation sustain essential metabolic and anabolic activities under unfavorable conditions.
    DOI:  https://doi.org/10.1016/j.cub.2022.04.071
  10. Trends Endocrinol Metab. 2022 Jun 17. pii: S1043-2760(22)00099-6. [Epub ahead of print]
      Citrin deficiency is a pan-ethnic and highly prevalent mitochondrial disease with three different stages: neonatal intrahepatic cholestasis (NICCD), a relatively mild adaptation stage, and type II citrullinemia in adulthood (CTLN2). The cause is the absence or dysfunction of the calcium-regulated mitochondrial aspartate/glutamate carrier 2 (AGC2/SLC25A13), also called citrin, which imports glutamate into the mitochondrial matrix and exports aspartate to the cytosol. In citrin deficiency, these missing transport steps lead to impairment of the malate-aspartate shuttle, gluconeogenesis, amino acid homeostasis, and the urea cycle. In this review, we describe the geological spread and occurrence of citrin deficiency, the metabolic consequences and use our current knowledge of the structure to predict the impact of the known pathogenic mutations on the calcium-regulatory and transport mechanism of citrin.
    Keywords:  SLC25A12; SLC25A13; calcium regulation; mitochondrial carrier family; transport; urea cycle disorders
    DOI:  https://doi.org/10.1016/j.tem.2022.05.002
  11. Proc Natl Acad Sci U S A. 2022 Jun 28. 119(26): e2121987119
      Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis in vivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.
    Keywords:  GPD2; cell death; ferroptosis; lipid peroxidation; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2121987119
  12. Mol Carcinog. 2022 Jun 21.
      Research has shown extensive metabolic remodeling in clear cell renal cell carcinoma (ccRCC), with increased glutathione (GSH) levels. We hypothesized that activating transcription factor-4 (ATF4) and the integrated stress response (ISR) induce a metabolic shift, including increased GSH accumulation, and that Vitamin A deficiency (VAD), found in ccRCCs, can also activate ATF4 signaling in the kidney. To determine the role of ATF4, we used publicly available RNA sequencing (RNA-seq) data sets from The Cancer Genomics Atlas. Subsequently, we performed RNA-seq and liquid chromatography-mass spectrometry-based metabolomics analysis of the murine TRAnsgenic Cancer of the Kidney (TRACK) model for early-stage ccRCC. To validate our findings, we generated RCC4 cell lines with ATF4 gene edits (ATF4-knockout [KO]) and subjected these cells to metabolic isotope tracing. Analysis of variance, the two-sided Student's t test, and gene set enrichment analysis were used (p < 0.05) to determine statistical significance. Here we show that most human ccRCC tumors exhibit activation of the transcription factor ATF4. Activation of ATF4 is concomitant with enrichment of the ATF4 gene set and elevated expression of ATF4 target genes ASNS, ALDH1L2, MTHFD2, DDIT3 (CHOP), DDIT4, TRIB3, EIF4EBP1, SLC7A11, and PPP1R15A (GADD34). Transcript profiling and metabolomics analyses show that activated hypoxia-inducible factor-1α (HIF1α) signaling in our TRACK ccRCC murine model also induces an ATF4-mediated ISR. Notably, both normoxic HIF1α signaling in TRACK kidneys and VAD in wild-type kidneys diminish amino acid levels, increase ASNS, TRIB3, and MTHFD2 messenger RNA levels, and increase levels of lipids and GSH. By metabolic isotope tracing in human RCC4 kidney cancer parental and ATF4 gene-edited (ATF4-KO) cell lines, we show that ATF4 increases GSH accumulation in part via activation of the mitochondrial one-carbon metabolism pathway. Our results demonstrate for the first time that activation of ATF4 enhances GSH accumulation, increases purine and pyrimidine biosynthesis, and contributes to transcriptional and metabolic remodeling in ccRCC. Moreover, constitutive HIF1α expressed only in murine kidney proximal tubules activates ATF4, leading to the metabolic changes associated with the ISR. Our data indicate that HIF1α can promote ccRCC via ATF4 activation. Moreover, lack of Vitamin A in the kidney recapitulates aspects of the ISR.
    Keywords:  HIF; glutathione; kidney cancer; nutrition; retinoids
    DOI:  https://doi.org/10.1002/mc.23437
  13. Membranes (Basel). 2022 Jun 15. pii: 625. [Epub ahead of print]12(6):
      Mitochondria are surrounded by two membranes; the outer mitochondrial membrane and the inner mitochondrial membrane. They are unique organelles since they have their own DNA, the mitochondrial DNA (mtDNA), which is replicated continuously. Mitochondrial membranes have direct interaction with mtDNA and are therefore involved in organization of the mitochondrial genome. They also play essential roles in mitochondrial dynamics and the supply of nucleotides for mtDNA synthesis. In this review, we will discuss how the mitochondrial membranes interact with mtDNA and how this interaction is essential for mtDNA maintenance. We will review different mtDNA maintenance disorders that result from defects in this crucial interaction. Finally, we will review therapeutic approaches relevant to defects in mitochondrial membranes.
    Keywords:  IMM; OMM; fission; fusion; mitochondria; mtDNA
    DOI:  https://doi.org/10.3390/membranes12060625
  14. Biomolecules. 2022 Jun 04. pii: 786. [Epub ahead of print]12(6):
      Mitochondrial function is dependent on molecular chaperones, primarily due to their necessity in the formation of respiratory complexes and clearance of misfolded proteins. Heat shock proteins (Hsps) are a subset of molecular chaperones that function in all subcellular compartments, both constitutively and in response to stress. The Hsp90 chaperone TNF-receptor-associated protein-1 (TRAP1) is primarily localized to the mitochondria and controls both cellular metabolic reprogramming and mitochondrial apoptosis. TRAP1 upregulation facilitates the growth and progression of many cancers by promoting glycolytic metabolism and antagonizing the mitochondrial permeability transition that precedes multiple cell death pathways. TRAP1 attenuation induces apoptosis in cellular models of cancer, identifying TRAP1 as a potential therapeutic target in cancer. Similar to cytosolic Hsp90 proteins, TRAP1 is also subject to post-translational modifications (PTM) that regulate its function and mediate its impact on downstream effectors, or 'clients'. However, few effectors have been identified to date. Here, we will discuss the consequence of TRAP1 deregulation in cancer and the impact of post-translational modification on the known functions of TRAP1.
    Keywords:  Hsp90; TRAP1; Warburg effect; cancer; chaperone; metabolism; mitochondria; post-translational modification
    DOI:  https://doi.org/10.3390/biom12060786
  15. Cell Metab. 2022 Jun 14. pii: S1550-4131(22)00223-6. [Epub ahead of print]
      In this review, we focus on recent developments in our understanding of nutrient-induced insulin secretion that challenge a key aspect of the "canonical" model, in which an oxidative phosphorylation-driven rise in ATP production closes KATP channels. We discuss the importance of intrinsic β cell metabolic oscillations; the phasic alignment of relevant metabolic cycles, shuttles, and shunts; and how their temporal and compartmental relationships align with the triggering phase or the secretory phase of pulsatile insulin secretion. Metabolic signaling components are assigned regulatory, effectory, and/or homeostatic roles vis-à-vis their contribution to glucose sensing, signal transmission, and resetting the system. Taken together, these functions provide a framework for understanding how allostery, anaplerosis, and oxidative metabolism are integrated into the oscillatory behavior of the secretory pathway. By incorporating these temporal as well as newly discovered spatial aspects of β cell metabolism, we propose a much-refined MitoCat-MitoOx model of the signaling process for the field to evaluate.
    DOI:  https://doi.org/10.1016/j.cmet.2022.06.003
  16. Cell Death Dis. 2022 Jun 22. 13(6): 561
      Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.
    DOI:  https://doi.org/10.1038/s41419-022-05016-z
  17. STAR Protoc. 2022 Sep 16. 3(3): 101454
      Membrane contact sites are recognized as critical means of intercompartmental communication. Here, we describe a protocol for engineering and validating a synthetic bridge between the inner and outer mitochondrial membranes to support functioning of the endogenous mitochondrial contact site and cristae organizing system (MICOS). A chimeric protein, MitoT, is stably expressed in cultured mammalian cells to bridge the mitochondrial membranes. This approach can be a valuable tool to study the function of the MICOS complex and associated proteins. For complete details on the use and execution of this protocol, please refer to Viana et al. (2021).
    Keywords:  Biotechnology and bioengineering; Cell Biology; Cell Membrane; Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Metabolism; Microscopy; Molecular Biology
    DOI:  https://doi.org/10.1016/j.xpro.2022.101454
  18. Acta Physiol (Oxf). 2022 Jun 20. e13852
      Mitochondria are complex small organelles of eukaryotic cells and build the cellular source of energy. Several morphological features of mitochondria such as the double membrane and the circular DNA structure support the thesis that they originated from a prokaryotic eubacterial ancestor that has been taken up by the eukaryotic cell very early during the eukaryotic evolution. Since this "uptake-event" mitochondria were integrated into cellular processes and regulation which was realized by the transfer of mitochondrial genes into the host cell genome. 1 The mitochondrial genome reduced to for instance 13 encoded protein subunits of the oxidative phosphorylation system in human cells. Mitochondria offer energy for the cell by producing about 95% of cellular ATP.2 Nutrients, mainly pyruvate from the glycolysis enter the tricarboxylic acid cycle and undergo iterative oxidations whereas electrons are transferred to the reduction equivalents NADH and FADH2 . These redox equivalents transport electrons to the electron transport chain located on the inner mitochondrial membrane and protons are pumped into the perimembranal room. The F1 F0 -ATP synthase generates ATP driven by protons flowing down an electrochemical gradient during a process named oxidative phosphorylation. As a byproduct reactive oxygen species are generated. Mitochondria are more than simple batteries for the cell, they are furthermore involved in numerous vital cellular processes, among them are calcium homeostasis, cell death, fatty acid oxidation, reactive oxygen species (ROS) signaling, cholesterol synthesis and nucleotide synthesis, topics that are frequently published in Acta Physiologica.
    DOI:  https://doi.org/10.1111/apha.13852
  19. Proc Natl Acad Sci U S A. 2022 Jun 28. 119(26): e2200923119
      All kingdoms of life produce essential nicotinamide dinucleotide NADP(H) using NAD kinases (NADKs). A panel of published NADK structures from bacteria, eukaryotic cytosol, and yeast mitochondria revealed similar tetrameric enzymes. Here, we present the 2.8-Å structure of the human mitochondrial kinase NADK2 with a bound substrate, which is an exception to this uniformity, diverging both structurally and biochemically from NADKs. We show that NADK2 harbors a unique tetramer disruptor/dimerization element, which is conserved in mitochondrial kinases of animals (EMKA) and absent from other NADKs. EMKA stabilizes the NADK2 dimer but prevents further NADK2 oligomerization by blocking the tetramerization interface. This structural change bears functional consequences and alters the activation mechanism of the enzyme. Whereas tetrameric NADKs undergo cooperative activation via oligomerization, NADK2 is a constitutively active noncooperative dimer. Thus, our data point to a unique regulation of NADP(H) synthesis in animal mitochondria achieved via structural adaptation of the NADK2 kinase.
    Keywords:  NADK; NADK2; cooperative; dimer; structure
    DOI:  https://doi.org/10.1073/pnas.2200923119
  20. Cell Death Differ. 2022 Jun 20.
      The ability of mitochondria to buffer a rapid rise in cytosolic Ca2+ is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca2+ imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca2+ release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca2+. When the latter exceeded the organelle buffering capacity, an abrupt mitochondrial inner membrane permeabilization (MIMP) occurred, releasing matrix contents into the cytosol. MIMP was followed by cell death that was independent of Bcl-2 family members and inhibitable by the intracellular Ca2+ chelator BAPTA-AM. Cyclosporin A (CsA), capable of blocking the mitochondrial permeability transition (MPT), completely prevented cell death induced by m-3M3FBS. However, CsA acted upstream of mitochondria by preventing Ca2+ release from ER stores. Therefore, loss of Ca2+ intracellular balance and mitochondrial Ca2+ overload followed by MIMP induced a cell death process that is distinct from Bcl-2 family-regulated mitochondrial outer membrane permeabilization (MOMP). Further, the inhibition of cell death by CsA or its analogues can be independent of effects on the MPT.
    DOI:  https://doi.org/10.1038/s41418-022-01025-9
  21. Proc Natl Acad Sci U S A. 2022 Jun 28. 119(26): e2123247119
      Mitochondria, a highly metabolically active organelle, have been shown to play an essential role in regulating innate immune function. Mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) is an essential process regulating mitochondrial metabolism by targeting key enzymes involved in the tricarboxylic acid cycle (TCA). Accumulative evidence suggests MCU-dependent mitochondrial Ca2+ signaling may bridge the metabolic reprogramming and regulation of immune cell function. However, the mechanism by which MCU regulates inflammation and its related disease remains elusive. Here we report a critical role of MCU in promoting phagocytosis-dependent activation of NLRP3 (nucleotide-binding domain, leucine-rich repeat containing family, pyrin domain-containing 3) inflammasome by inhibiting phagolysosomal membrane repair. Myeloid deletion of MCU (McuΔmye) resulted in an attenuated phagolysosomal rupture, leading to decreased caspase-1 cleavage and interleukin (IL)-1β release, in response to silica or alum challenge. In contrast, other inflammasome agonists such as adenosine triphosphate (ATP), nigericin, poly(dA:dT), and flagellin induced normal IL-1β release in McuΔmye macrophages. Mechanistically, we demonstrated that decreased NLRP3 inflammasome activation in McuΔmye macrophages was caused by improved phagolysosomal membrane repair mediated by ESCRT (endosomal sorting complex required for transport)-III complex. Furthermore, McuΔmye mice showed a pronounced decrease in immune cell recruitment and IL-1β production in alum-induced peritonitis, a typical IL-1-dependent inflammation model. In sum, our results identify a function of MCU in promoting phagocytosis-dependent NLRP3 inflammatory response via an ESCRT-mediated phagolysosomal membrane repair mechanism.
    Keywords:  ESCRT; MCU; inflammasome; phagosome
    DOI:  https://doi.org/10.1073/pnas.2123247119
  22. Nat Commun. 2022 Jun 20. 13(1): 3518
      System-wide metabolic homeostasis is crucial for maintaining physiological functions of living organisms. Stable-isotope tracing metabolomics allows to unravel metabolic activity quantitatively by measuring the isotopically labeled metabolites, but has been largely restricted by coverage. Delineating system-wide metabolic homeostasis at the whole-organism level remains challenging. Here, we develop a global isotope tracing metabolomics technology to measure labeled metabolites with a metabolome-wide coverage. Using Drosophila as an aging model organism, we probe the in vivo tracing kinetics with quantitative information on labeling patterns, extents and rates on a metabolome-wide scale. We curate a system-wide metabolic network to characterize metabolic homeostasis and disclose a system-wide loss of metabolic coordinations that impacts both intra- and inter-tissue metabolic homeostasis significantly during Drosophila aging. Importantly, we reveal an unappreciated metabolic diversion from glycolysis to serine metabolism and purine metabolism as Drosophila aging. The developed technology facilitates a system-level understanding of metabolic regulation in living organisms.
    DOI:  https://doi.org/10.1038/s41467-022-31268-6
  23. Methods Mol Biol. 2022 ;2493 153-165
      Mitochondria are cellular organelles that play an essential role in eukaryotes, producing the energy needed for a cell to survive. Beyond the ~3.2 Gb of nuclear genomic DNA, each human cell has hundreds of mitochondria which carry one or a few copies of the 16.5 kb circular mitochondria DNA (mtDNA). Despite its small size, the circular genome encodes 37 genes, including 13 proteins that generate respiratory chain complexes together with other proteins of nuclear origin. Similar to nuclear genome, mtDNA in cancer cells frequently harbor somatically acquired alterations. Whole-genome or whole-exome sequencing of the tumor and its matched normal tissues (frequently blood or adjacent non-tumor tissues) enables sensitive and efficient detection of somatic mtDNA mutations. Because each cancer cell commonly carries hundreds to thousands of mtDNA copies, detection of mtDNA mutations is dependent on the heteroplasmic level of each mutation. Here, we describe strategies to accurately identify somatic mtDNA mutations in cancer genome studies.
    Keywords:  Bioinformatics; Genome sequencing; Genomics; Heteroplasmy; Mitochondria; Next-generation sequencing; Somatic mutations
    DOI:  https://doi.org/10.1007/978-1-0716-2293-3_10
  24. Genes (Basel). 2022 Jun 07. pii: 1025. [Epub ahead of print]13(6):
      Despite two decades of paraganglioma-pheochromocytoma research, the fundamental question of how the different succinate dehydrogenase (SDH)-related tumor phenotypes are initiated has remained unanswered. Here, we discuss two possible scenarios by which missense (hypomorphic alleles) or truncating (null alleles) SDH gene variants determine clinical phenotype. Dysfunctional SDH is a major source of reactive oxygen species (ROS) but ROS are inhibited by rising succinate levels. In scenario 1, we propose that SDH missense variants disrupt electron flow, causing elevated ROS levels that are toxic in sympathetic PPGL precursor cells but well controlled in oxygen-sensing parasympathetic paraganglion cells. We also suggest that SDHAF2 variants, solely associated with HNPGL, may cause the reversal of succinate dehydrogenase to fumarate reductase, producing very high ROS levels. In scenario 2, we propose a modified succinate threshold model of tumor initiation. Truncating SDH variants cause high succinate accumulation and likely initiate tumorigenesis via disruption of 2-oxoglutarate-dependent enzymes in both PPGL and HNPGL precursor tissues. We propose that missense variants (including SDHAF2) cause lower succinate accumulation and thus initiate tumorigenesis only in very metabolically active tissues such as parasympathetic paraganglia, which naturally show very high levels of succinate.
    Keywords:  head and neck paraganglioma; neuroendocrine tumor; pheochromocytoma; reactive oxygen species; succinate dehydrogenase
    DOI:  https://doi.org/10.3390/genes13061025
  25. BMB Rep. 2022 Jun 21. pii: 5590. [Epub ahead of print]
      MitoNEET, a mitochondrial outer membrane protein containing the Asn-Glu-Glu-Thr (NEET) sequence, controls the formation of intermitochondrial junctions and confers autophagy resistance. Moreover, mitoNEET as a mitochondrial substrate undergoes ubiquitination by activated Parkin during the initiation of mitophagy. Therefore, mitoNEET is linked to the regulation of autophagy and mitophagy. Mitophagy is the selective removal of the damaged or unnecessary mitochondria, which is crucial to sustaining mitochondrial quality control. In numerous human diseases, the accumulation of damaged mitochondria by impaired mitophagy has been observed. However, the therapeutic strategy targeting of mitoNEET as a mitophagy-enhancing mediator requires further research. Herein, we confirmed that mitophagy is indeed activated by mitoNEET inhibition. CCCP (carbonyl cyanide m-chlorophenyl hydrazone), which leads to mitochondrial depolarization, induces mitochondrial dysfunction and superoxide production. This, in turn, contributes to the induction of mitophagy; mitoNEET protein levels were initially increased before an increase in LC3-Ⅱ protein following CCCP treatment. Pharmacological inhibition of mitoNEET using mitoNEET Ligand-1 (NL-1) promoted accumulation of Pink1 and Parkin, which are mitophagy-associated proteins, and activation of mitochondria-lysosome crosstalk, in comparison to CCCP alone. Inhibition of mitoNEET using NL-1, or mitoNEET shRNA transfected into RAW264.7 cells, abrogated CCCP-induced ROS and mitochondrial cell death; additionally, it activated the expression of PGC-1α and SOD2, regulators of oxidative metabolism. In particular, the increase in PGC-1α, which is a major regulator of mitochondrial biogenesis, promotes mitochondrial quality control. These results indicated that mitoNEET is a potential therapeutic target in numerous human diseases to enhance mitophagy and protect cells by maintaining a network of healthy mitochondria.
  26. Nat Commun. 2022 Jun 24. 13(1): 3615
      Mitochondrial cytochrome c oxidase (CcO) or respiratory chain complex IV is a heme aa3-copper oxygen reductase containing metal centers essential for holo-complex biogenesis and enzymatic function that are assembled by subunit-specific metallochaperones. The enzyme has two copper sites located in the catalytic core subunits. The COX1 subunit harbors the CuB site that tightly associates with heme a3 while the COX2 subunit contains the binuclear CuA site. Here, we report that in human cells the CcO copper chaperones form macromolecular assemblies and cooperate with several twin CX9C proteins to control heme a biosynthesis and coordinate copper transfer sequentially to the CuA and CuB sites. These data on CcO illustrate a mechanism that regulates the biogenesis of macromolecular enzymatic assemblies with several catalytic metal redox centers and prevents the accumulation of cytotoxic reactive assembly intermediates.
    DOI:  https://doi.org/10.1038/s41467-022-31413-1
  27. Proc Natl Acad Sci U S A. 2022 Jun 28. 119(26): e2111506119
      Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.
    Keywords:  fucosidosis; lysosomes; macroautophagy; α-l-fucosidase 1
    DOI:  https://doi.org/10.1073/pnas.2111506119
  28. Metabolites. 2022 Jun 08. pii: 527. [Epub ahead of print]12(6):
      In inborn errors of metabolism, such as amino acid breakdown disorders, loss of function mutations in metabolic enzymes within the catabolism pathway lead to an accumulation of the catabolic intermediate that is the substrate of the mutated enzyme. In patients of such disorders, dietarily restricting the amino acid(s) to prevent the formation of these catabolic intermediates has a therapeutic or even entirely preventative effect. This demonstrates that the pathology is due to a toxic accumulation of enzyme substrates rather than the loss of downstream products. Here, we provide an overview of amino acid metabolic disorders from the perspective of the 'toxic metabolites' themselves, including their mechanism of toxicity and whether they are involved in the pathology of other disease contexts as well. In the research literature, there is often evidence that such metabolites play a contributing role in multiple other nonhereditary (and more common) disease conditions, and these studies can provide important mechanistic insights into understanding the metabolite-induced pathology of the inborn disorder. Furthermore, therapeutic strategies developed for the inborn disorder may be applicable to these nonhereditary disease conditions, as they involve the same toxic metabolite. We provide an in-depth illustration of this cross-informing concept in two metabolic disorders, methylmalonic acidemia and hyperammonemia, where the pathological metabolites methylmalonic acid and ammonia are implicated in other disease contexts, such as aging, neurodegeneration, and cancer, and thus there are opportunities to apply mechanistic or therapeutic insights from one disease context towards the other. Additionally, we expand our scope to other metabolic disorders, such as homocystinuria and nonketotic hyperglycinemia, to propose how these concepts can be applied broadly across different inborn errors of metabolism and various nonhereditary disease conditions.
    Keywords:  ammonia; hyperammonemia; inborn error of metabolism (IEM); intoxification; metabolism; metabolites; methylmalonic acidemia; toxic metabolites
    DOI:  https://doi.org/10.3390/metabo12060527
  29. Curr Opin Biotechnol. 2022 Jun 20. pii: S0958-1669(22)00073-8. [Epub ahead of print]76 102739
      Biochemical characterization of metabolism provides molecular insights for understanding biology in health and disease. Over the past decades, metabolic perturbations have been implicated in cancer, neurodegeneration, and diabetes, among others. Isotope tracing is a technique that allows tracking of labeled atoms within metabolites through biochemical reactions. This technique has become an integral component of the contemporary metabolic research. Isotope tracing measures substrate contribution to downstream metabolites and indicates its utilization in cellular metabolic networks. In addition, isotopic labeling data are necessary for quantitative metabolic flux analysis. Here, we review recent work utilizing metabolic tracing to study health and disease, and highlight its application to interrogate subcellular, intercellular, and in vivo metabolism. We further discuss the current challenges and opportunities to expand the utility of isotope tracing to new research areas.
    DOI:  https://doi.org/10.1016/j.copbio.2022.102739
  30. J Biol Methods. 2022 ;9(2): e160
      The regulation of cellular energetics is a complex process that requires the coordinated function of multiple organelles. Historically, studies focused on understanding cellular energy utilization and production have been overwhelmingly concentrated on the mitochondria. While mitochondria account for the majority of intracellular energy production, they alone are incapable of maintaining the variable energetic demands of the cell. The peroxisome has recently emerged as a secondary metabolic organelle that complements and improves mitochondrial performance. Although mitochondria and peroxisomes are structurally distinct organelles, they share key functional similarities that allows for the potential to repurpose readily available tools initially developed for mitochondrial assessment to interrogate peroxisomal metabolic function in a novel manner. To this end, we report here on procedures for the isolation, purification and real-time metabolic assessment of peroxisomal β-oxidation using the Agilent Seahorse® system. When used together, these protocols provide a straightforward, reproducible and highly quantifiable method for measuring the contributions of peroxisomes to cellular and organismal metabolism.
    Keywords:  fatty acid metabolism; peroxisome; seahorse assay
    DOI:  https://doi.org/10.14440/jbm.2022.374
  31. Commun Biol. 2022 Jun 23. 5(1): 620
      Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance.
    DOI:  https://doi.org/10.1038/s42003-022-03568-6
  32. Cancer Res. 2022 Jun 24. pii: canres.4403.2021. [Epub ahead of print]
      The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth that controls cell homeostasis in response to nutrients, growth factors, and other environmental cues. Recent studies have emphasized the importance of lysosomes as a hub for nutrient sensing, especially amino acid sensing by mTORC1. This review highlights recent advances in understanding the amino acid-mTORC1 signaling axis and the role of mTORC1 in cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-4403
  33. Antioxidants (Basel). 2022 Jun 12. pii: 1151. [Epub ahead of print]11(6):
      Control of excessive mitochondrial oxidative stress could provide new targets for both preventive and therapeutic interventions in the treatment of chronic inflammation or any pathology that develops under an inflammatory scenario, such as rheumatoid arthritis (RA). Increasing evidence has demonstrated the role of mitochondrial alterations in autoimmune diseases mainly due to the interplay between metabolism and innate immunity, but also in the modulation of inflammatory response of resident cells, such as synoviocytes. Thus, mitochondrial dysfunction derived from several danger signals could activate tricarboxylic acid (TCA) disruption, thereby favoring a vicious cycle of oxidative/mitochondrial stress. Mitochondrial dysfunction can act through modulating innate immunity via redox-sensitive inflammatory pathways or direct activation of the inflammasome. Besides, mitochondria also have a central role in regulating cell death, which is deeply altered in RA. Additionally, multiple evidence suggests that pathological processes in RA can be shaped by epigenetic mechanisms and that in turn, mitochondria are involved in epigenetic regulation. Finally, we will discuss about the involvement of some dietary components in the onset and progression of RA.
    Keywords:  cell death; diet; epigenetic; inflammation; metabolism; mitochondria; oxidative stress; rheumatoid arthritis
    DOI:  https://doi.org/10.3390/antiox11061151
  34. Mol Oncol. 2022 Jun 21.
      Aging represents the major risk factor for the development of cancer and many other diseases. Recent findings show that normal tissues become riddled with expanded clones that are frequently driven by cancer-associated mutations in an aging-dependent fashion. Additional studies show how aged tissue microenvironments promote the initiation and progression of malignancies, while young healthy tissues actively suppress the outgrowth of malignant clones. Here, we discuss conserved mechanisms that eliminate poorly functioning or potentially malignant cells from our tissues to maintain organismal health and fitness. Natural selection acts to preserve tissue function and prevent disease to maximize reproductive success but these mechanisms wane as reproduction becomes less likely. The ensuing age-dependent tissue decline can impact the shape and direction of clonal somatic evolution, with lifestyle and exposures influencing its pace and intensity. We also consider how aging- and exposure-dependent clonal expansions of "oncogenic" mutations might both increase cancer risk late in life and contribute to tissue decline and non-malignant disease. Still, we can marvel at the ability of our bodies to avoid cancers and other diseases despite the accumulation of billions of cells with cancer-associated mutations.
    Keywords:  NOTCH1; aging; clonal hematopoiesis; life history theory; p53; somatic evolution
    DOI:  https://doi.org/10.1002/1878-0261.13275
  35. Proc Natl Acad Sci U S A. 2022 Jun 28. 119(26): e2200158119
      Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive. Here we determined the structure of TOM core complex at 2.53 Å and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 Å. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain. Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.
    Keywords:  TOM complex; Tom20; Tom22; cryo-EM; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2200158119
  36. Nat Rev Nephrol. 2022 Jun 20.
      Cellular hypoxia occurs when the demand for sufficient molecular oxygen needed to produce the levels of ATP required to perform physiological functions exceeds the vascular supply, thereby leading to a state of oxygen depletion with the associated risk of bioenergetic crisis. To protect against the threat of hypoxia, eukaryotic cells have evolved the capacity to elicit oxygen-sensitive adaptive transcriptional responses driven primarily (although not exclusively) by the hypoxia-inducible factor (HIF) pathway. In addition to the canonical regulation of HIF by oxygen-dependent hydroxylases, multiple other input signals, including gasotransmitters, non-coding RNAs, histone modifiers and post-translational modifications, modulate the nature of the HIF response in discreet cell types and contexts. Activation of HIF induces various effector pathways that mitigate the effects of hypoxia, including metabolic reprogramming and the production of erythropoietin. Drugs that target the HIF pathway to induce erythropoietin production are now approved for the treatment of chronic kidney disease-related anaemia. However, HIF-dependent changes in cell metabolism also have profound implications for functional responses in innate and adaptive immune cells, and thereby heavily influence immunity and the inflammatory response. Preclinical studies indicate a potential use of HIF therapeutics to treat inflammatory diseases, such as inflammatory bowel disease. Understanding the links between HIF, cellular metabolism and immunity is key to unlocking the full therapeutic potential of drugs that target the HIF pathway.
    DOI:  https://doi.org/10.1038/s41581-022-00587-8
  37. Science. 2022 Jun 24. 376(6600): eabh2841
      Tumor necrosis factor (TNF) is a critical host resistance factor against tuberculosis. However, excess TNF produces susceptibility by increasing mitochondrial reactive oxygen species (mROS), which initiate a signaling cascade to cause pathogenic necrosis of mycobacterium-infected macrophages. In zebrafish, we identified the mechanism of TNF-induced mROS in tuberculosis. Excess TNF in mycobacterium-infected macrophages elevates mROS production by reverse electron transport (RET) through complex I. TNF-activated cellular glutamine uptake leads to an increased concentration of succinate, a Krebs cycle intermediate. Oxidation of this elevated succinate by complex II drives RET, thereby generating the mROS superoxide at complex I. The complex I inhibitor metformin, a widely used antidiabetic drug, prevents TNF-induced mROS and necrosis of Mycobacterium tuberculosis-infected zebrafish and human macrophages; metformin may therefore be useful in tuberculosis therapy.
    DOI:  https://doi.org/10.1126/science.abh2841
  38. Elife. 2022 06 20. pii: e79941. [Epub ahead of print]11
      Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.
    Keywords:  AgrA; NADK; infection; infectious disease; macrophage; microbiology; staphylococcus; virulence; zebrafish
    DOI:  https://doi.org/10.7554/eLife.79941
  39. Mol Cell. 2022 Jun 21. pii: S1097-2765(22)00492-0. [Epub ahead of print]
      The tolerance of amino acid starvation is fundamental to robust cellular fitness. Asparagine depletion is lethal to some cancer cells, a vulnerability that can be exploited clinically. We report that resistance to asparagine starvation is uniquely dependent on an N-terminal low-complexity domain of GSK3α, which its paralog GSK3β lacks. In response to depletion of specific amino acids, including asparagine, leucine, and valine, this domain mediates supramolecular assembly of GSK3α with ubiquitin-proteasome system components in spatially sequestered cytoplasmic bodies. This effect is independent of mTORC1 or GCN2. In normal cells, GSK3α promotes survival during essential amino acid starvation. In human leukemia, GSK3α body formation predicts asparaginase resistance, and sensitivity to asparaginase combined with a GSK3α inhibitor. We propose that GSK3α body formation provides a cellular mechanism to maximize the catalytic efficiency of proteasomal protein degradation in response to amino acid starvation, an adaptive response co-opted by cancer cells for asparaginase resistance.
    Keywords:  GSK3; Wnt; asparaginase; protein degradation; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.molcel.2022.05.025
  40. Nature. 2022 Jun 22.
      
    Keywords:  Cancer; Medical research
    DOI:  https://doi.org/10.1038/d41586-022-01639-6
  41. Signal Transduct Target Ther. 2022 Jun 22. 7(1): 192
      Folic acid, served as dietary supplement, is closely linked to one-carbon metabolism and methionine metabolism. Previous clinical evidence indicated that folic acid supplementation displays dual effect on cancer development, promoting or suppressing tumor formation and progression. However, the underlying mechanism remains to be uncovered. Here, we report that high-folate diet significantly promotes cancer development in mice with hepatocellular carcinoma (HCC) induced by DEN/high-fat diet (HFD), simultaneously with increased expression of methionine adenosyltransferase 2A (gene name, MAT2A; protein name, MATIIα), the key enzyme in methionine metabolism, and acceleration of methionine cycle in cancer tissues. In contrast, folate-free diet reduces MATIIα expression and impedes HFD-induced HCC development. Notably, methionine metabolism is dynamically reprogrammed with valosin-containing protein p97/p47 complex-interacting protein (VCIP135) which functions as a deubiquitylating enzyme to bind and stabilize MATIIα in response to folic acid signal. Consistently, upregulation of MATIIα expression is positively correlated with increased VCIP135 protein level in human HCC tissues compared to adjacent tissues. Furthermore, liver-specific knockout of Mat2a remarkably abolishes the advocating effect of folic acid on HFD-induced HCC, demonstrating that the effect of high or free folate-diet on HFD-induced HCC relies on Mat2a. Moreover, folate and multiple intermediate metabolites in one-carbon metabolism are significantly decreased in vivo and in vitro upon Mat2a deletion. Together, folate promotes the integration of methionine and one-carbon metabolism, contributing to HCC development via hijacking MATIIα metabolic pathway. This study provides insight into folate-promoted cancer development, strongly recommending the tailor-made folate supplement guideline for both sub-healthy populations and patients with cancer expressing high level of MATIIα expression.
    DOI:  https://doi.org/10.1038/s41392-022-01017-8
  42. Cancer Res. 2022 Jun 24. pii: canres.1360.2021. [Epub ahead of print]
      Subunits from the chromatin remodelers mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) are mutated, deleted or amplified in more than 40% of cancers. Understanding their functions in normal cells and the consequences of cancerous alterations will provide insight into developing new targeted therapies. Here we examined whether mSWI/SNF mutations increase cellular sensitivity to specific drugs. Taking advantage of the DepMap studies, we demonstrate that cancer cells harboring mutations of specific mSWI/SNF subunits exhibit a genetic dependency on translation factors and are sensitive to translation pathway inhibitors. Furthermore, mSWI/SNF subunits were present in the cytoplasm and interacted with the translation initiation machinery, and short-term inhibition and depletion of specific subunits decreased global translation, implicating a direct role for these factors in translation. Depletion of specific mSWI/SNF subunits also increased sensitivity to mTOR-PI3K inhibitors. In patient-derived breast cancer samples, mSWI/SNF subunits expression in both the nucleus and the cytoplasm was substantially altered. In conclusion, an unexpected cytoplasmic role for mSWI/SNF complexes in translation suggests potential new therapeutic opportunities for patients afflicted by cancers demonstrating alterations in their subunits.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-1360
  43. Trends Mol Med. 2022 Jun 16. pii: S1471-4914(22)00154-X. [Epub ahead of print]
      Genetic or pharmacological inhibition of enzymes involved in GTP biosynthesis has substantial biological effects, underlining the need to better understand the function of GTP levels in regulation of cellular processes and the significance of targeting GTP biosynthesis enzymes for therapeutic intervention. Our current understanding of spatiotemporal regulation of GTP metabolism and its role in physiological and pathological cellular processes is far from complete. Novel methodologies such as genetically encoded sensors of free GTP offered insights into intracellular distribution and function of GTP molecules. In the current Review, we provide analysis of recent discoveries in the field of GTP metabolism and evaluate the key enzymes as molecular targets.
    Keywords:  GTP; biosynthesis; cancer; metabolism; signal transduction
    DOI:  https://doi.org/10.1016/j.molmed.2022.05.012
  44. Cell Mol Life Sci. 2022 Jun 21. 79(7): 375
      The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid β-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.
    Keywords:  Clubfoot; Dimethylglycine dehydrogenase; Hypoplasia of fibulae; Multiple acyl-coenzyme A dehydrogenation deficiency; Riboflavin-responsive exercise intolerance; Serine metabolism
    DOI:  https://doi.org/10.1007/s00018-022-04404-0
  45. Nature. 2022 Jun 22.
      
    Keywords:  Cancer; Health care; Medical research
    DOI:  https://doi.org/10.1038/d41586-022-01724-w
  46. Cell. 2022 Jun 23. pii: S0092-8674(22)00648-1. [Epub ahead of print]185(13): 2213-2233.e25
      The impact of apolipoprotein E ε4 (APOE4), the strongest genetic risk factor for Alzheimer's disease (AD), on human brain cellular function remains unclear. Here, we investigated the effects of APOE4 on brain cell types derived from population and isogenic human induced pluripotent stem cells, post-mortem brain, and APOE targeted replacement mice. Population and isogenic models demonstrate that APOE4 local haplotype, rather than a single risk allele, contributes to risk. Global transcriptomic analyses reveal human-specific, APOE4-driven lipid metabolic dysregulation in astrocytes and microglia. APOE4 enhances de novo cholesterol synthesis despite elevated intracellular cholesterol due to lysosomal cholesterol sequestration in astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co-cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain. Thus, APOE4 initiates glia-specific cell and non-cell autonomous dysregulation that may contribute to increased AD risk.
    Keywords:  APOE; Alzheimer; astrocytes; cholesterol; genetic heterogeneity; haplotypes; iPSC disease modeling; inflammation; matrisome; microglia
    DOI:  https://doi.org/10.1016/j.cell.2022.05.017
  47. Cell Rep. 2022 Jun 21. pii: S2211-1247(22)00779-3. [Epub ahead of print]39(12): 110993
      Although KRAS has long been considered undruggable, direct KRASG12C inhibitors have shown promising initial clinical efficacy. However, the majority of patients still fail to respond. Adaptive feedback reactivation of RAS-mitogen-activated protein kinase (MAPK) signaling has been proposed by our group and others as a key mediator of resistance, but the exact mechanism driving reactivation and the therapeutic implications are unclear. We find that upstream feedback activation of wild-type RAS, as opposed to a shift in KRASG12C to its active guanosine triphosphate (GTP)-bound state, is sufficient to drive RAS-MAPK reactivation in a KRASG12C-independent manner. Moreover, multiple receptor tyrosine kinases (RTKs) can drive feedback reactivation, potentially necessitating targeting of convergent signaling nodes for more universal efficacy. Even in colorectal cancer, where feedback is thought to be primarily epidermal growth factor receptor (EGFR)-mediated, alternative RTKs drive pathway reactivation and limit efficacy, but convergent upstream or downstream signal blockade can enhance activity. Overall, these data provide important mechanistic insight to guide therapeutic strategies targeting KRAS.
    Keywords:  CP: Cancer; KRAS; KRASG12C; adagrasib; adaptive resistance; sotorasib
    DOI:  https://doi.org/10.1016/j.celrep.2022.110993
  48. Oncogene. 2022 Jun 24.
      The dynamics of mitochondrial biogenesis regulation is critical in maintaining cellular homeostasis for immune regulation and tumor prevention. Here, we report that mitochondrial biogenesis disruption through TFAM reduction significantly impairs mitochondrial function, induces autophagy, and promotes esophageal squamous cell carcinoma (ESCC) growth. We found that TFAM protein reduction promotes mitochondrial DNA (mtDNA) release into the cytosol, induces cytosolic mtDNA stress, subsequently activates the cGAS-STING signaling pathway, thereby stimulating autophagy and ESCC growth. STING depletion or mtDNA degradation by DNase I abrogates mtDNA stress response, attenuates autophagy, and decreases the growth of TFAM depleted cells. In addition, autophagy inhibitor also ameliorates mitochondrial dysfunction-induced activation of the cGAS-STING signaling pathway and ESCC growth. In conclusion, our results indicate that mtDNA stress induced by mitochondria biogenesis perturbation activates the cGAS-STING pathway and autophagy to promote ESCC growth, revealing an underappreciated therapeutic strategy for ESCC.
    DOI:  https://doi.org/10.1038/s41388-022-02365-z
  49. Cancer Res. 2022 Jun 22. pii: canres.CAN-22-0562-E.2022. [Epub ahead of print]
      Metastasis is the main cause of cancer death, yet the evolutionary processes behind it remain largely unknown. Here, through analysis of large panel-based genomic datasets from the AACR GENIE project, including 40,979 primary and metastatic tumors across 25 distinct cancer types, we explore how the evolutionary pressure of cancer metastasis shapes the selection of genomic drivers of cancer. The most commonly affected genes were TP53, MYC, and CDKN2A, with no specific pattern associated with metastatic disease. This suggests that, on a driver mutation level, the selective pressure operating in primary and metastatic tumors is similar. The most highly enriched individual driver mutations in metastatic tumors were mutations known to drive resistance to hormone therapies in breast and prostate cancer (ESR1 and AR), anti-EGFR therapy in non-small cell lung cancer (EGFR T790M), and imatinib in gastrointestinal cancer (KIT V654A). Specific mutational signatures were also associated with treatment in three cancer types, supporting clonal selection following anti-cancer therapy. Overall, this implies that initial acquisition of driver mutations is predominantly shaped by the tissue of origin, where specific mutations define the developing primary tumor and drive growth, immune escape, and tolerance to chromosomal instability. However, acquisition of driver mutations that contribute to metastatic disease is less specific, with the main genomic drivers of metastatic cancer evolution associating with resistance to therapy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-0562