bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒06‒19
33 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Front Oncol. 2022 ;12 892195
      Changes in dynamics of ATP γ- and β-phosphoryl turnover and metabolic flux through phosphotransfer pathways in cancer cells are still unknown. Using 18O phosphometabolite tagging technology, we have discovered phosphotransfer dynamics in three breast cancer cell lines: MCF7 (non-aggressive), MDA-MB-231 (aggressive), and MCF10A (control). Contrary to high intracellular ATP levels, the 18O labeling method revealed a decreased γ- and β-ATP turnover in both breast cancer cells, compared to control. Lower β-ATP[18O] turnover indicates decreased adenylate kinase (AK) flux. Aggressive cancer cells had also reduced fluxes through hexokinase (HK) G-6-P[18O], creatine kinase (CK) [CrP[18O], and mitochondrial G-3-P[18O] substrate shuttle. Decreased CK metabolic flux was linked to the downregulation of mitochondrial MTCK1A in breast cancer cells. Despite the decreased overall phosphoryl flux, overexpression of HK2, AK2, and AK6 isoforms within cell compartments could promote aggressive breast cancer growth.
    Keywords:  18 O stable isotope labeling technology; adenylate kinase; creatine kinase; glycolysis; oxidative phosphorylation; phosphotransfer network; triple-negative breast cancer; γ-and β-ATP phosphoryl turnover
  2. Front Physiol. 2022 ;13 866590
      Adenine nucleotide translocases (ANTs) and uncoupling proteins (UCPs) are known to facilitate proton leak across the inner mitochondrial membrane. However, it remains to be unravelled whether UCP2/3 contribute to significant amount of proton leak in vivo. Reports are indicative of UCP2 dependent proton-coupled efflux of C4 metabolites from the mitochondrial matrix. Previous studies have suggested that UCP2/3 knockdown (KD) contributes to increased ANT-dependent proton leak. Here we investigated the hypothesis that interaction exists between the UCP2 and ANT2 proteins, and that such interaction is regulated by the cellular metabolic demand. Protein-protein interaction was evaluated using reciprocal co-immunoprecipitation and in situ proximity ligation assay. KD of ANT2 and UCP2 was performed by siRNA in human embryonic kidney cells 293A (HEK293A) cells. Mitochondrial and cellular respiration was measured by high-resolution respirometry. ANT2-UCP2 interaction was demonstrated, and this was dependent on cellular metabolism. Inhibition of ATP synthase promoted ANT2-UCP2 interaction whereas high cellular respiration, induced by adding the mitochondrial uncoupler FCCP, prevented interaction. UCP2 KD contributed to increased carboxyatractyloside (CATR) sensitive proton leak, whereas ANT2 and UCP2 double KD reduced CATR sensitive proton leak, compared to UCP2 KD. Furthermore, proton leak was reduced in double KD compared to UCP2 KD. In conclusion, our results show that there is an interaction between ANT2-UCP2, which appears to be dynamically regulated by mitochondrial respiratory activity. This may have implications in the regulation of mitochondrial efficiency or cellular substrate utilization as increased activity of UCP2 may promote a switch from glucose to fatty acid metabolism.
    Keywords:  adenine nucleotide translocase-2; mitochondria; protein interaction; proximity ligation assay; uncoupling protein-2
  3. Biochem Pharmacol. 2022 Jun 10. pii: S0006-2952(22)00226-X. [Epub ahead of print] 115132
      Mitochondria-lysosome crosstalk is an intercellular communication platform regulating mitochondrial quality control (MQC). Activated dynamin-related protein 1 (Drp1) with phosphorylation at serine 616 (p-Drp1Ser616) plays a critical role in mitophagy-dependent cell survival and anti-cancer therapy for hepatocellular carcinoma (HCC). However, the underlying mechanisms that p-Drp1Ser616 involved in regulating mitochondria-lysosome crosstalk and mediating anti-HCC therapy remain unknown. HCC cells and mouse xenograft models were conducted to evaluate the relationship between p-Drp1Ser616 and Ras-associated protein 7 (Rab7) and the underlying mechanism by protein phosphatase 2A (PP2A)-B56γ regulating mitophagy via dephosphorylation of p-Drp1Ser616 in HCC. Herein, we found that Drp1 was frequently upregulated and was associated with poor prognosis in HCC. Mitochondrial p-Drp1Ser616 was a novel inter-organelle tethering protein localized to mitochondrion and lysosome membrane contact sites (MCSs) via interaction with Rab7 to trigger an increase in the mitochondria-lysosome crosstalk, resulting in PINK1-Parkin-dependent mitophagy and anti-apoptosis in HCC cells under the treatment of chemotherapy drugs. Moreover, we demonstrate that B56γ-mediated direct dephosphorylation of p-Drp1Ser616 inhibited mitophagy and thus increased mitochondria-dependent apoptosis. Overall, our findings demonstrated that activation of B56γ sensitizes the anti-cancer effect of HCC chemoprevention via dephosphorylated regulation of p-Drp1Ser616 in inhibiting the interaction between p-Drp1Ser616 and Rab7, which may provide a novel mechanism underlying the theranostics for targeting intervention in HCC.
    Keywords:  Rab7-dependent mitochondria-lysosome crosstalk; anti-cancer therapy of HCC; mitophagy; p-Drp1(Ser616); protein phosphatase 2A-B56γ
  4. EMBO Rep. 2022 Jun 14. e54825
      The mitochondrial respiratory chain (MRC) is composed of four multiheteromeric enzyme complexes. According to the endosymbiotic origin of mitochondria, eukaryotic MRC derives from ancestral proteobacterial respiratory structures consisting of a minimal set of complexes formed by a few subunits associated with redox prosthetic groups. These enzymes, which are the "core" redox centers of respiration, acquired additional subunits, and increased their complexity throughout evolution. Cytochrome c oxidase (COX), the terminal component of MRC, has a highly interspecific heterogeneous composition. Mammalian COX consists of 14 different polypeptides, of which COX7B is considered the evolutionarily youngest subunit. We applied proteomic, biochemical, and genetic approaches to investigate the COX composition in the invertebrate model Drosophila melanogaster. We identified and characterized a novel subunit which is widely different in amino acid sequence, but similar in secondary and tertiary structures to COX7B, and provided evidence that this object is in fact replacing the latter subunit in virtually all protostome invertebrates. These results demonstrate that although individual structures may differ the composition of COX is functionally conserved between vertebrate and invertebrate species.
    Keywords:   D. melanogaster ; COX7B; cytochrome c oxidase; mitochondria; respiratory chain
  5. Mol Cell Biochem. 2022 Jun 17.
      Chemotherapy resistance is the main reason for the failure of cancer treatment. The mechanism of drug resistance is complex and diverse. In recent years, the role of glucose metabolism and mitochondrial function in cancer resistance has gathered considerable interest. The increase in metabolic plasticity of cancer cells' mitochondria and adaptive changes to the mitochondrial function are some of the mechanisms through which cancer cells resist chemotherapy. As a key molecule regulating the mitochondrial function and glucose metabolism, PGC-1α plays an indispensable role in cancer progression. However, the role of PGC-1α in chemotherapy resistance remains controversial. Here, we discuss the role of PGC-1α in glucose metabolism and mitochondrial function and present a comprehensive overview of PGC-1α in chemotherapy resistance.
    Keywords:  Cancer; Chemoresistance; Metabolism; Mitochondrion; PGC-1α
  6. Glycobiology. 2022 Jun 16. pii: cwac038. [Epub ahead of print]
      Co-targeting of O-GlcNAc transferase (OGT) and the transcriptional kinase CDK9 is toxic to prostate cancer cells. As OGT is an essential glycosyltransferase, identifying an alternative target showing similar effects is of great interest. Here, we used a multiomics approach (transcriptomics, metabolomics and proteomics) to better understand the mechanistic basis of the combinatorial lethality between OGT and CDK9 inhibition. CDK9 inhibition preferentially affected transcription. In contrast, depletion of OGT activity predominantly remodeled the metabolome. Using an unbiased systems biology approach (weighted gene correlation network analysis), we discovered that CDK9 inhibition alters mitochondrial activity / flux, and high OGT activity is essential to maintain mitochondrial respiration when CDK9 activity is depleted. Our metabolite profiling data revealed that pantothenic acid (vitamin B5) is the metabolite that is most robustly induced by both OGT and OGT+CDK9 inhibitor treatments, but not by CDK9 inhibition alone. Finally, supplementing prostate cancer cell lines with vitamin B5 in the presence of CDK9 inhibitor mimics the effects of co-targeting OGT and CDK9.
    Keywords:  Cyclin-dependent kinase 9; O-GlcNAc transferase; metabolism; prostate cancer; systems biology
  7. Prog Mol Subcell Biol. 2022 ;61 15-26
      In this chapter, the current understanding of the potential roles played by polyphosphate in mitochondrial function with a specific focus on energy metabolism and mitochondrial pathologies caused by stress is summarized. Here we will discuss details of the possible ion transporting mechanisms of mitochondria that might involve polyP and their role in mitochondrial physiology and pathology are discussed.
    Keywords:  ATP; Calcium; Mitochondrial function; Oxidative phosphorylation; Permeability transition pore; PolyP hydrolyzing enzyme; Polyhydroxybutyrate; Polyphosphate kinase
  8. Bioessays. 2022 Jun 16. e2200056
      A decade ago I postulated that ROS formation in mitochondria was influenced by different FADH2 /NADH (F/N) ratios of catabolic substrates. Thus, fatty acid oxidation (FAO) would give higher ROS formation than glucose oxidation. Both the emergence of peroxisomes and neurons not using FAO, could be explained thus. ROS formation in NADH:ubiquinone oxidoreductase (Complex I) comes about by reverse electron transport (RET) due to high QH2 levels, and scarcity of its electron-acceptor (Q) during FAO. The then new, unexpected, finding of an FAO enzyme, ACAD9, being involved in complex I biogenesis, hinted at connections in line with the hypothesis. Recent findings about ACAD9's role in regulation of respiration fit with predictions the model makes: cementing connections between ROS production and F/N ratios. I describe how ACAD9 might be central to reversing the oxidative damage in complex I resulting from FAO. This seems to involve two distinct, but intimately connected, ACAD9 characteristics: (i) its upregulation of complex I biogenesis, and (ii) releasing FADH2 , with possible conversion into FMN, the crucial prosthetic group of complex I.
    Keywords:  ACAD9; Beta-oxidation; ECSIT; FADH2/NADH ratio; MCIA complex; peroxisomes; reverse electron transport (RET)
  9. Nat Commun. 2022 Jun 17. 13(1): 3486
      Mitochondria generate ATP and play regulatory roles in various cellular activities. Cancer cells often exhibit fragmented mitochondria. However, the underlying mechanism remains elusive. Here we report that a mitochondrial protein FUN14 domain containing 2 (FUNDC2) is transcriptionally upregulated in primary mouse liver tumors, and in approximately 40% of human hepatocellular carcinoma (HCC). Importantly, elevated FUNDC2 expression inversely correlates with patient survival, and its knockdown inhibits liver tumorigenesis in mice. Mechanistically, the amino-terminal region of FUNDC2 interacts with the GTPase domain of mitofusin 1 (MFN1), thus inhibits its activity in promoting fusion of outer mitochondrial membrane. As a result, loss of FUNDC2 leads to mitochondrial elongation, decreased mitochondrial respiration, and reprogrammed cellular metabolism. These results identified a mechanism of mitochondrial fragmentation in cancer through MFN1 inhibition by FUNDC2, and suggested FUNDC2 as a potential therapeutic target of HCC.
  10. Cell Mol Gastroenterol Hepatol. 2022 Jun 14. pii: S2352-345X(22)00102-3. [Epub ahead of print]
      BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a multistep process whereby abnormally proliferating cancer cells undergo extensive metabolic reprogramming. Metabolic alterations in hepatocarcinogenesis depend on the activation of specific oncogenes, thus partially explaining HCC heterogeneity. C-Myc oncogene overexpression, frequently observed in human HCCs, leads to a metabolic rewiring toward a Warburg phenotype and production of lactate, resulting in the acidification of the extracellular space, favoring the emergence of an immune-permissive tumor microenvironment. Here, we investigated whether Lactate dehydrogenase alpha (Ldha) genetic ablation interferes with metabolic reprogramming and HCC development in the mouse.METHODS: We characterized the metabolic reprogramming in tumors induced in C57BL/6J mice hydrodynamically co-transfected with c-Myc and h-Ras. Using the same experimental model, we investigated the effect of Ldha inhibition - gained through the inducible and hepatocyte-specific Ldha knockout - on cancer cell metabolic reprogramming, number and size of HCC lesions, and TME alterations.
    RESULTS: C-Myc/h-Ras driven tumors display a striking glycolytic metabolism, suggesting a switch to a Warburg phenotype. The tumors also exhibited enhanced pentose phosphate pathway activity, the switch of glutamine to sustain glutathione synthesis instead of Tricarboxylic acid cycle, and the impairment of oxidative phosphorylation. In addition, Ldha abrogation significantly hampered tumor number and size together with an evident inhibition of the Warburg-like metabolic feature and a remarkable increase of CD4+ lymphocytes compared to Ldha wild-type livers.
    CONCLUSIONS: These results demonstrate that Ldha deletion significantly impairs mouse HCC development and suggest LDH as a potential target to enhance the efficacy of the current therapeutic options.
    Keywords:  C-Myc; HCC; Ldha; Metabolic Reprogramming; TME
  11. Sci Rep. 2022 Jun 13. 12(1): 9740
      We aimed to determine the mechanism by which the sodium glucose co-transporter 2 inhibitor, luseogliflozin, preserves pancreatic beta-cell mass and function in db/db mice. Six-week-old db/db mice were fed to standard chow or standard chow containing 0.01% luseogliflozin. After 4 weeks, DNA microarray analysis, real-time PCR analysis, and measurement of mitochondrial respiratory capacity and reactive oxygen species (ROS) generation were performed using isolated islets. Immunohistochemistry and electron microscopic analysis were performed using pancreatic tissues. Metabolites extracted from the islets were measured by capillary electrophoresis mass spectrometry. The expression of genes involved in the tricarboxylic acid (TCA) cycle and electron transport chain was upregulated by luseogliflozin. Luseogliflozin improved the mitochondrial complex II-linked oxidative phosphorylation capacity and reduced ROS generation. Mitochondrial morphology was normally maintained by luseogliflozin. Luseogliflozin increased NK6 homeobox 1 (NKX6.1) expression and TCA cycle metabolites. Relief of glucotoxicity by luseogliflozin may involve lower mitochondrial ROS generation and an improvement in complex II-linked mitochondrial respiration. Reducing ROS generation through preventing complex II damage likely increases NKX6.1 expression and ameliorate glucose metabolism in the TCA cycle, contributing to the protection of pancreatic beta-cells. Protection of complex II in pancreatic beta-cells represents a novel therapeutic target for type 2 diabetes.
  12. Int J Mol Med. 2022 Aug;pii: 104. [Epub ahead of print]50(2):
      Mitochondria are the critical organelles involved in various cellular functions. Mitochondrial biogenesis is activated by multiple cellular mechanisms which require a synchronous regulation between mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). The mitochondrial DNA copy number (mtDNA‑CN) is a proxy indicator for mitochondrial activity, and its alteration reflects mitochondrial biogenesis and function. Despite the precise mechanisms that modulate the amount and composition of mtDNA, which have not been fully elucidated, mtDNA‑CN is known to influence numerous cellular pathways that are associated with cancer and as well as multiple other diseases. In addition, the utility of current technology in measuring mtDNA‑CN contributes to its extensive assessment of diverse traits and tumorigenesis. The present review provides an overview of mtDNA‑CN variations across human cancers and an extensive summary of the existing knowledge on the regulation and machinery of mtDNA‑CN. The current information on the advanced methods used for mtDNA‑CN assessment is also presented.
    Keywords:  biomarker; cancer; mitochondrial DNA; mtDNA copy number alterations; mtDNA replication
  13. J Immunol. 2022 Jun 13. pii: ji2100567. [Epub ahead of print]
      Obesity is associated with increased cancer risk and weak responses to vaccination and sepsis treatment. Although dendritic cells (DCs) are fundamental for the initiation and maintenance of competent immune responses against pathogens and tumors, how obesity alters the normal physiology of these myeloid cells remains largely unexplored. In this study, we report that obesity caused by prolonged high-fat diet feeding disrupts the metabolic and functional status of mouse splenic DCs (SpDCs). High-fat diet-induced obesity drastically altered the global transcriptional profile of SpDCs, causing severe changes in the expression of gene programs implicated in lipid metabolism and mitochondrial function. SpDCs isolated from obese mice demonstrated enhanced mitochondrial respiration provoked by increased fatty acid oxidation (FAO), which drove the intracellular accumulation of reactive oxygen species that impaired Ag presentation to T cells. Accordingly, treatment with the FAO inhibitor etomoxir, or antioxidants such as vitamin E or N-acetyl-l-cysteine, restored the Ag-presenting capacity of SpDCs isolated from obese mice. Our findings reveal a major detrimental effect of obesity in DC physiology and suggest that controlling mitochondrial FAO or reactive oxygen species overproduction may help improve DC function in obese individuals.
  14. Exp Cell Res. 2022 Jun 09. pii: S0014-4827(22)00242-7. [Epub ahead of print] 113249
      The well-documented tumor suppressor p53 is also a major stress response factor for its diverse regulation on cellular energetics. However, the effect of p53 on mitochondrial biogenesis, which plays a predominant role in response to the elevated energy demands, appears to be pleiotropic in various conditions and has not reached agreement. Mitochondrial ribosomal protein L12 (MRPL12), reported as a bi-functional protein for its roles in both mitochondrial ribosomes and transcriptional complexes, is a core regulatory component in mitochondrial biogenesis. Here we proved that MRPL12 is transcriptionally regulated by p53. Furthermore, the p53/MRPL12 regulation of mitochondria is part of the signaling pathway that maintains the basal mitochondrial content and positively coordinates the mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) in response to metabolic perturbation. Since p53 serves as the'Guardian of the Genome', our findings may revealed a new mechanism underlying the conditions when more ATP is warranted to maintain the genome integrity and cell survival. Therefore the pharmacological intervention or metabolic modulation (e.g., through fasting or exercise) of the p53/MRPL12 pathway promises to be a therapeutic approach that can safeguard health.
    Keywords:  MRPL12(2); Mitochondrial DNA(3); Mitochondrial transcription(4); Oxidative phosphorylation(5); p53(1)
  15. Carbohydr Polym. 2022 Sep 01. pii: S0144-8617(22)00369-1. [Epub ahead of print]291 119464
      The anti-cancer effects of Aloe vera barbadensis extract C (AVBEC) have been demonstrated in a previous study. However, the specific functional ingredient and mechanism remain undefined. This study aimed to evaluate the function and associated mechanisms of purified polysaccharide (ABPA1) from AVBEC on colorectal cancer. Here, we identify that ABPA1 can induce colorectal cancer apoptosis. In vivo, ABPA1 significantly suppressed tumor growth in an orthotopic colon cancer model. Mechanistically, ABPA1 alters mitochondrial membrane permeability by promoting Bax translocation while causing cytochrome-c release, which initiates the caspase cascade reaction. Additionally, we found that ABPA1 exerted distinct impacts on the mitochondrial metabolism of colorectal cancer cells. Our study elucidated the mechanism by which the polysaccharide ABPA1 induces apoptosis in colorectal cancer cells through the regulation of Bax and cytochrome-c mediated mitochondrial pathway, indicating that ABPA1 may be developed as a mitochondrial-targeting anti-cancer drug.
    Keywords:  Aloe polysaccharide; Apoptosis; Colorectal cancer; Metabolism; Mitochondria
  16. J Hepatol. 2022 Jun 14. pii: S0168-8278(22)00364-6. [Epub ahead of print]
      BACKGROUND & AIMS: Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular metabolism, most of the SLC25 family members remain uncharacterized. We undertook a comprehensive tissue distribution analysis of all Slc25 family members across metabolic organs and identified SLC25A47 as a liver-specific mitochondrial carrier.METHOD: We used a murine loss-of-function model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. We performed extensive metabolic phenotyping and molecular characterization of newly generated Slc25a47hep-/- and Slc25a47-Fgf21hep-/- mice.
    RESULTS: Slc25a47hep-/- mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration in this organ. This mitochondrial phenotype was associated with an activation of the mitochondrial stress response (MSR) in the liver, and the development of fibrosis, which was exacerbated upon feeding a high-fat high-sucrose diet. The MSR induced the secretion of several mitokines, amongst which FGF21 played a preponderant role on systemic physiology. To dissect the FGF21-dependent and -independent physiological changes induced in Slc25a47hep-/- mice, we generated a double Slc25a47-Fgf21hep-/- mouse model and demonstrated that several aspects of the hypermetabolic state were driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility observed in Slc25a47hep-/- mice could not be rescued with the genetic removal of Fgf21.
    CONCLUSION: Collectively, our data place SLC25A47 at the center of mitochondrial homeostasis, which upon dysfunction triggers robust liver-specific and systemic adaptive stress responses. The prominent role of SLC25A47 in hepatic fibrosis identifies this carrier, or its transported metabolite, as a potential target for therapeutic intervention.
    LAY SUMMARY: SLC25A47 is a liver-specific mitochondrial carrier. Slc25a47hep-/- mice are unable to maintain mitochondrial homeostasis in hepatocytes and show impaired mitochondrial respiration resulting in chronic energy deficiency, mitochondrial stress, and fibrosis in hepatocytes. Hepatic mitochondrial stress is characterized by the secretion of the mitokine FGF21 which drives a strong and systemic hypermetabolic state impacting whole-body physiology.
    Keywords:  FGF21; Fibrosis; Liver; Metabolism; Mitochondrial solute carriers; Mitochondrial stress response
  17. J Biol Chem. 2022 Jun 10. pii: S0021-9258(22)00564-6. [Epub ahead of print] 102123
      High levels of redox enzymes have been commonly observed in various types of human cancer, although whether and how the enzymes contribute to cancer malignancy and therapeutic resistance have yet to be understood. Peroxiredoxin IV (Prx4) is an antioxidant with bona fide peroxidase and molecular chaperone functions. Here, we report that Prx4 is highly expressed in prostate cancer patient specimens as well as established prostate cancer cell lines, and that its levels can be further stimulated through the activation of androgen receptor (AR) signaling. We used lentivirus-mediated shRNA knockdown and CRISPR-Cas9 based knockout techniques to establish Prx4-depleted prostate cancer cells, which showed delayed cell cycle progression, reduced rate of cell proliferation, migration and invasion compared to control cells. In addition, we used proteome profiler phospho-kinase arrays to identify signaling changes in Prx4-depleted cells; we found that loss of Prx4 results in insufficient phosphorylation of both Akt and its downstream kinase GSK3α/β. Moreover, we demonstrate that Prx4-depleted cells are more sensitive to ionizing radiation as they display compromised ability to scavenge reactive oxygen species (ROS) and increased accumulation of DNA damage. In mouse xenograft models, we show depletion of Prx4 leads to significant suppression of tumor growth, and tumors formed by Prx4-depleted cells respond more effectively to radiation therapy. Our findings suggest that increased levels of Prx4 contribute to the malignancy and radioresistance of prostate cancer through the activation of Akt/GSK3 signaling pathways. Therefore, strategies targeting Prx4 may be utilized to potentially inhibit tumor growth and overcome radioresistance in prostate cancer.
    Keywords:  antioxidant; peroxiredoxin; prostate cancer; radioresistance
  18. Life Sci Alliance. 2022 Oct;pii: e202201478. [Epub ahead of print]5(10):
      Ion fluxes across the inner mitochondrial membrane control mitochondrial volume, energy production, and apoptosis. TMBIM5, a highly conserved protein with homology to putative pH-dependent ion channels, is involved in the maintenance of mitochondrial cristae architecture, ATP production, and apoptosis. Here, we demonstrate that overexpressed TMBIM5 can mediate mitochondrial calcium uptake. Under steady-state conditions, loss of TMBIM5 results in increased potassium and reduced proton levels in the mitochondrial matrix caused by attenuated exchange of these ions. To identify the in vivo consequences of TMBIM5 dysfunction, we generated mice carrying a mutation in the channel pore. These mutant mice display increased embryonic or perinatal lethality and a skeletal myopathy which strongly correlates with tissue-specific disruption of cristae architecture, early opening of the mitochondrial permeability transition pore, reduced calcium uptake capability, and mitochondrial swelling. Our results demonstrate that TMBIM5 is an essential and important part of the mitochondrial ion transport system machinery with particular importance for embryonic development and muscle function.
  19. Mol Metab. 2022 Jun 09. pii: S2212-8778(22)00095-3. [Epub ahead of print] 101526
      OBJECTIVE: Uncoupling protein 1 (UCP1) catalyses mitochondrial proton leak in brown adipose tissue to facilitate nutrient oxidation for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we set out to characterise activator binding to purified UCP1 to clarify the activation process, discern novel activators and the potential to target UCP1.METHODS: We assessed ligand binding to purified UCP1 by protein thermostability shift analysis, which unlike many conventional approaches can inform on the binding of hydrophobic ligands to membrane proteins. A detailed activator interaction analysis and screening approach was carried out, supported by investigations of UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1 expression-controlled cell lines.
    RESULTS: We reveal that fatty acids and other activators influence UCP1 through a specific destabilising interaction, behaving as transport substrates that shift the protein to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the over-the-counter drug ibuprofen, where ligand analysis indicates that UCP1 has a relatively wide structural specificity for interacting molecules. Ibuprofen successfully induced UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1-expressing HEK293 cells but not in cultured brown adipocytes, suggesting drug delivery differs in each cell type.
    CONCLUSIONS: These findings clarify the nature of the activator-UCP1 interaction and demonstrate that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.
    Keywords:  Brown adipose tissue; Differential scanning fluorimetry; Energy expenditure; Ligand binding; Mitochondrial carrier; Proton transport; Thermal stability assay
  20. J Biol Chem. 2022 Jun 14. pii: S0021-9258(22)00581-6. [Epub ahead of print] 102139
      Copper (Cu) and iron (Fe) are redox-active metals that serve as cofactors for many essential cellular enzymes. Disruption in the intracellular homeostasis of these metals results in debilitating and frequently fatal human disorders, such as Menkes disease and Friedreich's ataxia. Recently, we reported that an investigational anticancer drug, elesclomol (ES), can deliver Cu to critical mitochondrial cuproenzymes and has the potential to be repurposed for treatment of Cu deficiency disorders. Here, we sought to determine the specificity of ES and the ES-Cu complex in delivering Cu to cuproenzymes in different intracellular compartments. Using a combination of yeast genetics, subcellular fractionation, and inductively coupled plasma-mass spectrometry-based metal measurements, we showed that ES and ES-Cu treatment results in an increase in cellular and mitochondrial Fe content, along with the expected increase in Cu. Utilizing yeast mutants of Cu and Fe transporters, we demonstrate that ES-based elevation in cellular Fe levels is independent of the major cellular Cu importer, but is dependent on the Fe importer Ftr1 and its partner Fet3, a multicopper-oxidase. As Fet3 is metallated in the Golgi lumen, we sought to uncover the mechanism by which Fet3 receives Cu from ES. Using yeast knockouts of genes involved in Cu delivery to Fet3, we determined that ES can bypass Atx1, a metallochaperone involved in Cu delivery to the Golgi membrane Cu pump, Ccc2, but not Ccc2 itself. Taken together, our study provides a mechanism by which ES distributes Cu in cells and impacts cellular and mitochondrial Fe homeostasis.
    Keywords:  Ccc2; Copper; Fet3; Golgi; iron; mitochondria
  21. Stem Cell Res Ther. 2022 Jun 17. 13(1): 256
      The most frequent biochemical defect of inherited mitochondrial disease is isolated complex I deficiency. There is no cure for this disorder, and treatment is mainly supportive. In this study, we investigated the effects of human mesenchymal stem cells (MSCs) on skin fibroblast derived from three individuals with complex I deficiency carrying different pathogenic variants in mitochondrial DNA-encoded subunits (MT-ND3, MT-ND6). Complex I-deficient fibroblasts were transiently co-cultured with bone marrow-derived MSCs. Mitochondrial transfer was analysed by fluorescence labelling and validated by Sanger sequencing. Levels of reactive oxygen species (ROS) were measured using MitoSOX Red. Moreover, mitochondrial respiration was analysed by Seahorse XFe96 Extracellular Flux Analyzer. Levels of antioxidant proteins were investigated via immunoblotting. Co-culturing of complex I-deficient fibroblast with MSCs lowered cellular ROS levels. The effect on ROS production was more sustained compared to treatment of patient fibroblasts with culture medium derived from MSC cultures. Investigation of cellular antioxidant defence systems revealed an upregulation of SOD2 (superoxide dismutase 2, mitochondrial) and HO-1 (heme oxygenase 1) in patient-derived cell lines. This adaptive response was normalised upon MSC treatment. Moreover, Seahorse experiments revealed a significant improvement of mitochondrial respiration, indicating a mitigation of the oxidative phosphorylation defect. Experiments with repetitive MSC co-culture at two consecutive time points enhanced this effect. Our study indicates that MSC-based treatment approaches might constitute an interesting option for patients with mitochondrial DNA-encoded mitochondrial diseases. We suggest that this strategy may prove more promising for defects caused by mitochondrial DNA variants compared to nuclear-encoded defects.
    Keywords:  Complex I; Gene therapy; Mesenchymal stem cells; Mitochondrial DNA; Mitochondrial transfer; ND3; ND6
  22. J Clin Invest. 2022 Jun 14. pii: e157504. [Epub ahead of print]
      Mitochondrial stress triggers a response in the cell's mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a knock-in mouse model and found that mutant CHCHD10 aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, survival of CHCHD10 knock-in mice depended on a protective stress response mediated by OMA1. The OMA1 stress response acted both locally within mitochondria, causing mitochondrial fragmentation, and signaled outside the mitochondria, activating the integrated stress response through cleavage of DELE1. We additionally identified an isoform switch in the terminal complex of the electron transport chain as a component of this response. Our results demonstrate that OMA1 is critical for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.
    Keywords:  Cell Biology; Cell stress; Genetics; Mitochondria; Proteases
  23. Front Oncol. 2022 ;12 873516
      Background: Pyruvate dehydrogenase (PDH) complex converts pyruvate into acetyl-CoA by pyruvate decarboxylation, which drives energy metabolism during cell growth, including prostate cancer (PCa) cell growth. The major catalytic subunit of PDH, PDHA1, is regulated by phosphorylation/dephosphorylation by pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). There are four kinases, PDK1, PDK2, PDK3 and PDK4, which can phosphorylate and inactivate PDH; and two phosphatases, PDP1 and PDP2, that dephosphorylate and activate PDH.Methods: We have analyzed by immunohistochemistry the expression and clinicopathological correlations of PDHA1, PDP1, PDP2, PDK1, PDK2, PDK3, and PDK4, as well as of androgen receptor (AR), in a retrospective PCa cohort of patients. A total of 120 PCa samples of representative tumor areas from all patients were included in tissue microarray (TMA) blocks for analysis. In addition, we studied the subcellular localization of PDK2 and PDK3, and the effects of the PDK inhibitor dichloroacetate (DCA) in the growth, proliferation, and mitochondrial respiration of PCa cells.
    Results: We found heterogeneous expression of the PDH complex components in PCa tumors. PDHA1, PDP1, PDK1, PDK2, and PDK4 expression correlated positively with AR expression. A significant correlation of PDK2 immunostaining with biochemical recurrence and disease-free survival was revealed. In PCa tissue specimens, PDK2 displayed cytoplasmic and nuclear immunostaining, whereas PDK1, PDK3 and PDK4 showed mostly cytoplasmic staining. In cells, ectopically expressed PDK2 and PDK3 were mainly localized in mitochondria compartments. An increase in maximal mitochondrial respiration was observed in PCa cells upon PDK inhibition by DCA, in parallel with less proliferative capacity.
    Conclusion: Our findings support the notion that expression of specific PDH complex components is related with AR signaling in PCa tumors. Furthermore, PDK2 expression associated with poor PCa prognosis. This highlights a potential for PDH complex components as targets for intervention in PCa.
    Keywords:  androgen receptor (AR); dichloroacetate (DCA); prostate cancer (PCa); pyruvate dehydrogenase (PDH); pyruvate dehydrogenase kinase (PDK)
  24. Am J Physiol Cell Physiol. 2022 Jun 15.
      Mitochondria buffer cytosolic Ca2+increases following Ca2+ influx from extracellular spaces and Ca2+ release from intracellular Ca2+ store sites under physiological circumstances. Therefore, close contact of mitochondria with the sarcoplasmic reticulum (SR) is required for maintaining Ca2+ homeostasis. Mitofusin 2 (Mfn2) localizes in both mitochondrial and SR membranes, and is hypothesized to optimize the distance and Ca2+ transfer between these organelles. However, the physiological significance of Mfn2 in vascular smooth muscle cells (VSMCs) is poorly understood. In the present study, the role of Mfn2 in the physical and functional couplings between SR and mitochondria was examined in rat aortic smooth muscle cells (rASMCs) by confocal and electron microscope imaging. When Mfn2 was knocked-down using siRNA in rASMCs, the mean distance between these organelles was extended from 16.2 to 21.6 nm. The increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) induced by 100 nM arginine vasopressin (AVP) was not affected by Mfn2 siRNA knockdown, whereas cytosolic Ca2+ removal was slower after Mfn2 knockdown. Following the AVP-induced [Ca2+]cyt increase, mitochondrial Ca2+ uptake and Ca2+ refill into the SR were attenuated by Mfn2 knockdown. In addition, Mfn2-knockdown cells exhibited a loss of mitochondrial membrane potential (ΔΨmito) and lower ATP levels in mitochondria. Moreover, Mfn2 knockdown inhibited cell proliferation. In contrast, Mfn2 overexpression increased ΔΨmito and cell growth. This study strongly suggests that Mfn2 is responsible for SR-mitochondria Ca2+ signaling by tethering mitochondria to SR, thereby regulating ATP production and proliferation of VSMCs.
    Keywords:  calcium; mitochondria; mitofusin; sarcoplasmic reticulum; smooth muscle
  25. ChemMedChem. 2022 Jun 17.
      Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a mitochondrial 1-carbon metabolism enzyme, which is an attractive anticancer drug target as it is highly upregulated in cancer but is not expressed in healthy adult cells. Selective MTHFD2 inhibitors could therefore offer reduced side-effects during treatment, which are common with antifolate drugs that target other 1C-metabolism enzymes. This task is challenging however, as MTHFD2 shares high sequence identity with the constitutively expressed isozymes cytosolic MTHFD1 and mitochondrial MTHFD2L. In fact, one of the most potent MTHFD2 inhibitors reported to date, TH7299, is actually more active against MTHFD1 and MTHFD2L. While structures of MTHFD2 and MTHFD1 exist, no MTHFD2L structures are available. We determined the first structure of MTHFD2L and its complex with TH7299, which reveals the structural basis for its highly potent MTHFD2L inhibition. Detailed analysis of the MTHFD2L structure presented here clearly highlights the challenges associated with developing truly isoform-selective MTHFD2 inhibitors.
    Keywords:  1C-metabolism; cancer; enzyme inhibition; methylene tetrahydrofolate dehydrogenase; structural biology
  26. Sci Adv. 2022 Jun 17. 8(24): eabo4271
      Infection is one of the major causes of mortality in patients with systemic lupus erythematosus (SLE). We previously found that CD38, an ectoenzyme that regulates the production of NAD+, is up-regulated in CD8+ T cells of SLE patients and correlates with the risk of infection. Here, we report that CD38 reduces CD8+ T cell function by negatively affecting mitochondrial fitness through the inhibition of multiple steps of mitophagy, a process that is critical for mitochondria quality control. Using a murine lupus model, we found that administration of a CD38 inhibitor in a CD8+ T cell-targeted manner reinvigorated their effector function, reversed the defects in autophagy and mitochondria, and improved viral clearance. We conclude that CD38 represents a target to mitigate infection rates in people with SLE.
  27. PNAS Nexus. 2022 May;1(2): pgac056
      Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1 -/- mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.
  28. EMBO Mol Med. 2022 Jun 13. e16171
      Acute Myeloid Leukemia is one of the most aggressive blood cancers with a high frequency of relapse. While standard chemotherapy is able to target rapidly proliferating immature blasts, it fails to eradicate slowly proliferating Leukemic Stem Cells. Therefore, new therapeutic strategies that efficiently target LSCs are urgently needed. Recent studies suggest that LSCs have particular metabolic vulnerabilities, which would open the possibility of a therapeutic window with limited off-target effects on the normal hematopoietic system. In this issue of EMBO Molecular Medicine, So and colleagues investigate the mechanism of action of AG636, a new potent inhibitor of de novo pyrimidine synthesis, and discovered an unexpected link to AML protein translation essential for LSC function.
  29. Nature. 2022 Jun 15.
      Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1-5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanine occurs in CNDP2+ cells, including macrophages, monocytes and other immune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are also observed in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.
  30. Blood. 2022 Jun 15. pii: blood.2021014304. [Epub ahead of print]
      The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Since there is no common genetic alteration causing resistance to venetoclax in CLL and B cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole exome sequencing, methylated DNA immunoprecipitation sequencing and genome wide CRISPR/Cas9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter which is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity towards venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher OXPHOS and ATP production, resembling the metabolic phenotype that is seen upon venetoclax resistance. While PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity towards both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive DLBCL in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.
  31. Nat Chem Biol. 2022 Jun 16.
      Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.
  32. J Cancer. 2022 ;13(8): 2477-2489
      Backgrounds: Hepatocellular carcinoma (HCC) is a major type of death-causing cancer whose pathological mechanisms are not fully understood. In addition, the identification of effective biomarkers for HCC prognosis is in emergency. Although a variety of studies have shown that Complement C11 binding protein (C1QBP) may play a tumor-promoting or tumor-suppressive role in cancer, the functions and mechanisms of C1QBP in HCC progression are under-investigating. Methods and results: Bioinformatic approaches were employed for checking the expression of C1QBP in HCC patient samples and the association between C1QBP mRNA expression and survival rates of patients with HCC or the promoter methylation of C1QBP. MTT analysis, PI/Annexin V staining, transwell and metabolic flux assays were performed to examine the effects of C1QBP on proliferation, apoptosis, migration, invasion, and oxidative phosphorylation of HCC cells. In the present study, we observed that C1QBP is lower expressed in HCC samples and cell lines. Moreover, high levels of C1QBP were associated with unfavorable outcomes of HCC patients. Loss-of-function assays showed that proliferation, migration and invasion of HCC cells were mitigated while cell apoptosis was augmented upon the loos of C1QBP. Moreover, the oxidative phosphorylation was moderately decreased when C1QBP was depleted. Furthermore, we also investigated the methylation status and copy number variation of C1QBP and analyzed their correlation with its mRNA expression in HCC patients. Finally, we suggested that C1QBP is correlated with genes encoding ribosome RPL-related proteins and mitochondrial MRPL-related proteins in HCC patients. Conclusions: C1QBP is correlated with a poor prognosis of HCC patients and promotes the survival, migration and invasion of HCC cells.
    Keywords:  C1QBP; Hepatocellular carcinoma; Methylation; Migration; Survival
  33. Sci Rep. 2022 Jun 15. 12(1): 9977
      Inflammatory bowel disease (IBD) is a complex, chronic, relapsing and heterogeneous disease induced by environmental, genomic, microbial and immunological factors. MCJ is a mitochondrial protein that regulates the metabolic status of macrophages and their response to translocated bacteria. Previously, an acute murine model of DSS-induced colitis showed increased disease severity due to MCJ deficiency. Unexpectedly, we now show that MCJ-deficient mice have augmented tumor necrosis factor α converting enzyme (TACE) activity in the context of chronic inflammation. This adaptative change likely affects the balance between soluble and transmembrane TNF and supports the association of the soluble form and a milder phenotype. Interestingly, the general shifts in microbial composition previously observed during acute inflammation were absent in the chronic model of inflammation in MCJ-deficient mice. However, the lack of the mitochondrial protein resulted in increased alpha diversity and the reduction in critical microbial members associated with inflammation, such as Ruminococcus gnavus, which could be associated with TACE activity. These results provide evidence of the dynamic metabolic adaptation of the colon tissue to chronic inflammatory changes mediated by the control of mitochondrial function.