bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021‒02‒14
thirty-nine papers selected by
Kelsey Fisher-Wellman
East Carolina University


  1. J Physiol. 2021 Feb 12.
    Bartlett MF, Fitzgerald LF, Kent JA.
      NEW FINDINGS: The oxygen cost of high-intensity exercise at power outputs above an individual's lactate threshold (LT) is greater than would be predicted by the linear oxygen consumption-power relationship observed below the LT. However, whether these augmentations are caused by an increased ATP cost of force generation (ATPCOST ) or an increased oxygen cost of ATP synthesis is unclear. We used 31 P-MRS to measure changes in cytosolic [ADP] (intramyocellular marker of oxidative metabolism), oxidative ATP synthesis (ATPOX ), and ATPCOST during a 6-stage, stepwise knee extension protocol. ATPCOST was unchanged across stages. The relationship between [ADP] and muscle power output was augmented at workloads above the pH threshold (pHT ; proxy for LT), whereas increases in ATPOX were attenuated. These results suggest the greater oxygen cost of contractions at workloads beyond the pHT is not caused by mechanisms that increase ATPCOST , but rather mechanisms that alter intrinsic mitochondrial function or capacity.ABSTRACT: Increases in skeletal muscle metabolism and oxygen consumption are linearly related to muscle power output for workloads below the lactate threshold (LT), but are augmented (i.e., greater rate of increase relative to workload) thereafter. Presently, it is unclear whether these metabolic augmentations are caused by increases in the ATP cost of force generation (ATPCOST ) or changes in the efficiency of mitochondrial oxygen consumption and oxidative ATP synthesis (ATPOX ). To partition these two hypotheses in vivo, we used 31 P-MRS to calculate slopes relating step-changes in muscle work to concurrent changes in cytosolic phosphates and ATPOX before and after the pH threshold (pHT ; used here as a proxy for LT) within the vastus lateralis muscle of eight young adults during a stepwise knee extension test. Changes in muscle phosphates and ATPOX were linearly related to workload above and below the pHT . However, slopes above the pHT were greater for muscle phosphates (p<0.05) and lower for ATPOX (p<0.05) than were the slopes observed below the pHT . The maximal capacity for ATPOX (Vmax ) and ADP-specific ATPOX also declined beyond the pHT (p<0.05), whereas ATPCOST was unchanged (p = 0.10). These results oppose the hypothesis that high-intensity contractions increase ATPCOST and suggest that greater oxidative metabolism at workloads beyond the pHT is caused by mechanisms that affect intrinsic mitochondrial function or capacity, such as alterations in substrate selection or electron entry into the electron transport chain, temperature-mediated changes in mitochondrial permeability to protons, or stimulation of mitochondrial uncoupling by reactive oxygen species generation. This article is protected by copyright. All rights reserved.
    Keywords:  ATP cost; VO2 slow component; bioenergetics; metabolism; mitochondria; muscle; muscle fatigue; oxidative phosphorylation; uncoupling
    DOI:  https://doi.org/10.1113/JP280851
  2. Cells. 2021 Feb 10. pii: 369. [Epub ahead of print]10(2):
    Čunátová K, Reguera DP, Vrbacký M, Fernández-Vizarra E, Ding S, Fearnley IM, Zeviani M, Houštěk J, Mráček T, Pecina P.
      The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.
    Keywords:  COX; COX4; OXPHOS; biogenesis interdependency; cI; cIV; cIV assembly; complex I; complexome profiling; knock-out; mitochondria; mitochondrial protein synthesis
    DOI:  https://doi.org/10.3390/cells10020369
  3. Front Cell Dev Biol. 2020 ;8 609493
    Angebault C, Panel M, Lacôte M, Rieusset J, Lacampagne A, Fauconnier J.
      Besides skeletal muscle dysfunction, Duchenne muscular dystrophy (DMD) exhibits a progressive cardiomyopathy characterized by an impaired calcium (Ca2+) homeostasis and a mitochondrial dysfunction. Here we aimed to determine whether sarco-endoplasmic reticulum (SR/ER)-mitochondria interactions and mitochondrial function were impaired in dystrophic heart at the early stage of the pathology. For this purpose, ventricular cardiomyocytes and mitochondria were isolated from 3-month-old dystrophin-deficient mice (mdx mice). The number of contacts points between the SR/ER Ca2+ release channels (IP3R1) and the porine of the outer membrane of the mitochondria, VDAC1, measured using in situ proximity ligation assay, was greater in mdx cardiomyocytes. Expression levels of IP3R1 as well as the mitochondrial Ca2+ uniporter (MCU) and its regulated subunit, MICU1, were also increased in mdx heart. MICU2 expression was however unchanged. Furthermore, the mitochondrial Ca2+ uptake kinetics and the mitochondrial Ca2+ content were significantly increased. Meanwhile, the Ca2+-dependent pyruvate dehydrogenase phosphorylation was reduced, and its activity significantly increased. In Ca2+-free conditions, pyruvate-driven complex I respiration was decreased whereas in the presence of Ca2+, complex I-mediated respiration was boosted. Further, impaired complex I-mediated respiration was independent of its intrinsic activity or expression, which remains unchanged but is accompanied by an increase in mitochondrial reactive oxygen species production. Finally, mdx mice were treated with the complex I modulator metformin for 1 month. Metformin normalized the SR/ER-mitochondria interaction, decreased MICU1 expression and mitochondrial Ca2+ content, and enhanced complex I-driven respiration. In summary, before any sign of dilated cardiomyopathy, the DMD heart displays an aberrant SR/ER-mitochondria coupling with an increase mitochondrial Ca2+ homeostasis and a complex I dysfunction. Such remodeling could be reversed by metformin providing a novel therapeutic perspective in DMD.
    Keywords:  Duchenne muscular dystrophy cardiomyopathy; MICU1; calcium; mitochondria-associated ER membrane; mitochondrial calcium uniporter
    DOI:  https://doi.org/10.3389/fcell.2020.609493
  4. Cancers (Basel). 2021 Feb 09. pii: 698. [Epub ahead of print]13(4):
    Courtois S, de Luxán-Delgado B, Penin-Peyta L, Royo-García A, Parejo-Alonso B, Jagust P, Alcalá S, Rubiolo JA, Sánchez L, Sainz B, Heeschen C, Sancho P.
      Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to its intrinsic aggressiveness, metastatic potential, and chemoresistance of the contained cancer stem cells (CSCs). Pancreatic CSCs strongly rely on mitochondrial metabolism to maintain their stemness, therefore representing a putative target for their elimination. Since mitochondrial homeostasis depends on the tightly controlled balance between fusion and fission processes, namely mitochondrial dynamics, we aim to study this mechanism in the context of stemness. In human PDAC tissues, the mitochondrial fission gene DNM1L (DRP1) was overexpressed and positively correlated with the stemness signature. Moreover, we observe that primary human CSCs display smaller mitochondria and a higher DRP1/MFN2 expression ratio, indicating the activation of the mitochondrial fission. Interestingly, treatment with the DRP1 inhibitor mDivi-1 induced dose-dependent apoptosis, especially in CD133+ CSCs, due to the accumulation of dysfunctional mitochondria and the subsequent energy crisis in this subpopulation. Mechanistically, mDivi-1 inhibited stemness-related features, such as self-renewal, tumorigenicity, and invasiveness and chemosensitized the cells to the cytotoxic effects of Gemcitabine. In summary, mitochondrial fission is an essential process for pancreatic CSCs and represents an attractive target for designing novel multimodal treatments that will more efficiently eliminate cells with high tumorigenic potential.
    Keywords:  CD133; DRP1; PDAC; cancer stem cells; energy crisis; mitochondria; mitochondrial dynamics; mitochondrial fission; pancreatic cancer
    DOI:  https://doi.org/10.3390/cancers13040698
  5. Mol Ther Nucleic Acids. 2021 Mar 05. 23 657-666
    Liu Y, Zhou K, Guo S, Wang Y, Ji X, Yuan Q, Su L, Guo X, Gu X, Xing J.
      Mitochondrial DNA (mtDNA) mutations are closely implicated in the pathogenesis of multiple cancers, making circulating cell-free mtDNA (ccf-mtDNA) as a potential non-invasive tumor biomarker. However, an effective approach to comprehensively profile ccf-mtDNA mutations is still lacking. In this study, we first characterized ccf-mtDNA by low-depth whole-genome sequencing (WGS) and found that plasma DNA samples exhibited a dramatic decrease in mtDNA copy number when compared with fresh tumor tissues. Further analysis revealed that plasma ccf-mtDNA had a biased distribution of fragment size with a peak around 90 bp. Based on these insights, we developed a robust captured-based mtDNA deep-sequencing approach that enables accurate and efficient detection of plasma ccf-mtDNA mutations by systematic optimization of probe quantity and length, hybridization temperature, and PCR amplification cycles. Moreover, we found that placement of isolated plasma for 6 h at both 4°C and room temperature (RT) led to a dramatic decrease of ccf-mtDNA stability, highlighting the importance of proper plasma sample processing. We further showed that the optimized approach can successfully detect a substantial fraction of tumor-specific mtDNA mutations in plasma ccf-mtDNA specifically from hepatocellular carcinoma (HCC) patients but not from colorectal cancer (CRC) patients, suggesting the presence of a potential cancer-specific difference in the abundance of tumor-derived mtDNA in plasma.
    DOI:  https://doi.org/10.1016/j.omtn.2020.12.017
  6. Mol Cell Oncol. 2021 ;8(1): 1839341
    Silva-Pavez E, Ahumada-Castro U, Lovy A, Cárdenas JC.
      The inositol 1,4,5-triphosphate receptor (InsP3R)-mediated calcium (Ca2+) transfer to mitochondria is important to maintain mitochondrial respiration and bioenergetics in normal and cancer cells, even though cancer cells have defective oxidative phosphorylation (OXPHOS). Here, we discuss how tumor mitochondria could become a feasible therapeutic target to treat tumors that depend on reductive carboxylation.
    Keywords:  Calcium; OXPHOS; autophagy; cancer; cell survival
    DOI:  https://doi.org/10.1080/23723556.2020.1839341
  7. Cell Mol Life Sci. 2021 Feb 13.
    Poole LP, Macleod KF.
      Cells use mitophagy to remove dysfunctional or excess mitochondria, frequently in response to imposed stresses, such as hypoxia and nutrient deprivation. Mitochondrial cargo receptors (MCR) induced by these stresses target mitochondria to autophagosomes through interaction with members of the LC3/GABARAP family. There are a growing number of these MCRs, including BNIP3, BNIP3L, FUNDC1, Bcl2-L-13, FKBP8, Prohibitin-2, and others, in addition to mitochondrial protein targets of PINK1/Parkin phospho-ubiquitination. There is also an emerging link between mitochondrial lipid signaling and mitophagy where ceramide, sphingosine-1-phosphate, and cardiolipin have all been shown to promote mitophagy. Here, we review the upstream signaling mechanisms that regulate mitophagy, including components of the mitochondrial fission machinery, AMPK, ATF4, FoxOs, Sirtuins, and mtDNA release, and address the significance of these pathways for stress responses in tumorigenesis and metastasis. In particular, we focus on how mitophagy modulators intersect with cell cycle control and survival pathways in cancer, including following ECM detachment and during cell migration and metastasis. Finally, we interrogate how mitophagy affects tissue atrophy during cancer cachexia and therapy responses in the clinic.
    Keywords:  AMPK; ATF4; Autophagy; BCL2-L-13; BNIP3/BNIP3L; Cachexia; DRP1; Electron transport chain; FUNDC1; Fission; FoxOs; LC3/GABARAP; Metabolism; Metastasis; Mitochondria; Mitohormesis; Mitophagy; NAD+; PARP; PINK1/Parkin; ROS; Respiration; Sirtuins; UPRmt
    DOI:  https://doi.org/10.1007/s00018-021-03774-1
  8. Mol Genet Metab Rep. 2021 Mar;26 100721
    Vamecq J, Pigny P.
      
    Keywords:  2-oxoglutarate-malate antiport; Aspartate; Chromaffin cells; Citrin; Complex II, SDHx; Cytosolic glutamine reductive pathway; Familial pheochromocytoma; Glutamine; NADH redox status; Paraganglioma; SCL25A13; SLC25A11; Succinate dehydrogenase
    DOI:  https://doi.org/10.1016/j.ymgmr.2021.100721
  9. Cell Mol Life Sci. 2021 Feb 13.
    Sessions DT, Kashatus DF.
      Many tumors are now understood to be heterogenous cell populations arising from a minority of epithelial-like cancer stem cells (CSCs). CSCs demonstrate distinctive metabolic signatures from the more differentiated surrounding tumor bulk that confer resistance to traditional chemotherapeutic regimens and potential for tumor relapse. Many CSC phenotypes including metabolism, epithelial-to-mesenchymal transition, cellular signaling pathway activity, and others, arise from altered mitochondrial function and turnover, which are regulated by constant cycles of mitochondrial fusion and fission. Further, recycling of mitochondria through mitophagy in CSCs is associated with maintenance of reactive oxygen species levels that dictate gene expression. The protein machinery that drives mitochondrial dynamics is surprisingly simple and may represent attractive new therapeutic avenues to target CSC metabolism and selectively eradicate tumor-generating cells to reduce the risks of metastasis and relapse for a variety of tumor types.
    Keywords:  Cancer stem cells; EMT; Metabolism; Mitochondrial dynamics; Mitochondrial morphology; Signaling; Therapeutic resistance
    DOI:  https://doi.org/10.1007/s00018-021-03773-2
  10. Cell Death Discov. 2020 Jul 24. 6(1): 64
    Vial J, Huchedé P, Fagault S, Basset F, Rossi M, Geoffray J, Soldati H, Bisaccia J, Elsensohn MH, Creveaux M, Neves D, Blay JY, Fauvelle F, Bouquet F, Streichenberger N, Corradini N, Bergeron C, Maucort-Boulch D, Castets P, Carré M, Weber K, Castets M.
      Rhabdomyosarcoma (RMS) is the most frequent form of pediatric soft-tissue sarcoma. It is divided into two main subtypes: ERMS (embryonal) and ARMS (alveolar). Current treatments are based on chemotherapy, surgery, and radiotherapy. The 5-year survival rate has plateaued at 70% since 2000, despite several clinical trials. RMS cells are thought to derive from the muscle lineage. During development, myogenesis includes the expansion of muscle precursors, the elimination of those in excess by cell death and the differentiation of the remaining ones into myofibers. The notion that these processes may be hijacked by tumor cells to sustain their oncogenic transformation has emerged, with RMS being considered as the dark side of myogenesis. Thus, dissecting myogenic developmental programs could improve our understanding of RMS molecular etiology. We focused herein on ANT1, which is involved in myogenesis and is responsible for genetic disorders associated with muscle degeneration. ANT1 is a mitochondrial protein, which has a dual functionality, as it is involved both in metabolism via the regulation of ATP/ADP release from mitochondria and in regulated cell death as part of the mitochondrial permeability transition pore. Bioinformatics analyses of transcriptomic datasets revealed that ANT1 is expressed at low levels in RMS. Using the CRISPR-Cas9 technology, we showed that reduced ANT1 expression confers selective advantages to RMS cells in terms of proliferation and resistance to stress-induced death. These effects arise notably from an abnormal metabolic switch induced by ANT1 downregulation. Restoration of ANT1 expression using a Tet-On system is sufficient to prime tumor cells to death and to increase their sensitivity to chemotherapy. Based on our results, modulation of ANT1 expression and/or activity appears as an appealing therapeutic approach in RMS management.
    DOI:  https://doi.org/10.1038/s41420-020-00302-1
  11. Biosci Biotechnol Biochem. 2020 Dec 28. pii: zbaa119. [Epub ahead of print]
    Amai T, Tsuji T, Ueda M, Kuroda K.
      Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a "mito-CRISPR system" that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.
    Keywords:  CRISPR-Cas9 system; Saccharomyces cerevisiae; mitochondrial DNA; mitochondrial target sequence; ρ° cells
    DOI:  https://doi.org/10.1093/bbb/zbaa119
  12. Oncol Lett. 2021 Feb;21(2): 124
    Zeng X, Zhao L, Chen S, Li X.
      Cancer cachexia is a life-threatening syndrome characterized by muscle atrophy. Cancer cachectic muscle atrophy (CCMA) is associated with mitochondrial injury. Mitochondrial calpains have been reported to induce mitochondrial injury in mouse cardiomyocytes and pulmonary smooth muscle. In the present study, the presence of calpain in the mitochondria of skeletal muscle and its potential role in CCMA were investigated. Transwell plates were used to develop a myotube-carcinoma cell co-culture model to simulate the cancer cachexia environment in vitro. The calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used to inhibit calpain activity in myotubes during co-culture. Calpain-1, calpain-2 and CAST were found to be present in mouse myotube mitochondria. Co-culture activated calpain in both cytoplasm and mitochondria, which caused myotube atrophy. CAST and CAPT treatment prevented calpain activation in both cytoplasm and mitochondria, which inhibited myotube atrophy during co-culture. Additionally, CAST and CAPT treatment increased mitochondrial complex I activity, decreased mitochondrial permeability transition pore opening and improved mitochondrial membrane potential in myotubes during co-culture. In addition, CAST and CAPT treatment increased AKT/mTOR activity, inhibited FoxO3a activity and decreased atrogin-1 content in myotubes during co-culture. The present findings provide new insights to understand the mechanism of CCMA and further help the development of focused approaches to treat CCMA by manipulating the mitochondrial and cytosolic calpain activity.
    Keywords:  calpain; cancer cachexia; co-culture; mitochondria; muscle atrophy
    DOI:  https://doi.org/10.3892/ol.2020.12385
  13. EMBO Rep. 2021 Feb 08. e50629
    Liu L, Li Y, Wang J, Zhang D, Wu H, Li W, Wei H, Ta N, Fan Y, Liu Y, Wang X, Wang J, Pan X, Liao X, Zhu Y, Chen Q.
      Mitophagy is an essential cellular autophagic process that selectively removes superfluous and damaged mitochondria, and it is coordinated with mitochondrial biogenesis to fine tune the quantity and quality of mitochondria. Coordination between these two opposing processes to maintain the functional mitochondrial network is of paramount importance for normal cellular and organismal metabolism. However, the underlying mechanism is not completely understood. Here we report that PGC-1α and nuclear respiratory factor 1 (NRF1), master regulators of mitochondrial biogenesis and metabolic adaptation, also transcriptionally upregulate the gene encoding FUNDC1, a previously characterized mitophagy receptor, in response to cold stress in brown fat tissue. NRF1 binds to the classic consensus site in the promoter of Fundc1 to upregulate its expression and to enhance mitophagy through its interaction with LC3. Specific knockout of Fundc1 in BAT results in reduced mitochondrial turnover and accumulation of functionally compromised mitochondria, leading to impaired adaptive thermogenesis. Our results demonstrate that FUNDC1-dependent mitophagy is directly coupled with mitochondrial biogenesis through the PGC-1α/NRF1 pathway, which dictates mitochondrial quantity, quality, and turnover and contributes to adaptive thermogenesis.
    Keywords:  adaptive thermogenesis; brown adipose tissue; mitochondrial biogenesis; mitophagy
    DOI:  https://doi.org/10.15252/embr.202050629
  14. Cells. 2021 Feb 05. pii: 325. [Epub ahead of print]10(2):
    Venturoli C, Piga I, Curtarello M, Verza M, Esposito G, Venuto S, Navaglia F, Grassi A, Indraccolo S.
      Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways-comprising both oxidative phosphorylation and glycolysis-in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment.
    Keywords:  PDK1; angiogenesis; glycolysis; metabolism; ovarian cancer
    DOI:  https://doi.org/10.3390/cells10020325
  15. Cancers (Basel). 2021 Jan 29. pii: 524. [Epub ahead of print]13(3):
    Santiappillai NT, Abuhammad S, Slater A, Kirby L, McArthur GA, Sheppard KE, Smith LK.
      Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are being tested in numerous clinical trials and are currently employed successfully in the clinic for the treatment of breast cancers. Understanding their mechanism of action and interaction with other therapies is vital in their clinical development. CDK4/6 regulate the cell cycle via phosphorylation and inhibition of the tumour suppressor RB, and in addition can phosphorylate many cellular proteins and modulate numerous cellular functions including cell metabolism. Metabolic reprogramming is observed in melanoma following standard-of-care BRAF/MEK inhibition and is involved in both therapeutic response and resistance. In preclinical models, CDK4/6 inhibitors overcome BRAF/MEK inhibitor resistance, leading to sustained tumour regression; however, the metabolic response to this combination has not been explored. Here, we investigate how CDK4/6 inhibition reprograms metabolism and if this alters metabolic reprogramming observed upon BRAF/MEK inhibition. Although CDK4/6 inhibition has no substantial effect on the metabolic phenotype following BRAF/MEK targeted therapy in melanoma, CDK4/6 inhibition alone significantly enhances mitochondrial metabolism. The increase in mitochondrial metabolism in melanoma cells following CDK4/6 inhibition is fuelled in part by both glutamine metabolism and fatty acid oxidation pathways and is partially dependent on p53. Collectively, our findings identify new p53-dependent metabolic vulnerabilities that may be targeted to improve response to CDK4/6 inhibitors.
    Keywords:  BRAF; CDK4; melanoma; metabolism; targeted therapy
    DOI:  https://doi.org/10.3390/cancers13030524
  16. Biochim Biophys Acta Bioenerg. 2021 Feb 04. pii: S0005-2728(21)00026-8. [Epub ahead of print] 148393
    Kuznetsov AV, Javadov S, Margreiter R, Grimm M, Hagenbuchner J, Ausserlechner MJ.
      Cancer cells bioenergetics is more dependent on glycolysis than mitochondrial oxidative phosphorylation, a phenomenon known as the Warburg Effect. It has been proposed that inhibition of glycolysis may selectively affect cancer cells. However, the effects of glycolysis inhibition on mitochondrial function and structure in cancer cells are not completely understood. Here, we investigated the comparative effects of 2-deoxy-D-glucose (2-DG, a glucose analogue, which suppresses cellular glycolysis) on cellular bioenergetics in human colon cancer DLD-1 cells, smooth muscle cells, human umbilical vein endothelial cells and HL-1 cardiomyocytes. In all cells, 2-DG treatment resulted in significant ATP depletion, however, the cell viability remained unchanged. Also, we did not observe the synergistic effects of 2-DG with anticancer drugs doxorubicin and 5-fluorouracil. Instead, after 2-DG treatment and ATP depletion, mitochondrial respiration and membrane potential were significantly enhanced and mitochondrial morphology changed in the direction of more network organization. Analysis of protein expression demonstrated that 2-DG treatment induced an activation of AMPK (elevated pAMPK/AMPK ratio), increased mitochondrial fusion (mitofusins 1 and 2) and decreased fission (Drp1) proteins. In conclusion, this study suggests a strong link between respiratory function and structural organization of mitochondria in the cell. We propose that the functionality of the mitochondrial network is enhanced compared to disconnected mitochondria.
    Keywords:  2-Deoxy-D-glucose; Cancer cells; Cellular ATP; Energy stress; Glucose metabolism; Mitochondria; Mitochondrial dynamics/network; Mitochondrial function; Mitochondrial membrane potential
    DOI:  https://doi.org/10.1016/j.bbabio.2021.148393
  17. Mol Cell Proteomics. 2021 Feb 06. pii: S1535-9476(21)00026-8. [Epub ahead of print] 100053
    Guo Z, Pan F, Peng L, Tian S, Jiao J, Liao L, Lu C, Zhai G, Wu Z, Dong H, Xu X, Wu J, Chen P, Bai X, Lin D, Xu L, Li E, Zhang K.
      Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and post-translational modifications (PTMs) leading to the pathogenesis of ESCC remains unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and post-translational modification (PTM) pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.
    Keywords:  Esophageal squamous; Migration; Post-translational modification; Succinylation
    DOI:  https://doi.org/10.1074/mcp.RA120.002150
  18. Phytomedicine. 2021 Jan 29. pii: S0944-7113(21)00025-8. [Epub ahead of print]83 153483
    Kim TJ, Kim HJ, Kang M, Cho JH, Kim YG, Lee SM, Byun JS, Kim DY.
      BACKGROUND: Glioblastoma (GBM) is the most aggressive tumor residing within the central nervous system, with extremely poor prognosis. Although the cytotoxic effects of ginsenoside F2 (GF2) on GBM were previously suggested, the precise anti-GBM mechanism of GF2 remains unclear. The aim of this study was to explore the anti-cancer molecular mechanism of GF2 toward human GBM.METHODS: GF2-driven cellular toxicity was confirmed in two different GBM cells, U373 and Hs683. To test mitochondrial impairment driven by GF2, we examined the mitochondrial membrane potential, OCR, and ATP production. An intracellular redox imbalance was identified by measuring the relative ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), glutaredoxin (GLRX) mRNA expression, intracellular NAD+ level, and AMPK phosphorylation status.
    RESULTS: GF2 increased the percentage of cleaved caspase 3-positive cells and γH2AX signal intensities, confirming that GF2 shows the cytotoxicity against GBM. GO enrichment analysis suggested that the mitochondrial function could be negatively influenced by GF2. GF2 reduced the mitochondrial membrane potential, basal mitochondrial respiratory rate, and ATP production capacity. Our results showed that GF2 downregulated the relative GSH/GSSG, intracellular NAD+ level, and GLRX expression, suggesting that GF2 may alter the intracellular redox balance that led to mitochondrial impairment.
    CONCLUSION: GF2 reduces mitochondrial membrane potential, inhibits cellular oxygen consumption, activates AMPK signaling, and induces cell death. Our study examined the potential vulnerability of mitochondrial activity in GBM, and this may hold therapeutic promise.
    Keywords:  Ginsenoside F2; Glioblastoma; Mitochondria
    DOI:  https://doi.org/10.1016/j.phymed.2021.153483
  19. Nat Cancer. 2020 ;1 653-664
    Parsa S, Ortega-Molina A, Ying HY, Jiang M, Teater M, Wang J, Zhao C, Reznik E, Pasion JP, Kuo D, Mohan P, Wang S, Camarillo JM, Thomas PM, Jain N, Garcia-Bermudez J, Cho BK, Tam W, Kelleher NL, Socci N, Dogan A, De Stanchina E, Ciriello G, Green MR, Li S, Birsoy K, Melnick AM, Wendel HG.
      Cancer cells adapt their metabolic activities to support growth and proliferation. However, increased activity of metabolic enzymes is not usually considered an initiating event in the malignant process. Here, we investigate the possible role of the enzyme serine hydroxymethyltransferase-2 (SHMT2) in lymphoma initiation. SHMT2 localizes to the most frequent region of copy number gains at chromosome 12q14.1 in lymphoma. Elevated expression of SHMT2 cooperates with BCL2 in lymphoma development; loss or inhibition of SHMT2 impairs lymphoma cell survival. SHMT2 catalyzes the conversion of serine to glycine and produces an activated one-carbon unit that can be used to support S-adenosyl methionine synthesis. SHMT2 induces changes in DNA and histone methylation patterns leading to promoter silencing of previously uncharacterized mutational genes, such as SASH1 and PTPRM. Together, our findings reveal that amplification of SHMT2 in cooperation with BCL2 is sufficient in the initiation of lymphomagenesis through epigenetic tumor suppressor silencing.
    DOI:  https://doi.org/10.1038/s43018-020-0080-0
  20. Free Radic Biol Med. 2021 Feb 09. pii: S0891-5849(21)00073-3. [Epub ahead of print]
    Chen X, Xu W, Zhuo S, Chen X, Chen P, Guan S, Huang D, Sun X, Cheng Y.
      Syntaphilin (SNPH) halts mitochondrial movements and regulates proliferation-motility phenotype switching of cancer cells. We sought to investigate the significance of SNPH-mediated mitochondria distribution in radioresistant (RR) phenotype switching in esophageal squamous cell carcinoma (ESCC). RR ESCC cells were established by long-term exposure to radiation. Effects of SNPH on proliferation, migration, mitochondrial distribution, radiation-induced oxidative damage and radiosensitivity were investigated by overexpressing or silencing SNPH. The mechanisms regulating SNPH expression and the potential molecules mediating the SNPH-re-expression-induced radiosensitization were explored. SNPH expression in specimens from 156 patients was analyzed to evaluate its clinical significance. We found that RR ESCC cells had a sparse mitochondrial network and lower SNPH level. SNPH reconstitution in RR ESCC cells inhibited migration, induced proliferation and mitochondrial aggregation, exacerbated the radiation-induced oxidative damage and ultimately promoted radiosensitization. Mechanistically, ubiquitin-proteasomal degradation and histone modification contributed to SNPH downregulation in RR ESCC cells. Subsequently, we found that CREB dephosphorylation facilitated the SNPH re-expression-induced radiosensitization. Furthermore, SNPH expression was correlated with the radiotherapeutic efficacy and served as an independent prognostic factor for survival of ESCC patients. Our study revealed that low SNPH expression was a novel indicator for radioresistance, and targeting SNPH could be a promising regimen to improve the radiotherapeutic efficiency in ESCC patients.
    Keywords:  Esophageal squamous cell carcinoma; Invasion; Mitochondrial distribution; Radioresistance; Syntaphilin
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.01.056
  21. Free Radic Res. 2021 Feb 08. 1-11
    Canizal-García M, Olmos-Orizaba BE, Moreno-Jiménez M, Calderón-Cortés E, Saavedra-Molina A, Cortés-Rojo C.
      Glutathione peroxidase 4 (Gpx4) counteracts mitochondrial lipid peroxidation in mammals. In yeast, Gpx2 is orthologous of Gpx4, is localized in mitochondria, and reduces both inorganic and organic peroxides. However, a phenotype of oxidative stress hypersensitivity has not been observed with gpx2 deletion. We hypothesized that the absence of polyunsaturated fatty acids (PUFA) in yeast membranes may mask an antioxidant role of Gpx2 in mitochondria. Thus, we tested the effects of PUFA on cell viability, mitochondrial function, ROS production, and mitochondrial fatty acid composition of a gpx2Δ mutant subjected to chronological aging. As expected, gpx2Δ mutation did not alter these parameters with respect to wild-type (WT) cells after 30 h growth, even in the presence of linolenic acid (C18:3), except for some activities of the electron transport chain (ETC) complexes. Conversely, aged gpx2Δ cells exhibited lower viability, impaired respiration, decreased ETC activities, and increased ROS generation in comparison to aged WT cells. These effects were exacerbated by C18:3, as gpx2Δ cells displayed residual respiration, full inhibition of ETC complexes, and a burst in ROS production on day 15 that decreased on day 30, although ROS remained several-fold higher than in WT cells. gpx2 was not involved in the preservation of PUFA levels, as no differences in mitochondrial C18:3 content were observed between WT and gpx2Δ cells. These results indicate that gpx2 is a late - acting antioxidant system that decreases mitochondrial ROS production and preserves ETC function, without being involved in the preservation of PUFA levels in mitochondria.
    Keywords:  Yeast; aging; electron transport chain; glutathione peroxidase; polyunsaturated fatty acids
    DOI:  https://doi.org/10.1080/10715762.2021.1882677
  22. Elife. 2021 Feb 10. pii: e61798. [Epub ahead of print]10
    Rosenkranz SC, Shaposhnykov AA, Träger S, Engler JB, Witte ME, Roth V, Vieira V, Paauw N, Bauer S, Schwencke-Westphal C, Schubert C, Bal LC, Schattling B, Pless O, van Horssen J, Freichel M, Friese MA.
      While transcripts of neuronal mitochondrial genes are strongly suppressed in central nervous system inflammation, it is unknown whether this results in mitochondrial dysfunction and whether an increase of mitochondrial function can rescue neurodegeneration. Here we show that predominantly genes of the electron transport chain are suppressed in inflamed mouse neurons resulting in impaired mitochondrial complex IV activity. This was associated with posttranslational inactivation of the transcriptional co-regulator PGC-1α. In mice, neuronal overexpression of Ppargc1a, which encodes for PGC-1α, led to increased numbers of mitochondria, complex IV activity and maximum respiratory capacity. Moreover, Ppargc1a overexpressing neurons showed a higher mitochondrial membrane potential that related to an improved calcium buffering capacity. Accordingly, neuronal deletion of Ppargc1a aggravated neurodegeneration during experimental autoimmune encephalomyelitis (EAE), while neuronal overexpression of Ppargc1a ameliorated it. Our study provides systemic insights into mitochondrial dysfunction in neurons during inflammation and commends elevation of mitochondrial activity as a promising neuroprotective strategy.
    Keywords:  immunology; inflammation; mouse; neuroscience
    DOI:  https://doi.org/10.7554/eLife.61798
  23. iScience. 2021 Feb 19. 24(2): 102034
    Hsieh JY, Yang HP, Tewary SK, Cheng HC, Liu YL, Tai SC, Chen WL, Hsu CH, Huang TJ, Chou CJ, Huang YN, Peng CT, Ho MC, Liu GY, Hung HC.
      Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is well recognized to associate with cancer cell metabolism, and the single nucleotide variants (SNVs) of ME2 may play a role in enzyme regulation. Here we reported that the SNVs of ME2 occurring in the allosteric sites lead to inactivation or overactivation of ME2. Two ME2-SNVs, ME2_R67Q and ME2-R484W, that demonstrated inactivating or overactivating enzyme activities of ME2, respectively, have different impact toward the cells. The cells with overactivating SNV enzyme, ME2_R484W, grow more rapidly and are more resistant to cellular senescence than the cells with wild-type or inactivating SNV enzyme, ME2_R67Q. Crystal structures of these two ME2-SNVs reveal that ME2_R67Q was an inactivating "dead form," and ME2_R484W was an overactivating "closed form" of the enzyme. The resolved ME2-SNV structures provide a molecular basis to explain the abnormal kinetic properties of these SNV enzymes.
    Keywords:  Biological Sciences; Cancer; Cell Biology; Genetics; Structural Biology
    DOI:  https://doi.org/10.1016/j.isci.2021.102034
  24. J Mol Cell Cardiol. 2021 Feb 05. pii: S0022-2828(21)00025-0. [Epub ahead of print]154 41-59
    Aravamudhan S, Türk C, Bock T, Keufgens L, Nolte H, Lang F, Krishnan RK, König T, Hammerschmidt P, Schindler N, Brodesser S, Rozsivalova DH, Rugarli E, Trifunovic A, Brüning J, Langer T, Braun T, Krüger M.
      Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1-/- mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.
    Keywords:  Heart development; Lipid metabolism; Mitochondria; PERM1; Phosphoproteomics; SILAC
    DOI:  https://doi.org/10.1016/j.yjmcc.2021.01.010
  25. Int J Mol Sci. 2021 Feb 07. pii: 1665. [Epub ahead of print]22(4):
    Chimienti G, Picca A, Fracasso F, Russo F, Orlando A, Riezzo G, Leeuwenburgh C, Pesce V, Lezza AMS.
      Calorie restriction (CR) is the most efficacious treatment to delay the onset of age-related changes such as mitochondrial dysfunction. However, the sensitivity of mitochondrial markers to CR and the age-related boundaries of CR efficacy are not fully elucidated. We used liver samples from ad libitum-fed (AL) rats divided in: 18-month-old (AL-18), 28-month-old (AL-28), and 32-month-old (AL-32) groups, and from CR-treated (CR) 28-month-old (CR-28) and 32-month-old (CR-32) counterparts to assay the effect of CR on several mitochondrial markers. The age-related decreases in citrate synthase activity, in TFAM, MFN2, and DRP1 protein amounts and in the mtDNA content in the AL-28 group were prevented in CR-28 counterparts. Accordingly, CR reduced oxidative mtDNA damage assessed through the incidence of oxidized purines at specific mtDNA regions in CR-28 animals. These findings support the anti-aging effect of CR up to 28 months. Conversely, the protein amounts of LonP1, Cyt c, OGG1, and APE1 and the 4.8 Kb mtDNA deletion content were not affected in CR-28 rats. The absence of significant differences between the AL-32 values and the CR-32 counterparts suggests an age-related boundary of CR efficacy at this age. However, this only partially curtails the CR benefits in counteracting the generalized aging decline and the related mitochondrial involvement.
    Keywords:  age-sensitive efficacy of CR; aging; calorie restriction; mitochondrial biogenesis; mtDNA damage; rat liver
    DOI:  https://doi.org/10.3390/ijms22041665
  26. Cell Metab. 2021 Feb 04. pii: S1550-4131(21)00014-0. [Epub ahead of print]
    Kosaisawe N, Sparta B, Pargett M, Teragawa CK, Albeck JG.
      Cell-to-cell heterogeneity in metabolism plays an unknown role in physiology and pharmacology. To functionally characterize cellular variability in metabolism, we treated cells with inhibitors of oxidative phosphorylation (OXPHOS) and monitored their responses with live-cell reporters for ATP, ADP/ATP, or activity of the energy-sensing kinase AMPK. Across multiple OXPHOS inhibitors and cell types, we identified a subpopulation of cells resistant to activation of AMPK and reduction of ADP/ATP ratio. This resistant state persists transiently for at least several hours and can be inherited during cell divisions. OXPHOS inhibition suppresses the mTORC1 and ERK growth signaling pathways in sensitive cells, but not in resistant cells. Resistance is linked to a multi-factorial combination of increased glucose uptake, reduced protein biosynthesis, and G0/G1 cell-cycle status. Our results reveal dynamic fluctuations in cellular energetic balance and provide a basis for measuring and predicting the distribution of cellular responses to OXPHOS inhibition.
    Keywords:  AKT; FRET; PI3K; adenosine mono-phosphate-regulated protein kinase; electron transport chain; insulin signaling; mammalian target of rapamycin; metabolic cycle; oligomycin; oscillation; translation regulation
    DOI:  https://doi.org/10.1016/j.cmet.2021.01.014
  27. Cancers (Basel). 2021 Feb 06. pii: 654. [Epub ahead of print]13(4):
    Kang SH, Oh SY, Lee HJ, Kwon TG, Kim JW, Lee ST, Choi SY, Hong SH.
      Background: The critical effect of the tumor microenvironment on cancer progression is well recognized. Recent research suggests that the cancer-promoting properties of the tumor stroma may be attributed to fibroblasts. However, the effect of cancer-associated fibroblast (CAF) on the progression of head and neck squamous cell carcinoma (HNSCC) is not well known. Methods: From the immunohistochemical analysis of head and neck squamous cell carcinoma (HNSCC) tissues, we divided CAF into two groups depending on the presence or absence of a well-demarcated boundary between epithelial cancer cells and the surrounding extracellular matrix (ECM). Primary culture of CAF was performed, followed by co-transplantation with HNSCC cells into mice oral mucosa, and the tumorigenesis was compared. The mRNA expression patterns between these two CAF groups were compared using DNA microarray analysis. Results: CAFs from cancer tissues that showed no demarcation between ECM and epithelial cancer cells (CAF-Promote) tended to stimulate Matrigel invasion of HNSCC cells. Conversely, CAFs from cancer tissues that showed a boundary with epithelial cancer cells (CAF-Delay) caused no remarkable increase in Matrigel invasion. Compared with CAF-P, CAF-D is less effective in promoting FaDu tumorigenicity in the mouse model. In DNA microarray analysis, COL3A1 and COL6A6 showed particularly high expression in the CAF-D group. Conclusions: These cancer stroma-derived collagen proteins might delay the HNSCC progression. These findings are expected to provide vital information for predicting HNSCC prognosis and developing drug targets in the future.
    Keywords:  DNA microarray; cancer-associated fibroblast; collagen; head and neck squamous cell carcinoma
    DOI:  https://doi.org/10.3390/cancers13040654
  28. JACS Au. 2021 Jan 25. 1(1): 23-30
    Dzijak R, Galeta J, Vázquez A, Kozák J, Matoušová M, Fulka H, Dračínský M, Vrabel M.
      The development of abiotic chemical reactions that can be performed in an organelle-specific manner can provide new opportunities in drug delivery and cell and chemical biology. However, due to the complexity of the cellular environment, this remains a significant challenge. Here, we introduce structurally redesigned bioorthogonal tetrazine reagents that spontaneously accumulate in mitochondria of live mammalian cells. The attributes leading to their efficient accumulation in the organelle were optimized to include the right combination of lipophilicity and positive delocalized charge. The best performing mitochondriotropic tetrazines enable subcellular chemical release of TCO-caged compounds as we show using fluorogenic substrates and mitochondrial uncoupler niclosamide. Our work demonstrates that a shrewd redesign of common bioorthogonal reagents can lead to their transformation into organelle-specific probes, opening the possibility to activate prodrugs and manipulate biological processes at the subcellular level by using purely chemical tools.
    DOI:  https://doi.org/10.1021/jacsau.0c00053
  29. Free Radic Biol Med. 2021 Feb 06. pii: S0891-5849(21)00064-2. [Epub ahead of print]
    Su Y, Ahn B, Macpherson PCD, Ranjit R, Claflin DR, Van Remmen H, Brooks SV.
      Aging is accompanied by loss of muscle mass and force, known as sarcopenia. Muscle atrophy, weakness, and neuromuscular junction (NMJ) degeneration reminiscent of normal muscle aging are observed early in adulthood for mice deficient in Cu, Zn-superoxide dismutase (SOD, Sod1-/-). Muscles of Sod1-/- mice also display impaired mitochondrial ATP production and increased mitochondrial reactive oxygen species (ROS) generation implicating oxidative stress in sarcopenia. Restoration of CuZnSOD specifically in neurons of Sod1-/- mice (SynTgSod1-/-) prevents muscle atrophy and loss of force, but whether muscle mitochondrial function is preserved is not known. To establish links among CuZnSOD expression, mitochondrial function, and sarcopenia, we examined contractile properties, mitochondrial function and ROS production, intracellular calcium transients (ICT), and NMJ morphology in lumbrical muscles of 7-9 month wild type (WT), Sod1-/-, and SynTgSod1-/- mice. Compared with WT values, mitochondrial ROS production was increased 2.9-fold under basal conditions and 2.2-fold with addition of glutamate and malate in Sod1-/- muscle fibers while oxygen consumption was not significantly altered. In addition, NADH recovery was blunted following contraction and the peak of the ICT was decreased by 25%. Mitochondrial function, ROS generation and calcium handling were restored to WT values in SynTgSod1-/- mice, despite continued lack of CuZnSOD in muscle. NMJ denervation and fragmentation were also fully rescued in SynTgSod1-/- mice suggesting that muscle mitochondrial and calcium handling defects in Sod1-/- mice are secondary to neuronal oxidative stress and its effects on the NMJ rather than the lack of muscle CuZnSOD. We conclude that intact neuronal function and innervation are key to maintaining excitation-contraction coupling and muscle mitochondrial function.
    Keywords:  Aging; CuZnSOD; Excitation contraction coupling; Mitochondria; Oxidative stress; Sarcopenia
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.01.047
  30. Front Physiol. 2020 ;11 542950
    Haskins N, Bhuvanendran S, Anselmi C, Gams A, Kanholm T, Kocher KM, LoTempio J, Krohmaly KI, Sohai D, Stearrett N, Bonner E, Tuchman M, Morizono H, Jaiswal JK, Caldovic L.
      Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other, perhaps in a cluster. This view is supported by mutations in surface residues of the urea cycle proteins that impair ureagenesis in the patients, but do not affect protein stability or catalytic activity. We find the NAGS, CPS1, and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM) and can be co-immunoprecipitated. Our in-silico analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a protein-protein interaction region present only in the mammalian NAGS protein-"variable segment," which mediates the interaction of NAGS with CPS1. Use of super resolution microscopy showed that NAGS, CPS1 and OTC are organized into clusters in the hepatocyte mitochondria. These results indicate that mitochondrial urea cycle proteins cluster, instead of functioning either independently or in a rigid multienzyme complex.
    Keywords:  N-acetylglutamate synthase; carbamylphosphate synthetase 1; enzyme cluster; metabolite channeling; mitochondria; ornithine transcarbamylase; super-resolution imaging; urea cycle
    DOI:  https://doi.org/10.3389/fphys.2020.542950
  31. Nat Commun. 2021 02 09. 12(1): 891
    Davydova E, Shimazu T, Schuhmacher MK, Jakobsson ME, Willemen HLDM, Liu T, Moen A, Ho AYY, Małecki J, Schroer L, Pinto R, Suzuki T, Grønsberg IA, Sohtome Y, Akakabe M, Weirich S, Kikuchi M, Olsen JV, Dohmae N, Umehara T, Sodeoka M, Siino V, McDonough MA, Eijkelkamp N, Schofield CJ, Jeltsch A, Shinkai Y, Falnes PØ.
      Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.
    DOI:  https://doi.org/10.1038/s41467-020-20670-7
  32. Oncogene. 2021 Feb 09.
    Ma Y, Zha J, Yang X, Li Q, Zhang Q, Yin A, Beharry Z, Huang H, Huang J, Bartlett M, Ye K, Yin H, Cai H.
      Fatty acid metabolism is essential for the biogenesis of cellular components and ATP production to sustain proliferation of cancer cells. Long-chain fatty acyl-CoA synthetases (ACSLs), a group of rate-limiting enzymes in fatty acid metabolism, catalyze the bioconversion of exogenous or de novo synthesized fatty acids to their corresponding fatty acyl-CoAs. In this study, systematical analysis of ACSLs levels and the amount of fatty acyl-CoAs illustrated that ACSL1 were significantly associated with the levels of a broad spectrum of fatty acyl-CoAs, and were elevated in human prostate tumors. ACSL1 increased the biosynthesis of fatty acyl-CoAs including C16:0-, C18:0-, C18:1-, and C18:2-CoA, triglycerides and lipid accumulation in cancer cells. Mechanistically, ACSL1 modulated mitochondrial respiration, β-oxidation, and ATP production through regulation of CPT1 activity. Knockdown of ACSL1 inhibited the cell cycle, and suppressed the proliferation and migration of prostate cancer cells in vitro, and growth of prostate xenograft tumors in vivo. Our study implicates ACSL1 as playing an important role in prostate tumor progression, and provides a therapeutic strategy of targeting fatty acid metabolism for the treatment of prostate cancer.
    DOI:  https://doi.org/10.1038/s41388-021-01667-y
  33. Acta Physiol (Oxf). 2021 Feb 11. e13625
    Jacobs RA, Lundby C.
      AIM: This study sought to provide a statistically robust reference for measures of mitochondrial function from standardized high-resolution respirometry with permeabilized human skeletal muscle (ex vivo), compare analogous values obtained via indirect calorimetry, arterial-venous O2 differences, and 31 P magnetic resonance spectroscopy (in vivo), and attempt to resolve differences across complementary methodologies as necessary.METHODS: Data derived from 831 study participants across research published throughout March 2009 to November 2019 was amassed to examine the biological relevance of ex vivo assessments under standard conditions, i.e. physiological temperatures of 37 °C and respiratory chamber oxygen concentrations of ~250-500 μM.
    RESULTS: Standard ex vivo-derived measures are lower (Z ≥ 3.01, p ≤ 0.0258) en masse than corresponding in vivo-derived values. Correcting respiratory values to account for mitochondrial temperatures 10 °C higher than skeletal muscle temperatures at maximal exercise (~ 50 °C): i.) transforms data to resemble (Z ≤ 0.8, p > 0.9999) analogous yet context-specific in vivo measures, e.g. data collected during maximal 1-leg knee extension exercise; and ii.) supports the position that maximal skeletal muscle respiratory rates exceed (Z ≥ 13.2, p < 0.0001) those achieved during maximal whole-body exercise, e.g. maximal cycling efforts.
    CONCLUSION: This study outlines and demonstrates necessary considerations when actualizing the biological relevance of human skeletal muscle respiratory control, metabolic flexibility, and bioenergetics from standard ex vivo-derived assessments using permeabilized human muscle. These findings detail how cross-procedural comparisons of human skeletal muscle mitochondrial function may be collectively scrutinized in their relationship to human health and lifespan.
    Keywords:  carbohydrate oxidation rates; fatty acid oxidation rates; human bioenergetics; metabolic flexibility; skeletal muscle mitochondria; skeletal muscle temperature
    DOI:  https://doi.org/10.1111/apha.13625
  34. Elife. 2021 Feb 08. pii: e64821. [Epub ahead of print]10
    Díaz-García CM, Meyer DJ, Nathwani N, Rahman M, Martínez-François JR, Yellen G.
      When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca2+ in the mitochondrial responses has been debated. Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca2+ uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca2+ activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca2+/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na+ or Ca2+ extrusion, and in mouse dentate granule cells, Ca2+ creates the majority of this demand.
    Keywords:  brain metabolism; mitochondrial calcium; mitochondrial calcium uniporter; mouse; neuronal glycolysis; neuroscience
    DOI:  https://doi.org/10.7554/eLife.64821
  35. Cell Death Discov. 2020 Mar 30. 6(1): 18
    Doccini S, Morani F, Nesti C, Pezzini F, Calza G, Soliymani R, Signore G, Rocchiccioli S, Kanninen KM, Huuskonen MT, Baumann MH, Simonati A, Lalowski MM, Santorelli FM.
      CLN5 disease is a rare form of late-infantile neuronal ceroid lipofuscinosis (NCL) caused by mutations in the CLN5 gene that encodes a protein whose primary function and physiological roles remains unresolved. Emerging lines of evidence point to mitochondrial dysfunction in the onset and progression of several forms of NCL, offering new insights into putative biomarkers and shared biological processes. In this work, we employed cellular and murine models of the disease, in an effort to clarify disease pathways associated with CLN5 depletion. A mitochondria-focused quantitative proteomics approach followed by functional validations using cell biology and immunofluorescence assays revealed an impairment of mitochondrial functions in different CLN5 KO cell models and in Cln5-/- cerebral cortex, which well correlated with disease progression. A visible impairment of autophagy machinery coupled with alterations of key parameters of mitophagy activation process functionally linked CLN5 protein to the process of neuronal injury. The functional link between impaired cellular respiration and activation of mitophagy pathways in the human CLN5 disease condition was corroborated by translating organelle-specific proteome findings to CLN5 patients' fibroblasts. Our study highlights the involvement of CLN5 in activation of mitophagy and mitochondrial homeostasis offering new insights into alternative strategies towards the CLN5 disease treatment.
    DOI:  https://doi.org/10.1038/s41420-020-0250-y
  36. Free Radic Biol Med. 2021 Feb 06. pii: S0891-5849(21)00067-8. [Epub ahead of print]
    Varmazyad M, Modi MM, Kalen AL, Sarsour EH, Wagner B, Du J, Schultz MK, Buettner GR, Pigge FC, Goswami PC.
      Dihydroartemisinin (DHA) is an FDA-approved antimalarial drug that has been repurposed for cancer therapy because of its preferential antiproliferative effects on cancer versus normal cells. Mitochondria represent an attractive target for cancer therapy based on their regulatory role in proliferation and cell death. This study investigates whether DHA conjugated to innately fluorescent N-alkyl triphenylvinylpyridinium (TPVP) perturbs mitochondrial functions resulting in a differential toxicity of cancer versus normal cells. TPVP-DHA treatments resulted in a dose-dependent toxicity of human melanoma and pancreatic cancer cells, whereas normal human fibroblasts were resistant to this treatment. TPVP-DHA treatments resulted in a G1-delay of the cancer cell cycle, which was also associated with a significant inhibition of the mTOR-metabolic and ERK1/2-proliferative signaling pathways. TPVP-DHA treatments perturbed mitochondrial functions, which correlated with increases in mitochondrial fission. In summary, TPVP mediated mitochondrial targeting of DHA enhanced cancer cell toxicity by perturbing mitochondrial functions and morphology.
    Keywords:  Dihydroartemisinin; ERK1/2; Melanoma; Mitochondria-targeted DHA; N-alkyl triphenylvinylpyridinium; Pancreatic cancer; mTOR; mitochondria-fission
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.01.050
  37. Nat Commun. 2021 Feb 12. 12(1): 979
    Sachamitr P, Ho JC, Ciamponi FE, Ba-Alawi W, Coutinho FJ, Guilhamon P, Kushida MM, Cavalli FMG, Lee L, Rastegar N, Vu V, Sánchez-Osuna M, Coulombe-Huntington J, Kanshin E, Whetstone H, Durand M, Thibault P, Hart K, Mangos M, Veyhl J, Chen W, Tran N, Duong BC, Aman AM, Che X, Lan X, Whitley O, Zaslaver O, Barsyte-Lovejoy D, Richards LM, Restall I, Caudy A, Röst HL, Bonday ZQ, Bernstein M, Das S, Cusimano MD, Spears J, Bader GD, Pugh TJ, Tyers M, Lupien M, Haibe-Kains B, Artee Luchman H, Weiss S, Massirer KB, Prinos P, Arrowsmith CH, Dirks PB.
      Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.
    DOI:  https://doi.org/10.1038/s41467-021-21204-5
  38. STAR Protoc. 2021 Mar 19. 2(1): 100297
    Kasashima H, Duran A, Cid-Diaz T, Muta Y, Kinoshita H, Batlle E, Diaz-Meco MT, Moscat J.
      In vivo interrogation of the functional role of genes implicated in colorectal cancer (CRC) is limited by the need for physiological models that mimic the disease. Here, we describe a protocol that provides the steps required for the orthotopic co-implantation of tumoral and stromal cells into the cecum and rectum to investigate the crosstalk between the tumor and its microenvironment. This protocol recapitulates metastases to the lymph nodes, liver, and lungs observed in human CRC. For complete details on the use and execution of this protocol, please refer to Kasashima et al. (2020).
    Keywords:  Cancer; Model organisms; Organoids
    DOI:  https://doi.org/10.1016/j.xpro.2021.100297
  39. Nat Chem Biol. 2021 Feb 11.
    Amponsah PS, Yahya G, Zimmermann J, Mai M, Mergel S, Mühlhaus T, Storchova Z, Morgan B.
      Redox cycles have been reported in ultradian, circadian and cell cycle-synchronized systems. Redox cycles persist in the absence of transcription and cyclin-CDK activity, indicating that cells harbor multiple coupled oscillators. Nonetheless, the causal relationships and molecular mechanisms by which redox cycles are embedded within ultradian, circadian or cell division cycles remain largely elusive. Yeast harbor an ultradian oscillator, the yeast metabolic cycle (YMC), which comprises metabolic/redox cycles, transcriptional cycles and synchronized cell division. Here, we reveal the existence of robust cycling of H2O2 and peroxiredoxin oxidation during the YMC and show that peroxiredoxin inactivation disrupts metabolic cycling and abolishes coupling with cell division. We find that thiol-disulfide oxidants and reductants predictably modulate the switching between different YMC metabolic states, which in turn predictably perturbs cell cycle entry and exit. We propose that oscillatory H2O2-dependent protein thiol oxidation is a key regulator of metabolic cycling and its coordination with cell division.
    DOI:  https://doi.org/10.1038/s41589-020-00728-9