bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2024‒07‒28
twenty-one papers selected by
Kelsey Fisher-Wellman, East Carolina University
  1. Arginine methylation of DDX3 by PRMT1 mediates mitochondrial homeostasis to promote breast cancer metastasis.
  2. Role of mitochondrial and cytosolic folylpolyglutamate synthetase in one-carbon metabolism and antitumor efficacy of mitochondrial-targeted antifolates.
  3. Metastatic breast cancer cells are metabolically reprogrammed to maintain redox homeostasis during metastasis.
  4. Metabolism and HSC fate: what NADPH is made for.
  5. SLC25A48 influences plasma levels of choline and localizes to the inner mitochondrial membrane.
  6. Cardiolipin remodeling maintains the inner mitochondrial membrane in cells with saturated lipidomes.
  7. An NAD+-dependent metabolic checkpoint regulates hematopoietic stem cell activation and aging.
  8. Preferred inhibition of pro-apoptotic Bak by BclxL via a two-step mechanism.
  9. 100 years of the Warburg effect: A cancer metabolism endeavor.
  10. Vitamin K2 sensitizes the efficacy of venetoclax in acute myeloid leukemia by targeting the NOXA-MCL-1 pathway.
  11. Efficient Elimination of mtDNA from Mammalian Cells with 2',3'-Dideoxycytidine.
  12. Targeting ferritinophagy impairs quiescent cancer stem cells in acute myeloid leukemia in vitro and in vivo models.
  13. Sweetening mitochondria: Hexokinase shields mitochondria from fission when glucose is low.
  14. BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains.
  15. Separation of reproductive decline from lifespan extension during methionine restriction.
  16. Auranofin inhibition of thioredoxin reductase sensitizes lung neuroendocrine tumor cells (NETs) and small cell lung cancer (SCLC) cells to sorafenib as well as inhibiting SCLC xenograft growth.
  17. The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs.
  18. Evaluation of mtDNA common deletion in esophageal squamous cell carcinoma.
  19. Mitochondrial DNA mosaicism in normal human somatic cells.
  20. Cyclic fasting-mimicking diet in cancer treatment: Preclinical and clinical evidence.
  21. Glutathione synthesis in the mouse liver supports lipid abundance through NRF2 repression.
  1. Cancer Res. 2024 Jul 23.
    Arginine methylation of DDX3 by PRMT1 mediates mitochondrial homeostasis to promote breast cancer metastasis.
    Wen-Jing Hsu, Ming-Chen Chiang, Yi-Chun Chao, Yu-Chu Chang, Ming-Chien Hsu, Chu-Hung Chung, I-Lin Tsai, Cheng-Ying Chu, Han-Chung Wu, Ching-Chieh Yang, Chi-Ching Lee, Cheng-Wei Lin.
      Dysregulated mitochondrial dynamics and metabolism play important roles in tumorigenesis. Metastasizing tumor cells predominantly utilize mitochondrial metabolism, and regulators of metabolic reprogramming may provide reliable biomarkers for diagnosing cancer metastasis. Here, we identified a PRMT1-DDX3 axis that promotes breast cancer metastasis by coordinating mitochondrial biogenesis and mitophagy to ensure mitochondrial quality control. Mechanistically, PRMT1 induces arginine methylation of DDX3, which enhances its protein stability and prevents proteasomal degradation. DDX3 mediates mitochondrial homeostasis by translocating to mitochondria where it facilitates PINK1 translation in response to mitochondrial stress. Inhibition of DDX3 suppresses mitochondrial biogenesis and mitophagy, resulting in diminished cancer stemness and metastatic properties. Overall, this study uncovers a mechanism by which the PRMT1-DDX3 axis regulates mitochondrial homeostasis to support breast cancer metastasis, suggesting strategies for targeting metabolic vulnerabilities to treat metastatic breast cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-23-3829
  2. Mol Pharmacol. 2024 Jul 24. pii: MOLPHARM-AR-2024-000912. [Epub ahead of print]
    Role of mitochondrial and cytosolic folylpolyglutamate synthetase in one-carbon metabolism and antitumor efficacy of mitochondrial-targeted antifolates.
    Carrie O'Connor, Mathew Schneider, Jade M Katinas, Md Junayed Nayeen, Khushbu Shah, Tejashree Magdum, Abhishekh Sharma, Seongho Kim, Xun Bao, Jing Li, Charles E Dann, Aleem Gangjee, Larry H Matherly, Zhanjun Hou.
      Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange between serine, glycine and formate. In both the cytosol and mitochondria, folates exist as polyglutamates with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets SHMT2 in the mitochondria, and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ~19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (~30-fold) more than cFPGS (~4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the non-classical SHMT2/SHMT1 inhibitor, SHIN1. These results suggest a critical role of mFPGS levels in determining anti-tumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. Significance Statement AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347 Mitochondrial FPGS levels play important roles in determining the anti-tumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.
    Keywords:  metabolomics; mitochondria; purines; transporters
    DOI:  https://doi.org/10.1124/molpharm.124.000912
  3. Redox Biol. 2024 Jul 20. pii: S2213-2317(24)00254-4. [Epub ahead of print]75 103276
    Metastatic breast cancer cells are metabolically reprogrammed to maintain redox homeostasis during metastasis.
    Marco Biondini, Camille Lehuédé, Sébastien Tabariès, Matthew G Annis, Alain Pacis, Eric H Ma, Christine Tam, Brian E Hsu, Yannick Audet-Delage, Afnan Abu-Thuraia, Charlotte Girondel, Valerie Sabourin, Stephanie P Totten, Mariana de Sá Tavares Russo, Gaëlle Bridon, Daina Avizonis, Marie-Christine Guiot, Julie St-Pierre, Josie Ursini-Siegel, Russell Jones, Peter M Siegel.
      Metabolic rewiring is essential for tumor growth and progression to metastatic disease, yet little is known regarding how cancer cells modify their acquired metabolic programs in response to different metastatic microenvironments. We have previously shown that liver-metastatic breast cancer cells adopt an intrinsic metabolic program characterized by increased HIF-1α activity and dependence on glycolysis. Here, we confirm by in vivo stable isotope tracing analysis (SITA) that liver-metastatic breast cancer cells retain a glycolytic profile when grown as mammary tumors or liver metastases. However, hepatic metastases exhibit unique metabolic adaptations including elevated expression of genes involved in glutathione (GSH) biosynthesis and reactive oxygen species (ROS) detoxification when compared to mammary tumors. Accordingly, breast-cancer-liver-metastases exhibited enhanced de novo GSH synthesis. Confirming their increased capacity to mitigate ROS-mediated damage, liver metastases display reduced levels of 8-Oxo-2'-deoxyguanosine. Depletion of the catalytic subunit of the rate-limiting enzyme in glutathione biosynthesis, glutamate-cysteine ligase (GCLC), strongly reduced the capacity of breast cancer cells to form liver metastases, supporting the importance of these distinct metabolic adaptations. Loss of GCLC also affected the early steps of the metastatic cascade, leading to decreased numbers of circulating tumor cells (CTCs) and impaired metastasis to the liver and the lungs. Altogether, our results indicate that GSH metabolism could be targeted to prevent the dissemination of breast cancer cells.
    Keywords:  Breast cancer; GCLC; Glutathione; Glycolysis; HIF-1α; Liver metastasis; Metabolism; Oxidative stress
    DOI:  https://doi.org/10.1016/j.redox.2024.103276
  4. Trends Cell Biol. 2024 Jul 24. pii: S0962-8924(24)00141-7. [Epub ahead of print]
    Metabolism and HSC fate: what NADPH is made for.
    Claudia Morganti, Massimo Bonora, Keisuke Ito.
      Mitochondrial metabolism plays a central role in the regulation of hematopoietic stem cell (HSC) biology. Mitochondrial fatty acid oxidation (FAO) is pivotal in controlling HSC self-renewal and differentiation. Herein, we discuss recent evidence suggesting that NADPH generated in the mitochondria can influence the fate of HSCs. Although NADPH has multiple functions, HSCs show high levels of NADPH that are preferentially used for cholesterol biosynthesis. Endogenous cholesterol supports the biogenesis of extracellular vesicles (EVs), which are essential for maintaining HSC properties. We also highlight the significance of EVs in hematopoiesis through autocrine signaling. Elucidating the mitochondrial NADPH-cholesterol axis as part of the metabolic requirements of healthy HSCs will facilitate the development of new therapies for hematological disorders.
    Keywords:  FAO; HSC self-renewal; cholesterol; exosome; hematopoiesis; mitochondrial metabolism
    DOI:  https://doi.org/10.1016/j.tcb.2024.07.003
  5. Mol Genet Metab. 2024 Jun 20. pii: S1096-7192(24)00402-5. [Epub ahead of print]143(1-2): 108518
    SLC25A48 influences plasma levels of choline and localizes to the inner mitochondrial membrane.
    David J Bernard, Faith Pangilinan, Caitlin Mendina, Tara Desporte, Stephen M Wincovitch, Darren J Walsh, Richard K Porter, Anne M Molloy, Barry Shane, Lawrence C Brody.
      Choline contributes to the biogenesis of methyl groups, neurotransmitters, and cell membranes. Our genome-wide association study (GWAS) of circulating choline in 2228 college students found that alleles in SLC25A48 (rs6596270) influence choline concentrations in men (p = 9.6 × 10-8), but not women. Previously, the subcellular location and function of SLC25A48 were unknown. Using super-resolution immunofluorescence microscopy, we localized SLC25A48 to the inner mitochondrial membrane. Our results suggest that SLC25A48 transports choline across the inner mitochondrial membrane.
    Keywords:  Betaine; Choline; Inner mitochondrial membrane transport; Mitochondria; One carbon metabolism; SLC25A48
    DOI:  https://doi.org/10.1016/j.ymgme.2024.108518
  6. J Lipid Res. 2024 Jul 20. pii: S0022-2275(24)00106-8. [Epub ahead of print] 100601
    Cardiolipin remodeling maintains the inner mitochondrial membrane in cells with saturated lipidomes.
    Kailash Venkatraman, Itay Budin.
      Cardiolipin (CL) is a unique, four-chain phospholipid synthesized in the inner mitochondrial membrane (IMM). The acyl chain composition of CL is regulated through a remodeling pathway, whose loss causes mitochondrial dysfunction in Barth syndrome (BTHS). Yeast has been used extensively as a model system to characterize CL metabolism, but mutants lacking its two remodeling enzymes, Cld1p and Taz1p, exhibit mild structural and respiratory phenotypes compared to mammalian cells. Here we show the essential role of CL remodeling in the structure and function of the IMM in yeast grown under reduced oxygenation. Microaerobic fermentation, which mimics natural yeast environments, caused the accumulation of saturated fatty acids and, under these conditions, remodeling mutants showed a loss of IMM ultrastructure. We extended this observation to HEK293 cells, where iPLA2 inhibition by Bromoenol lactone resulted in respiratory dysfunction and cristae loss upon mild treatment with exogenous saturated fatty acids. In microaerobic yeast, remodeling mutants accumulated unremodeled, saturated CL, but also displayed reduced total CL levels, highlighting the interplay between saturation and CL biosynthesis and breakdown. We identified the mitochondrial phospholipase A1 Ddl1p as a regulator of CL levels, and those of its precursors phosphatidylglycerol and phosphatidic acid, under these conditions. Loss of DDL1 partially rescued IMM structure in cells unable to initiate CL remodeling and had differing lipidomic effects depending on oxygenation. These results introduce a revised yeast model for investigating CL remodeling and suggest that its structural functions are dependent on the overall lipid environment in the mitochondrion.
    Keywords:  Barth Syndrome; Cardiolipin; Lipid saturation; Mitochondria; Phospholipids
    DOI:  https://doi.org/10.1016/j.jlr.2024.100601
  7. Nat Aging. 2024 Jul 23.
    An NAD+-dependent metabolic checkpoint regulates hematopoietic stem cell activation and aging.
    Zehan Song, Sang Hee Park, Wei-Chieh Mu, Yufan Feng, Chih-Ling Wang, Yifei Wang, Marine Barthez, Ayane Maruichi, Jiayue Guo, Fanghan Yang, Anita Wong Lin, Kartoosh Heydari, Claudia C S Chini, Eduardo N Chini, Cholsoon Jang, Danica Chen.
      How hematopoietic stem cells (HSCs) maintain metabolic homeostasis to support tissue repair and regeneration throughout the lifespan is elusive. Here, we show that CD38, an NAD+-dependent metabolic enzyme, promotes HSC proliferation by inducing mitochondrial Ca2+ influx and mitochondrial metabolism in young mice. Conversely, aberrant CD38 upregulation during aging is a driver of HSC deterioration in aged mice due to dysregulated NAD+ metabolism and compromised mitochondrial stress management. The mitochondrial calcium uniporter, a mediator of mitochondrial Ca2+ influx, also supports HSC proliferation in young mice yet drives HSC decline in aged mice. Pharmacological inactivation of CD38 reverses HSC aging and the pathophysiological changes of the aging hematopoietic system in aged mice. Together, our study highlights an NAD+ metabolic checkpoint that balances mitochondrial activation to support HSC proliferation and mitochondrial stress management to enhance HSC self-renewal throughout the lifespan, and links aberrant Ca2+ signaling to HSC aging.
    DOI:  https://doi.org/10.1038/s43587-024-00670-8
  8. Cell Rep. 2024 Jul 22. pii: S2211-1247(24)00855-6. [Epub ahead of print]43(8): 114526
    Preferred inhibition of pro-apoptotic Bak by BclxL via a two-step mechanism.
    Kira D Leitl, Laura E Sperl, Franz Hagn.
      Bak is a pore-forming Bcl2 protein that induces apoptosis at the outer mitochondrial membrane, which can either proceed via Bak oligomerization or be inhibited by anti-apoptotic Bcl2 proteins, such as BclxL. BclxL is very efficient in inhibiting Bak pore formation, but the mechanistic basis of this preferred interaction has remained enigmatic. Here, we identify Bakα1 as a second binding site for BclxL and show that it specifically interacts with the Bcl2-homology (BH)3 binding groove of BclxL. The affinity between BclxL and Bakα1 is weaker than with Bak-BH3, suggesting that Bakα1, being exposed early in the pore-forming trajectory, transiently captures BclxL, which subsequently transitions to the proximal BH3 site. Bak variants where the initial transient interaction with BclxL is modulated show a markedly altered response to BclxL inhibition. This work contributes to a better mechanistic understanding of the fine-tuned interactions between different players of the Bcl2 protein family.
    Keywords:  CP: Cell biology; CP: Molecular biology; HDX-MS; NMR; apoptosis; inhibition; lipid nanodiscs; membrane
    DOI:  https://doi.org/10.1016/j.celrep.2024.114526
  9. Cell. 2024 Jul 25. pii: S0092-8674(24)00700-1. [Epub ahead of print]187(15): 3824-3828
    100 years of the Warburg effect: A cancer metabolism endeavor.
    Sarah-Maria Fendt.
      If you are a scientist and you only know one thing about tumor metabolism, it's likely the Warburg effect. But who was Otto Warburg, and how did his discoveries regarding the metabolism of tumors shape our current thinking about the metabolic needs of cancer cells?
    DOI:  https://doi.org/10.1016/j.cell.2024.06.026
  10. PLoS One. 2024 ;19(7): e0307662
    Vitamin K2 sensitizes the efficacy of venetoclax in acute myeloid leukemia by targeting the NOXA-MCL-1 pathway.
    Tetsuzo Tauchi, Shota Moriya, Seiichi Okabe, Hiromi Kazama, Keisuke Miyazawa, Naoharu Takano.
      Promising outcomes have been reported in elder patients with acute myeloid leukemia (AML) using combined therapy of venetoclax (VEN) and azacytidine (AZA) in recent years. However, approximately one-third of patients appear to be refractory to this therapy. Vitamin K2 (VK2) shows apoptosis-inducing activity in AML cells, and daily oral VK2 (menaquinone-4, GlakayR) has been approved for patients with osteoporosis in Japan. We observed a high response rate to AZA plus VEN therapy, with no 8-week mortality in the newly diagnosed AML patients consuming daily VK2 in our hospital. The median age of the patients was 75.9 years (range 66-84) with high-risk features. Patients received AZA 75 mg/m2 on D1-7, VEN 400 mg on D1-28, and daily VK2 45 mg. The CR/CRi ratio was 94.7% (18/19), with a CR rate of 79%. Complete cytogenetic CR was achieved in 15 of 19 (79%) patients, and MRD negativity in 2 of 15 (13%) evaluable CR patients. Owing to the extremely high response rate in clinical settings, we further attempted to investigate the underlying mechanisms. The combination of VK2 and VEN synergistically induced apoptosis in all five AML cell lines tested. VK2, but not VEN, induced mitochondrial reactive oxygen species (ROS), leading to the transcriptional upregulation of NOXA, followed by MCL-1 repression. ROS scavengers repressed VK2 induced-NOXA expression and led to the cancellation of pronounced apoptosis and the downregulation of MCL-1 by VK2 plus VEN. Additionally, knockdown and knockout of NOXA resulted in abrogation of the MCL-1 repression as well as enhanced cytotoxicity by the two-drug combination, indicating that VK2 suppresses MCL-1 via ROS-mediated NOXA induction. These data suggest that the dual inhibition of BCL-2 by VEN and MCL-1 by VK2 is responsible for the remarkable clinical outcomes in our patients. Therefore, large-scale clinical trials are required.
    DOI:  https://doi.org/10.1371/journal.pone.0307662
  11. DNA (Basel). 2024 Sep;4(3): 201-211
    Efficient Elimination of mtDNA from Mammalian Cells with 2',3'-Dideoxycytidine.
    Natalya Kozhukhar, Mikhail F Alexeyev.
      Mammalian cell lines devoid of mitochondrial DNA (mtDNA) are indispensable in studies aimed at elucidating the contribution of mtDNA to various cellular processes or interactions between nuclear and mitochondrial genomes. However, the repertoire of tools for generating such cells (also known as rho-0 or ρ0 cells) remains limited, and approaches remain time- and labor-intensive, ultimately limiting their availability. Ethidium bromide (EtBr), which is most commonly used to induce mtDNA loss in mammalian cells, is cytostatic and mutagenic as it affects both nuclear and mitochondrial genomes. Therefore, there is growing interest in new tools for generating ρ0 cell lines. Here, we examined the utility of 2',3'-dideoxycytidine (ddC, zalcitabine) alone or in combination with EtBr for generating ρ0 cell lines of mouse and human origin as well as inducing the ρ0 state in mouse/human somatic cell hybrids. We report that ddC is superior to EtBr in both immortalized mouse fibroblasts and human 143B cells. Also, unlike EtBr, ddC exhibits no cytostatic effects at the highest concentration tested (200 μM), making it more suitable for general use. We conclude that ddC is a promising new tool for generating mammalian ρ0 cell lines.
    Keywords:  A549; HT1080; HeLa; ddC; mtDNA depletion; rho-0 cells
    DOI:  https://doi.org/10.3390/dna4030013
  12. Sci Transl Med. 2024 Jul 24. 16(757): eadk1731
    Targeting ferritinophagy impairs quiescent cancer stem cells in acute myeloid leukemia in vitro and in vivo models.
    Clement Larrue, Sarah Mouche, Paolo Angelino, Maxime Sajot, Rudy Birsen, Olivier Kosmider, Thomas Mckee, François Vergez, Christian Recher, Véronique Mansat-De Mas, Qiong Gu, Jun Xu, Petros Tsantoulis, Jean-Emmanuel Sarry, Jerome Tamburini.
      Acute myeloid leukemia (AML) remains a challenging hematological malignancy with poor prognosis and limited treatment options. Leukemic stem cells (LSCs) contribute to therapeutic failure, relapse, and adverse outcome. This study investigates the role of quiescence and related molecular mechanisms in AML pathogenesis and LSC functions to identify potential therapeutic targets. Transcriptomic analysis revealed that the LSC-enriched quiescent cell population has a distinct gene signature with prognostic relevance in patients with AML. Mechanistically, quiescent blasts exhibit increased autophagic activity, which contributes to their sustained viability. Proteomic profiling uncovered differential requirements for iron metabolism between quiescent and cycling cells, revealing a unique dependence of quiescent cells on ferritinophagy, a selective form of autophagy mediated by nuclear receptor coactivator 4 (NCOA4), which regulates iron bioavailability. We evaluated the therapeutic potential of inhibiting NCOA4-mediated ferritinophagy using genetic knockdown and chemical inhibition approaches. In vitro assays showed that suppression of NCOA4 was toxic to leukemic blasts, particularly the CD34+CD38- LSC-enriched population, without affecting normal CD34+ hematopoietic progenitors. In vivo studies using murine patient-derived xenograft (PDX) models of AML confirmed that NCOA4 inhibition reduced tumor burden and impaired LSC viability and self-renewal, indicating a specific vulnerability of these cells to ferritinophagy disruption. Our findings underscore the role of NCOA4-mediated ferritinophagy in maintaining LSC quiescence and function and suggest that targeting this pathway may be an effective therapeutic strategy for AML. This study highlights the potential of NCOA4 inhibition to improve AML outcomes and paves the way for future research and clinical development.
    DOI:  https://doi.org/10.1126/scitranslmed.adk1731
  13. Mol Cell. 2024 Jul 25. pii: S1097-2765(24)00542-2. [Epub ahead of print]84(14): 2593-2595
    Sweetening mitochondria: Hexokinase shields mitochondria from fission when glucose is low.
    Antigoni Diokmetzidou, Luca Scorrano.
      In this issue of Molecular Cell, Pilic et al.1 show that hexokinase, the first enzyme of glycolysis, forms perimitochondrial rings that prevent mitochondrial fragmentation when ATP levels drop.
    DOI:  https://doi.org/10.1016/j.molcel.2024.06.035
  14. EMBO Rep. 2024 Jul 24.
    BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains.
    Tobias B Beigl, Alexander Paul, Thomas P Fellmeth, Dang Nguyen, Lynn Barber, Sandra Weller, Benjamin Schäfer, Bernhard F Gillissen, Walter E Aulitzky, Hans-Georg Kopp, Markus Rehm, David W Andrews, Kristyna Pluhackova, Frank Essmann.
      The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.
    Keywords:  Apoptosis; BCL-2; BOK; Endoplasmic Reticulum; Transmembrane Domain
    DOI:  https://doi.org/10.1038/s44319-024-00206-6
  15. Nat Aging. 2024 Jul 26.
    Separation of reproductive decline from lifespan extension during methionine restriction.
    Fangchao Wei, Shiyu Liu, Juan Liu, Yudong Sun, Annamarie E Allen, Michael A Reid, Jason W Locasale.
      Lifespan-extending interventions are generally thought to result in reduced fecundity. The generality of this principle and how it may extend to nutrition and metabolism is not understood. We considered dietary methionine restriction (MR), a lifespan-extending intervention linked to Mediterranean and plant-based diets. Using a chemically defined diet that we developed for Drosophila melanogaster, we surveyed the nutritional landscape in the background of MR and found that folic acid, a vitamin linked to one-carbon metabolism, notably was the lone nutrient that restored reproductive capacity while maintaining lifespan extension. In vivo isotope tracing, metabolomics and flux analysis identified the tricarboxylic cycle and redox coupling as major determinants of the MR-folic acid benefits, in part, as they related to sperm function. Together these findings suggest that dietary interventions optimized for longevity may be separable from adverse effects such as reproductive decline.
    DOI:  https://doi.org/10.1038/s43587-024-00674-4
  16. Cancer Biol Ther. 2024 Dec 31. 25(1): 2382524
    Auranofin inhibition of thioredoxin reductase sensitizes lung neuroendocrine tumor cells (NETs) and small cell lung cancer (SCLC) cells to sorafenib as well as inhibiting SCLC xenograft growth.
    Spenser S Johnson, Dijie Liu, Jordan T Ewald, Claudia Robles-Planells, Casey Pulliam, Keegan A Christensen, Khaliunaa Bayanbold, Brian R Wels, Shane R Solst, M Sue O'Dorisio, Yusuf Menda, Douglas R Spitz, Melissa A Fath.
      Thioredoxin Reductase (TrxR) functions to recycle thioredoxin (Trx) during hydroperoxide metabolism mediated by peroxiredoxins and is currently being targeted using the FDA-approved anti-rheumatic drug, auranofin (AF), to selectively sensitize cancer cells to therapy. AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the H727 atypical lung carcinoid cell line. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, an FDA-approved multi-kinase inhibitor that depleted intracellular glutathione (GSH). The pharmacokinetic, pharmacodynamic, and safety profile of AF was examined in nude mice with DMS273 xenografts administered AF intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1-5 d. Plasma levels of AF were 10-20 μM (determined by mass spectrometry of gold), and the optimal inhibition of TrxR activity was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. This AF treatment extended for 14 d, inhibited TrxR (>75%), and resulted in a significant prolongation of median overall survival from 19 to 23 d (p = .04, N = 30 controls, 28 AF). In this experiment, there were no observed changes in animal bodyweight, complete blood counts (CBCs), bone marrow toxicity, blood urea nitrogen, or creatinine. These results support the hypothesis that AF effectively inhibits TrxR both in vitro and in vivo in SCLC, sensitizes NETs and SCLC to sorafenib, and could be repurposed as an adjuvant therapy with targeted agents that induce disruptions in thiol metabolism.
    Keywords:  auranofin; dms273; glutathione; h727; lung neuroendocrine tumor; small cell lung cancer; sorafenib; thioredoxin reductase
    DOI:  https://doi.org/10.1080/15384047.2024.2382524
  17. Cell Death Discov. 2024 Jul 23. 10(1): 335
    The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs.
    Akandé Rouchidane Eyitayo, Laetitia Daury, Muriel Priault, Stéphen Manon.
      Membrane insertion of the pro-apoptotic protein Bax was investigated by setting up cell-free synthesis of full-length Bax in the presence of pre-formed nanodiscs. While Bax was spontaneously poorly inserted in nanodiscs, co-synthesis with the mitochondrial receptor Tom22 stimulated Bax membrane insertion. The initial interaction of Bax with the lipid bilayer exposed the hydrophobic GALLL motif in Hα1 leading to Bax precipitation through hydrophobic interactions. The same motif was recognized by Tom22, triggering conformational changes leading to the extrusion and the ensuing membrane insertion of the C-terminal hydrophobic Hα9. Tom22 was also required for Bax-membrane insertion after Bax was activated either by BH3-activators or by its release from Bcl-xL by WEHI-539. The effect of Tom22 was impaired by D154Y substitution in Bax-Hα7 and T174P substitution in Bax-Hα9, which are found in several tumors. Conversely, a R9E substitution promoted a spontaneous insertion of Bax in nanodiscs, in the absence of Tom22. Both Tom22-activated Bax and BaxR9E alone permeabilized liposomes to dextran-10kDa and formed ~5-nm-diameter pores in nanodiscs. The concerted regulation of Bax membrane insertion by Tom22 and BH3-activators is discussed.
    DOI:  https://doi.org/10.1038/s41420-024-02108-x
  18. Indian J Cancer. 2024 Jul 23.
    Evaluation of mtDNA common deletion in esophageal squamous cell carcinoma.
    Fatemeh Ghadyani, Shahrbanoo Sharif, Saeid Morovvati.
      BACKGROUND: Mitochondrial defects are thought to play a role in cancer initiation and progression for a long time. Because of the absence of protective histones and an inefficiency in the DNA repair process, mitochondrial DNA is known to be prone to mutations. The deletion of 4977bp is one of the most common mutations in human cancers. This study aimed to investigate the relationship between 4977bp common deletion and Esophageal Squamous Cell Carcinoma Disease (SCC) to provide prognostic information.METHODS: By using a PCR protocol, this study identified the 4977bp deletion of mtDNA. A PCR method was used on tumor samples from 41 squamous cell carcinoma patients and blood samples from 50 healthy individuals to detect DNA.
    RESULTS: Among the 41 tumor samples (80.5%), 33 were found to have the 4977bp deletion, while none of the blood samples from healthy individuals contained it.
    CONCLUSIONS: It is shown that the deletion of 4977bp of mtDNA correlates significantly with SCC in this study. A 4977bp deletion could be used as an effective cancer screening indicator and biomarker for early diagnosis and prevention of cancer.
    DOI:  https://doi.org/10.4103/ijc.ijc_324_23
  19. Nat Genet. 2024 Jul 22.
    Mitochondrial DNA mosaicism in normal human somatic cells.
    Jisong An, Chang Hyun Nam, Ryul Kim, Yunah Lee, Hyein Won, Seongyeol Park, Won Hee Lee, Hansol Park, Christopher J Yoon, Yohan An, Jie-Hyun Kim, Jong Kwan Jun, Jeong Mo Bae, Eui-Cheol Shin, Bun Kim, Yong Jun Cha, Hyun Woo Kwon, Ji Won Oh, Jee Yoon Park, Min Jung Kim, Young Seok Ju.
      Somatic cells accumulate genomic alterations with age; however, our understanding of mitochondrial DNA (mtDNA) mosaicism remains limited. Here we investigated the genomes of 2,096 clones derived from three cell types across 31 donors, identifying 6,451 mtDNA variants with heteroplasmy levels of ≳0.3%. While the majority of these variants were unique to individual clones, suggesting stochastic acquisition with age, 409 variants (6%) were shared across multiple embryonic lineages, indicating their origin from heteroplasmy in fertilized eggs. The mutational spectrum exhibited replication-strand bias, implicating mtDNA replication as a major mutational process. We evaluated the mtDNA mutation rate (5.0 × 10-8 per base pair) and a turnover frequency of 10-20 per year, which are fundamental components shaping the landscape of mtDNA mosaicism over a lifetime. The expansion of mtDNA-truncating mutations toward homoplasmy was substantially suppressed. Our findings provide comprehensive insights into the origins, dynamics and functional consequences of mtDNA mosaicism in human somatic cells.
    DOI:  https://doi.org/10.1038/s41588-024-01838-z
  20. Cell Metab. 2024 Jul 23. pii: S1550-4131(24)00270-5. [Epub ahead of print]
    Cyclic fasting-mimicking diet in cancer treatment: Preclinical and clinical evidence.
    Claudio Vernieri, Francesca Ligorio, Debu Tripathy, Valter D Longo.
      In preclinical tumor models, cyclic fasting and fasting-mimicking diets (FMDs) produce antitumor effects that become synergistic when combined with a wide range of standard anticancer treatments while protecting normal tissues from treatment-induced adverse events. More recently, results of phase 1/2 clinical trials showed that cyclic FMD is safe, feasible, and associated with positive metabolic and immunomodulatory effects in patients with different tumor types, thus paving the way for larger clinical trials to investigate FMD anticancer activity in different clinical contexts. Here, we review the tumor-cell-autonomous and immune-system-mediated mechanisms of fasting/FMD antitumor effects, and we critically discuss new metabolic interventions that could synergize with nutrient starvation to boost its anticancer activity and prevent or reverse tumor resistance while minimizing toxicity to patients. Finally, we highlight potential future applications of FMD approaches in combination with standard anticancer strategies as well as strategies to implement the design and conduction of clinical trials.
    Keywords:  cancer therapy; clinical trials; combination treatments; fasting-mimicking diet; resistance mechanisms
    DOI:  https://doi.org/10.1016/j.cmet.2024.06.014
  21. Nat Commun. 2024 Jul 21. 15(1): 6152
    Glutathione synthesis in the mouse liver supports lipid abundance through NRF2 repression.
    Gloria Asantewaa, Emily T Tuttle, Nathan P Ward, Yun Pyo Kang, Yumi Kim, Madeline E Kavanagh, Nomeda Girnius, Ying Chen, Katherine Rodriguez, Fabio Hecht, Marco Zocchi, Leonid Smorodintsev-Schiller, TashJaé Q Scales, Kira Taylor, Fatemeh Alimohammadi, Renae P Duncan, Zachary R Sechrist, Diana Agostini-Vulaj, Xenia L Schafer, Hayley Chang, Zachary R Smith, Thomas N O'Connor, Sarah Whelan, Laura M Selfors, Jett Crowdis, G Kenneth Gray, Roderick T Bronson, Dirk Brenner, Alessandro Rufini, Robert T Dirksen, Aram F Hezel, Aaron R Huber, Joshua Munger, Benjamin F Cravatt, Vasilis Vasiliou, Calvin L Cole, Gina M DeNicola, Isaac S Harris.
      Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we have developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are highest in liver tissue, which is also a hub for lipid production. While the loss of GSH does not cause liver failure, it decreases lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we find that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.
    DOI:  https://doi.org/10.1038/s41467-024-50454-2
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