bims-celmim 2024-11-03 papers

bims-celmim

Biomed News

on Cellular and mitochondrial metabolism

Issue of 2024‒11‒03
twenty-two papers selected by
Marc Segarra Mondejar

Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.
Metabolic gatekeepers: harnessing tumor-derived metabolites to optimize T cell-based immunotherapy efficacy in the tumor microenvironment.
Monitoring mitochondrial protein import by live cell imaging.
Isolation of yeast mitochondria by differential centrifugation.
Ubiquitination regulates autophagy in cancer: simple modifications, promising targets.
Glutamine and serum starvation alters the ATP production, oxidative stress, and abundance of mitochondrial RNAs in extracellular vesicles produced by cancer cells.
Role of Exogenous Pyruvate in Maintaining Adenosine Triphosphate Production under High-Glucose Conditions through PARP-Dependent Glycolysis and PARP-Independent Tricarboxylic Acid Cycle.
Noncanonical TCA cycle fosters canonical TCA cycle and mitochondrial integrity in acute myeloid leukemia.
Methods to monitor mitochondrial disulfide bonds.
MICU2 up-regulation enhances tumor aggressiveness and metabolic reprogramming during colorectal cancer development.
Integrated systems biology identifies disruptions in mitochondrial function and metabolism as key contributors to heart failure with preserved ejection fraction (HFpEF).
Mass Spectrometry Probe Combined with Machine Learning to Capture the Relationship between Metabolites and Mitochondrial Complex Activity at the Whole-Cell Level.
Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells.
Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP.
HIF1α-regulated glycolysis promotes activation-induced cell death and IFN-γ induction in hypoxic T cells.
TrkB signalling regulates dopamine circuits and motor function through metabolic pathways.
Mitophagy: insights into its signaling molecules, biological functions, and therapeutic potential in breast cancer.
The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.
Cell-cell junctions in focus - imaging junctional architectures and dynamics at high resolution.
A Novel Cyanine-Based Fluorescent Dye for Targeted Mitochondrial Imaging in Neurotoxic Conditions and In Vivo Brain Studies.
Fluorescent non-canonical amino acid provides insight into the human serotonin transporter.
Accumulation of F-actin drives brain aging and limits healthspan in Drosophila.

EMBO Mol Med. 2024 Oct 28.

Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.

Louise Nuyttens, Jolien Vandewalle, Claude Libert.

  Sepsis is a heterogeneous syndrome resulting from a dysregulated host response to infection. It is considered as a global major health priority. Sepsis is characterized by significant metabolic perturbations, leading to increased circulating metabolites such as lactate. In mammals, pyruvate is the primary substrate for lactate production. It plays a critical role in metabolism by linking glycolysis, where it is produced, with the mitochondrial oxidative phosphorylation pathway, where it is oxidized. Here, we provide an overview of all cytosolic and mitochondrial enzymes involved in pyruvate metabolism and how their activities are disrupted in sepsis. Based on the available data, we also discuss potential therapeutic strategies targeting these pyruvate-related enzymes leading to enhanced survival.

Keywords:  Lactate; Metabolism; Mitochondria; Pyruvate; Sepsis

DOI:  https://doi.org/10.1038/s44321-024-00155-6
Cell Death Dis. 2024 Oct 26. 15(10): 775

Metabolic gatekeepers: harnessing tumor-derived metabolites to optimize T cell-based immunotherapy efficacy in the tumor microenvironment.

Yucheng Zheng, Rongwei Xu, Xu Chen, Ye Lu, Jiarong Zheng, Yunfan Lin, Pei Lin, Xinyuan Zhao, Li Cui.

The tumor microenvironment (TME) orchestrates a complex interplay between tumor cells and immune cells, crucially modulating the immune response. This review delves into the pivotal role of metabolic reprogramming in the TME, highlighting how tumor-derived metabolites influence T lymphocyte functionality and the efficacy of cancer immunotherapies. Focusing on the diverse roles of these metabolites, we examine how lactate, lipids, amino acids, and other biochemical signals act not only as metabolic byproducts but as regulatory agents that can suppress or potentiate T cell-mediated immunity. By integrating recent findings, we underscore the dual impact of these metabolites on enhancing tumor progression and inhibiting immune surveillance. Furthermore, we propose innovative therapeutic strategies that target metabolic pathways to restore immune function within the TME. The insights provided in this review pave the way for the development of metabolic interventions aimed at enhancing the success of immunotherapies in oncology, offering new hope for precision medicine in the treatment of cancer.

DOI: https://doi.org/10.1038/s41419-024-07122-6
Methods Enzymol. 2024 ;pii: S0076-6879(24)00363-X. [Epub ahead of print]706 437-447

Monitoring mitochondrial protein import by live cell imaging.

F X Reymond Sutandy.

  The majority of mitochondrial proteins are synthesized in the cytosol and must be imported into mitochondria to attain their mature forms and execute their functions. Disruption of mitochondrial functions, whether caused by external or internal stress, may compromise mitochondrial protein import. Therefore, monitoring mitochondrial protein import has become a standard approach to assess mitochondrial health and gain insights into mitochondrial biology, especially during stress. This chapter describes a detailed protocol for monitoring mitochondrial import in live cells using microscopy. Co-localization between mitochondria and a genetic reporter of mitochondrially targeted enhanced GFP (eGFP) is employed to evaluate mitochondrial protein import efficiency under different physiological conditions. Overall, this technique provides a simple and robust approach to assess mitochondrial protein import efficiency within its native cellular environment.

Keywords:  MTS; mitochondria; protein import; stress response

DOI:  https://doi.org/10.1016/bs.mie.2024.07.027
Methods Enzymol. 2024 ;pii: S0076-6879(24)00360-4. [Epub ahead of print]706 3-18

Isolation of yeast mitochondria by differential centrifugation.

Kuo Song, Heike Rampelt.

  The isolation of intact and functional mitochondria is a powerful approach to characterize and study this organelle. The classical biochemical method of differential centrifugation is routinely used to isolate mitochondria. This method has several advantages, such as a high yield and easy adaptability. The isolated mitochondria are physiologically active and can be used for a variety of follow-up experiments, for example protein import and respiration measurements. Here, we describe the procedure to purify mitochondria from the budding yeast Saccharomyces cerevisiae. In addition, two approaches are introduced to assess the quality of isolated mitochondria, by limited proteinase K digestion or measurement of the membrane potential.

Keywords:  Mitochondrial preparation; fractionation; limited proteolysis; membrane potential; organelle isolation

DOI:  https://doi.org/10.1016/bs.mie.2024.07.024
J Transl Med. 2024 Oct 31. 22(1): 985

Ubiquitination regulates autophagy in cancer: simple modifications, promising targets.

Yihui Wu, Yifei Chen, Xianyan Tian, Genbao Shao, Qiong Lin, Aiqin Sun.

  Autophagy is an important lysosomal degradation process that digests and recycles bio-molecules, protein or lipid aggregates, organelles, and invaded pathogens. Autophagy plays crucial roles in regulation of metabolic and oxidative stress and multiple pathological processes. In cancer, the role of autophagy is dual and paradoxical. Ubiquitination has been identified as a key regulator of autophagy that can influence various steps in the autophagic process, with autophagy-related proteins being targeted for ubiquitination, thus impacting cancer progression and the effectiveness of therapeutic interventions. This review will concentrate on mechanisms underlying autophagy, ubiquitination, and their interactions in cancer, as well as explore the use of drugs that target the ubiquitin-proteasome system (UPS) and ubiquitination process in autophagy as part of cancer therapy.

Keywords:  Autophagy; Cancer; E3 ligase; UPS; Ubiquitination

DOI:  https://doi.org/10.1186/s12967-024-05565-1
Sci Rep. 2024 10 28. 14(1): 25815

Glutamine and serum starvation alters the ATP production, oxidative stress, and abundance of mitochondrial RNAs in extracellular vesicles produced by cancer cells.

Maria Bugajova, Martina Raudenska, Klara Hanelova, Jiri Navratil, Jaromir Gumulec, Frantisek Petrlak, Tomas Vicar, Sarka Hrachovinova, Michal Masarik, David Kalfert, Marek Grega, Jan Plzak, Jan Betka, Jan Balvan.

Induction of autophagy represents an effective survival strategy for nutrient-deprived or stressed cancer cells. Autophagy contributes to the modulation of communication within the tumor microenvironment. Here, we conducted a study of the metabolic and signaling implications associated with autophagy induced by glutamine (Gln) and serum starvation and PI3K/mTOR inhibitor and autophagy inducer NVP-BEZ235 (BEZ) in the head and neck squamous cell carcinoma (HNSCC) cell line FaDu. We compared the effect of these different types of autophagy induction on ATP production, lipid peroxidation, mitophagy, RNA cargo of extracellular vesicles (EVs), and EVs-associated cytokine secretome of cancer cells. Both BEZ and starvation resulted in a decline in ATP production. Simultaneously, Gln starvation enhanced oxidative damage of cancer cells by lipid peroxidation. In starved cells, there was a discernible fragmentation of the mitochondrial network coupled with an increase in the presence of tumor susceptibility gene 101 (TSG101) on the mitochondrial membrane, indicative of the sorting of mitochondrial cargo into EVs. Consequently, the abundance of mitochondrial RNAs (mtRNAs) in EVs released by FaDu cells was enhanced. Notably, mtRNAs were also detectable in EVs isolated from the serum of both HNSCC patients and healthy controls. Starvation and BEZ reduced the production of EVs by cancer cells, yet the characteristic molecular profile of these EVs remained unchanged. We also found that alterations in the release of inflammatory cytokines constitute a principal response to autophagy induction. Importantly, the specific mechanism driving autophagy induction significantly influenced the composition of the EVs-associated cytokine secretome.

DOI: https://doi.org/10.1038/s41598-024-73943-2
Int J Mol Sci. 2024 Oct 15. pii: 11089. [Epub ahead of print]25(20):

Role of Exogenous Pyruvate in Maintaining Adenosine Triphosphate Production under High-Glucose Conditions through PARP-Dependent Glycolysis and PARP-Independent Tricarboxylic Acid Cycle.

Hideji Yako, Naoko Niimi, Shizuka Takaku, Ayako Kato, Koichi Kato, Kazunori Sango.

  Pyruvate serves as a key metabolite in energy production and as an anti-oxidant. In our previous study, exogenous pyruvate starvation under high-glucose conditions induced IMS32 Schwann cell death because of the reduced glycolysis-tricarboxylic acid (TCA) cycle flux and adenosine triphosphate (ATP) production. Thus, this study focused on poly-(ADP-ribose) polymerase (PARP) to investigate the detailed molecular mechanism of cell death. Rucaparib, a PARP inhibitor, protected Schwann cells against cell death and decreased glycolysis but not against an impaired TCA cycle under high-glucose conditions in the absence of pyruvate. Under such conditions, reduced pyruvate dehydrogenase (PDH) activity and glycolytic and mitochondrial ATP production were observed but not oxidative phosphorylation or the electric transfer chain. In addition, rucaparib supplementation restored glycolytic ATP production but not PDH activity and mitochondrial ATP production. No differences in the increased activity of caspase 3/7 and the localization of apoptosis-inducing factor were found among the experimental conditions. These results indicate that Schwann cells undergo necrosis rather than apoptosis or parthanatos under the aforementioned conditions. Exogenous pyruvate plays a pivotal role in maintaining the flux in PARP-dependent glycolysis and the PARP-independent TCA cycle in Schwann cells under high-glucose conditions.

Keywords:  PARP; Schwann cells; adenosine triphosphate depletion; cell death; exogenous pyruvate; glycolysis; high-glucose; tricarboxylic acid cycle

DOI:  https://doi.org/10.3390/ijms252011089
Cancer Sci. 2024 Oct 31.

Noncanonical TCA cycle fosters canonical TCA cycle and mitochondrial integrity in acute myeloid leukemia.

Atsushi Watanabe, Chartsiam Tipgomut, Haruhito Totani, Kentaro Yoshimura, Tomohiko Iwano, Hamed Bashiri, Lee Hui Chua, Chong Yang, Toshio Suda.

  Cancer cells rely on mitochondrial oxidative phosphorylation (OXPHOS) and the noncanonical tricarboxylic acid (TCA) cycle. In this paper, we shed light on the vital role played by the noncanonical TCA cycle in a host-side concession to mitochondria, especially in highly energy-demanding malignant tumor cells. Inhibition of ATP-citrate lyase (ACLY), a key enzyme in the noncanonical TCA cycle, induced apoptosis by increasing reactive oxygen species levels and DNA damage while reducing mitochondrial membrane potential. The mitochondrial membrane citrate transporter inhibitor, CTPI2, synergistically enhanced these effects. ACLY inhibition reduced cytosolic citrate levels and CTPI2 lowered ACLY activity, suggesting that the noncanonical TCA cycle is sustained by a positive feedback mechanism. These inhibitions impaired ATP production, particularly through OXPHOS. Metabolomic analysis of mitochondrial and cytosolic fractions revealed reduced levels of glutathione pathway-related and TCA cycle-related metabolite, except fumarate, in mitochondria following noncanonical TCA cycle inhibition. Despite the efficient energy supply to the cell by mitochondria, this symbiosis poses challenges related to reactive oxygen species and mitochondrial maintenance. In conclusion, the noncanonical TCA cycle is indispensable for the canonical TCA cycle and mitochondrial integrity, contributing to mitochondrial domestication.

Keywords:  ATP‐citrate lyase; antimetabolites; apoptosis; cancer metabolism; cell lines; hematopoietic organ; mitochondria; noncanonical TCA cycle; others; reactive oxygen species

DOI:  https://doi.org/10.1111/cas.16347
Methods Enzymol. 2024 ;pii: S0076-6879(24)00379-3. [Epub ahead of print]706 125-158

Methods to monitor mitochondrial disulfide bonds.

Ben Hur Marins Mussulini, Michal Wasilewski, Agnieszka Chacinska.

  Mitochondria contain numerous proteins that utilize the chemistry of cysteine residues, which can be reversibly oxidized. These proteins are involved in mitochondrial biogenesis, protection against oxidative stress, metabolism, energy transduction to adenosine triphosphate, signaling and cell death among other functions. Many proteins located in the mitochondrial intermembrane space are imported by the mitochondrial import and assembly pathway the activity of which is based on the reversible oxidation of cysteine residues and oxidative trapping of substrates. Oxidative modifications of cysteine residues are particularly difficult to study because of their labile character. Here we present techniques that allow for monitoring the oxidative state of mitochondrial proteins as well as to investigate the mitochondrial import and assembly pathway. This chapter conveys basic concepts on sample preparation and techniques to monitor the redox state of cysteine residues in mitochondrial proteins as well as the strategies to study mitochondrial import and assembly pathway.

Keywords:  Direct thiol trap; Import assay; Indirect thiol trap; MIA40; Mitochondria

DOI:  https://doi.org/10.1016/bs.mie.2024.07.039
PLoS Biol. 2024 Oct 28. 22(10): e3002854

MICU2 up-regulation enhances tumor aggressiveness and metabolic reprogramming during colorectal cancer development.

Alison Robert, David Crottès, Jérôme Bourgeais, Naig Gueguen, Arnaud Chevrollier, Jean-François Dumas, Stéphane Servais, Isabelle Domingo, Stéphanie Chadet, Julien Sobilo, Olivier Hérault, Thierry Lecomte, Christophe Vandier, William Raoul, Maxime Guéguinou.

The mitochondrial Ca2+ uniporter (MCU) plays crucial role in intramitochondrial Ca2+ uptake, allowing Ca2+-dependent activation of oxidative metabolism. In recent decades, the role of MCU pore-forming proteins has been highlighted in cancer. However, the contribution of MCU-associated regulatory proteins mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2) to pathophysiological conditions has been poorly investigated. Here, we describe the role of MICU2 in cell proliferation and invasion using in vitro and in vivo models of human colorectal cancer (CRC). Transcriptomic analysis demonstrated an increase in MICU2 expression and the MICU2/MICU1 ratio in advanced CRC and CRC-derived metastases. We report that expression of MICU2 is necessary for mitochondrial Ca2+ uptake and quality of the mitochondrial network. Our data reveal the interplay between MICU2 and MICU1 in the metabolic flexibility between anaerobic glycolysis and OXPHOS. Overall, our study sheds light on the potential role of the MICUs in diseases associated with metabolic reprogramming.

DOI: https://doi.org/10.1371/journal.pbio.3002854
bioRxiv. 2024 Oct 25. pii: 2024.10.25.619450. [Epub ahead of print]

Integrated systems biology identifies disruptions in mitochondrial function and metabolism as key contributors to heart failure with preserved ejection fraction (HFpEF).

Andrew A Gibb, Kyle LaPenna, Ryan B Gaspar, Nadina R Latchman, Yinfei Tan, Carmen Choya-Foces, Jake E Doiron, Zhen Li, Huijing Xia, Michael P Lazaropoulos, Mariell Conwell, Thomas E Sharp, Traci T Goodchild, David J Lefer, John W Elrod.

Background: Heart failure with preserved ejection fraction (HFpEF) accounts for ∼50% of HF cases, with no effective treatments. The ZSF1-obese rat model recapitulates numerous clinical features of HFpEF including hypertension, obesity, metabolic syndrome, exercise intolerance, and LV diastolic dysfunction. Here, we utilized a systems-biology approach to define the early metabolic and transcriptional signatures to gain mechanistic insight into the pathways contributing to HFpEF development.Methods: Male ZSF1-obese, ZSF1-lean hypertensive controls, and WKY (wild-type) controls were compared at 14w of age for extensive physiological phenotyping and LV tissue harvesting for unbiased metabolomics, RNA-sequencing, and assessment of mitochondrial morphology and function. Utilizing ZSF1-lean and WKY controls enabled a distinction between hypertension-driven molecular changes contributing to HFpEF pathology, versus hypertension + metabolic syndrome.
Results: ZSF1-obese rats displayed numerous clinical features of HFpEF. Comparison of ZSF1-lean vs WKY (i.e., hypertension-exclusive effects) revealed metabolic remodeling suggestive of increased aerobic glycolysis, decreased β-oxidation, and dysregulated purine and pyrimidine metabolism with few transcriptional changes. ZSF1-obese rats displayed worsened metabolic remodeling and robust transcriptional remodeling highlighted by the upregulation of inflammatory genes and downregulation of the mitochondrial structure/function and cellular metabolic processes. Integrated network analysis of metabolomic and RNAseq datasets revealed downregulation of nearly all catabolic pathways contributing to energy production, manifesting in a marked decrease in the energetic state (i.e., reduced ATP/ADP, PCr/ATP). Cardiomyocyte ultrastructure analysis revealed decreased mitochondrial area, size, and cristae density, as well as increased lipid droplet content in HFpEF hearts. Mitochondrial function was also impaired as demonstrated by decreased substrate-mediated respiration and dysregulated calcium handling.
Conclusions: Collectively, the integrated omics approach applied here provides a framework to uncover novel genes, metabolites, and pathways underlying HFpEF, with an emphasis on mitochondrial energy metabolism as a potential target for intervention.

DOI: https://doi.org/10.1101/2024.10.25.619450
Anal Chem. 2024 Nov 01.

Mass Spectrometry Probe Combined with Machine Learning to Capture the Relationship between Metabolites and Mitochondrial Complex Activity at the Whole-Cell Level.

Jia-Yi Zheng, Xiao-Yuan Ji, An-Qi Zhao, Fang-Yuan Sun, Li-Fang Liu, Gui-Zhong Xin.

Mitochondrial complex activity controls a multitude of physiological processes by regulating the cellular metabolism. Current methods for evaluating mitochondrial complex activity mainly focus on single metabolic reactions within mitochondria. These methods often require fresh samples in large quantities for mitochondria purification or intact mitochondrial membranes for real-time monitoring. Confronting these limitations, we shifted the analytical perspective toward interactive metabolic networks at the whole-cell level to reflect mitochondrial complex activity. To this end, we compiled a panel of mitochondrial respiratory chain-mapped metabolites (MRCMs), whose perturbations theoretically provide an overall reflection on mitochondrial complex activity. By introducing N-dimethyl-p-phenylenediamine and N-methyl-p-phenylenediamine as a pair of mass spectrometry probes, an ultraperformance liquid chromatography-tandem mass spectrometry method with high sensitivity (LLOQ as low as 0.2 fmol) was developed to obtain accurate quantitative data of MRCMs. Machine learning was then combined to capture the relationship between MRCMs and mitochondrial complex activity. Using Complex I as a proof-of-concept, we identified NADH, alanine, and phosphoenolpyruvate as metabolites associated with Complex I activity based on the whole-cell level. The effectiveness of using their concentrations to reflect Complex I activity was further validated in external data sets. Hence, by capturing the relationship between metabolites and mitochondrial complex activity at the whole-cell level, this study explores a novel analytical paradigm for the interrogation of mitochondrial complex activity, offering a favorable complement to existing methods particularly when sample quantities, type, and treatment timeliness pose challenges. More importantly, it shifts the focus from individual metabolic reactions within mitochondria to a more comprehensive view of an interactive metabolic network, which should serve as a promising direction for future research into the functional architecture between mitochondrial complexes and metabolites.

DOI: https://doi.org/10.1021/acs.analchem.4c04376
J Vis Exp. 2024 Oct 11.

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells.

Connie Chen, Katherine A Rafael, Ginam Cho, Youngshin Lim.

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

DOI: https://doi.org/10.3791/66862
Methods Enzymol. 2024 ;pii: S0076-6879(24)00364-1. [Epub ahead of print]706 75-95

Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP.

Solène Zuttion, Bruno Senger, Chiranjit Panja, Sylvie Friant, Róża Kucharczyk, Hubert Dominique Becker.

  Even if a myriad of approaches has been developed to identify the subcellular localization of a protein, the easiest and fastest way remains to fuse the protein to Green Fluorescent Protein (GFP) and visualize its location using fluorescence microscopy. However, this strategy is not well suited to visualize the organellar pools of proteins that are simultaneously localized both in the cytosol and in organelles because the GFP signal of a cytosolic pool of the protein (cytosolic echoform) will inevitably mask or overlay the GFP signal of the organellar pool of the protein (organellar echoform). To solve this issue, we engineered a dedicated yeast strain expressing a Bi-Genomic Mitochondrial-Split-GFP. This split-GFP is bi-genomic because the first ten ß-strands of GFP (GFPß1-10) are encoded by the mitochondrial genome and translated by mitoribosomes whereas the remaining ß-strand of GFP (GFPß11) is fused to the protein of interest encoded by the nucleus and expressed by cytosolic ribosomes. Consequently, if the GFPß11-tagged protein localizes into mitochondria, GFP will be reconstituted by self-assembly GFPß1-10 and GFPß11 thereby generating a GFP signal restricted to mitochondria and detectable by regular fluorescence microscopy. In addition, because mitochondrial translocases and import mechanisms are evolutionary well conserved, the BiG Mito-Split-GFP yeast strain can be used to probe mitochondrial importability of proteins regardless of their organismal origins and can thus serve to identify unsuspected mitochondrial echoforms readily from any organism.

Keywords:  Dual-localized; Microscopy; Mitochondria; Protein import; Saccharomyces cerevisiae; Split-GFP

DOI:  https://doi.org/10.1016/bs.mie.2024.07.028
Nat Commun. 2024 Oct 30. 15(1): 9394

HIF1α-regulated glycolysis promotes activation-induced cell death and IFN-γ induction in hypoxic T cells.

Hongxing Shen, Oluwagbemiga A Ojo, Haitao Ding, Logan J Mullen, Chuan Xing, M Iqbal Hossain, Abdelrahman Yassin, Vivian Y Shi, Zach Lewis, Ewa Podgorska, Shaida A Andrabi, Maciek R Antoniewicz, James A Bonner, Lewis Zhichang Shi.

Hypoxia is a common feature in various pathophysiological contexts, including tumor microenvironment, and IFN-γ is instrumental for anti-tumor immunity. HIF1α has long been known as a primary regulator of cellular adaptive responses to hypoxia, but its role in IFN-γ induction in hypoxic T cells is unknown. Here, we show that the HIF1α-glycolysis axis controls IFN-γ induction in both human and mouse T cells, activated under hypoxia. Specific deletion of HIF1α in T cells (Hif1α-/-) and glycolytic inhibition suppresses IFN-γ induction. Conversely, HIF1α stabilization by hypoxia and VHL deletion in T cells (Vhl-/-) increases IFN-γ production. Hypoxic Hif1α-/- T cells are less able to kill tumor cells in vitro, and tumor-bearing Hif1α-/- mice are not responsive to immune checkpoint blockade (ICB) therapy in vivo. Mechanistically, loss of HIF1α greatly diminishes glycolytic activity in hypoxic T cells, resulting in depleted intracellular acetyl-CoA and attenuated activation-induced cell death (AICD). Restoration of intracellular acetyl-CoA by acetate supplementation re-engages AICD, rescuing IFN-γ production in hypoxic Hif1α-/- T cells and re-sensitizing Hif1α-/- tumor-bearing mice to ICB. In summary, we identify HIF1α-regulated glycolysis as a key metabolic control of IFN-γ production in hypoxic T cells and ICB response.

DOI: https://doi.org/10.1038/s41467-024-53593-8
Nat Metab. 2024 Oct 28.

TrkB signalling regulates dopamine circuits and motor function through metabolic pathways.

DOI: https://doi.org/10.1038/s42255-024-01154-0
Cell Death Discov. 2024 Oct 29. 10(1): 457

Mitophagy: insights into its signaling molecules, biological functions, and therapeutic potential in breast cancer.

Cong Chen, Aizhai Xiang, Xia Lin, Jufeng Guo, Jian Liu, Shufang Hu, Tao Rui, Qianwei Ye.

Mitophagy, a form of selective autophagy that removes damaged or dysfunctional mitochondria, plays a crucial role in maintaining mitochondrial and cellular homeostasis. Recent findings suggest that defective mitophagy is closely associated with various diseases, including breast cancer. Moreover, a better understanding of the multifaceted roles of mitophagy in breast cancer progression is crucial for the treatment of this disease. Here, we will summarize the molecular mechanisms of mitophagy process. In addition, we highlight the expression patterns and roles of mitophagy-related signaling molecules in breast cancer progression and the potential implications of mitophagy for the development of breast cancer, aiming to provide better therapeutic strategies for breast cancer treatment.

DOI: https://doi.org/10.1038/s41420-024-02226-6
Methods Enzymol. 2024 ;pii: S0076-6879(24)00369-0. [Epub ahead of print]706 407-436

The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.

Hope I Needs, Youmian Yan, Natalie M Niemi, Ian Collinson.

  The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.

Keywords:  Cell culture; Luciferase; MitoLuc assay; Mitochondrial biogenesis; Mitochondrial protein import; NanoLuc; Protein translocation; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.033
J Cell Sci. 2024 Oct 15. pii: jcs262041. [Epub ahead of print]137(20):

Cell-cell junctions in focus - imaging junctional architectures and dynamics at high resolution.

Vera Janssen, Stephan Huveneers.

  Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.

Keywords:  Adhesion; Intercalated disc; Junctions; Mechanotransduction; Microscopy; Optogenetics; Super-resolution; Volume electron microscopy; Zonula adherens

DOI:  https://doi.org/10.1242/jcs.262041
bioRxiv. 2024 Oct 26. pii: 2024.10.24.619902. [Epub ahead of print]

A Novel Cyanine-Based Fluorescent Dye for Targeted Mitochondrial Imaging in Neurotoxic Conditions and In Vivo Brain Studies.

Xin Yan, Xinqian Chen, Shanshan Hou, Xiaoxu Li, Jingyue Ju, Zhiying Shan, Lanrong Bi.

Mitochondrial dysfunction is a key feature of neurodegenerative diseases, often preceding symptoms and influencing disease progression. However, real-time in vivo imaging of mitochondria in the brain is limited by existing dyes like MitoTrackers, which struggle with poor tissue penetration, phototoxicity, and inability to cross the blood-brain barrier (BBB). This study introduces Cy5-PEG4, a novel mitochondrial-targeting dye that overcomes these limitations, enabling high-resolution, non-invasive imaging of mitochondrial dynamics. Cy5-PEG4 effectively labels mitochondria in primary neuronal cells exposed to the SARS-CoV-2 RNYIAQVD peptide, revealing dose-dependent alterations in mitochondrial function that may contribute to COVID-19-related neurodegeneration. Importantly, Cy5-PEG4 crosses the BBB without causing neuroinflammation or toxicity, making it a safe tool for in vivo brain imaging and detailed studies of mitochondrial responses. In 3D cultured cells, Cy5-PEG4 captures dynamic changes in mitochondrial distribution and morphology as cell structures mature, highlighting its potential in neurobiological research, diagnostics, and therapeutic development. These findings support Cy5-PEG4 as a powerful tool for studying disease progression, identifying early biomarkers, and evaluating therapeutic strategies in neurodegenerative disorders and COVID-19.

DOI: https://doi.org/10.1101/2024.10.24.619902
Nat Commun. 2024 Oct 27. 15(1): 9267

Fluorescent non-canonical amino acid provides insight into the human serotonin transporter.

Andreas Nygaard, Linda G Zachariassen, Kathrine S Larsen, Anders S Kristensen, Claus J Loland.

The serotonin transporter (SERT), responsible for the reuptake of released serotonin, serves as a major target for antidepressants and psychostimulants. Nevertheless, refining the mechanistic models for SERT remains challenging. Here, we expand the molecular understanding of the binding of ions, substrates, and inhibitors to SERT by incorporating the fluorescent non-canonical amino acid Anap through genetic code expansion. We elucidate steady-state changes in conformational dynamics of purified SERT with Anap inserted at intracellular- or extracellular sites. This uncovers the competitive mechanisms underlying cation binding and assigns distinct binding- and allosteric coupling patterns for several inhibitors and substrates. Finally, we track in real-time conformational transitions in response to the interaction with Na+ or serotonin. In this work, we present a methodological platform reporting on SERT conformational dynamics, which together with other approaches will deepen our insights into the molecular mechanisms of SERT.

DOI: https://doi.org/10.1038/s41467-024-53584-9
Nat Commun. 2024 Oct 25. 15(1): 9238

Accumulation of F-actin drives brain aging and limits healthspan in Drosophila.

Edward T Schmid, Joseph M Schinaman, Naomi Liu-Abramowicz, Kylie S Williams, David W Walker.

The actin cytoskeleton is a key determinant of cell structure and homeostasis. However, possible tissue-specific changes to actin dynamics during aging, notably brain aging, are not understood. Here, we show that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, decreasing F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, we show that autophagy, a recycling process required for neuronal homeostasis, is disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. Our data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan.

DOI: https://doi.org/10.1038/s41467-024-53389-w