bims-celmim 2024-07-21 papers

bims-celmim

Biomed News

on Cellular and mitochondrial metabolism

Issue of 2024–07–21
eightteen papers selected by
Marc Segarra Mondejar

Robustness of mitochondrial biogenesis and respiration explain aerobic glycolysis.
The role of NAD-dependent deacetylase sirtuin-2 in liver metabolic stress through regulating pyruvate kinase M2 ubiquitination.
Machine learning of cellular metabolic rewiring.
Analysis and Quantification of the Mitochondrial-ER Lipidome.
Metabolic Reprogramming Is an Initial Step in Pancreatic Carcinogenesis That Can Be Targeted to Inhibit Acinar-to-Ductal Metaplasia.
Targeting valine catabolism to inhibit metabolic reprogramming in prostate cancer.
Amino acids trigger MDC-dependent mitochondrial remodeling by altering mitochondrial function.
Imaging Tumor Metabolism.
Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.
Metabolism changes caused by glucose in normal and cancer human brain cell lines by Raman imaging and chemometric methods.
Metabolic imaging across scales reveals distinct prostate cancer phenotypes.
Role of vaccinia virus growth factor in stimulating the mTORC1-CAD axis of the de novo pyrimidine pathway under different nutritional cues.
Regulation of and challenges in targeting NAD+ metabolism.
Acetyl-CoA synthetase activity is enzymatically regulated by lysine acetylation using acetyl-CoA or acetyl-phosphate as donor molecule.
Therapeutic targeting of immunometabolism reveals a critical reliance on hexokinase 2 dosage for microglial activation and Alzheimer's progression.
Structural basis of nucleotide selectivity in pyruvate kinase.
Mfn2-dependent fusion pathway of PE-enriched micron-sized vesicles.
The metabolic benefits of thermogenic stimulation are preserved in aging.

bioRxiv. 2024 Jul 05. pii: 2024.07.04.601975. [Epub ahead of print]

Robustness of mitochondrial biogenesis and respiration explain aerobic glycolysis.

Easun Arunachalam, Felix C Keber, Richard C Law, Chirag K Kumar, Yihui Shen, Junyoung O Park, Martin Wühr, Daniel J Needleman.

A long-standing observation is that in fast-growing cells, respiration rate declines with increasing growth rate and is compensated by an increase in fermentation, despite respiration being more efficient than fermentation. This apparent preference for fermentation even in the presence of oxygen is known as aerobic glycolysis, and occurs in bacteria, yeast, and cancer cells. Considerable work has focused on understanding the potential benefits that might justify this seemingly wasteful metabolic strategy, but its mechanistic basis remains unclear. Here we show that aerobic glycolysis results from the saturation of mitochondrial respiration and the decoupling of mitochondrial biogenesis from the production of other cellular components. Respiration rate is insensitive to acute perturbations of cellular energetic demands or nutrient supplies, and is explained simply by the amount of mitochondria per cell. Mitochondria accumulate at a nearly constant rate across different growth conditions, resulting in mitochondrial amount being largely determined by cell division time. In contrast, glucose uptake rate is not saturated, and is accurately predicted by the abundances and affinities of glucose transporters. Combining these models of glucose uptake and respiration provides a quantitative, mechanistic explanation for aerobic glycolysis. The robustness of specific respiration rate and mitochondrial biogenesis, paired with the flexibility of other bioenergetic and biosynthetic fluxes, may play a broad role in shaping eukaryotic cell metabolism.

DOI: https://doi.org/10.1101/2024.07.04.601975
J Transl Med. 2024 Jul 14. 22(1): 656

The role of NAD-dependent deacetylase sirtuin-2 in liver metabolic stress through regulating pyruvate kinase M2 ubiquitination.

Jingru Guo, Junshu Nie, Dongni Li, Huaixiu Zhang, Tianrui Zhao, Shoufeng Zhang, Li Ma, Jingjing Lu, Hong Ji, Shize Li, Sha Tao, Bin Xu.

  NAD-dependent deacetylase Sirt2 is involved in mammalian metabolic activities, matching energy demand with energy production and expenditure, and is relevant to a variety of metabolic diseases. Here, we constructed Sirt2 knockout and adeno-associated virus overexpression mice and found that deletion of hepatic Sirt2 accelerated primary obesity and insulin resistance in mice with concomitant hepatic metabolic dysfunction. However, the key targets of Sirt2 are unknown. We identified the M2 isoform of pyruvate kinase (PKM2) as a key Sirt2 target involved in glycolysis in metabolic stress. Through yeast two-hybrid and mass spectrometry combined with multi-omics analysis, we identified candidate acetylation modification targets of Sirt2 on PKM2 lysine 135 (K135). The Sirt2-mediated deacetylation-ubiquitination switch of PKM2 regulated the development of glycolysis. Here, we found that Sirt2 deficiency led to impaired glucose tolerance and insulin resistance and induced primary obesity. Sirt2 severely disrupted liver function in mice under metabolic stress, exacerbated the metabolic burden on the liver, and affected glucose metabolism. Sirt2 underwent acetylation modification of lysine 135 of PKM2 through a histidine 187 enzyme active site-dependent effect and reduced ubiquitination of the K48 ubiquitin chain of PKM2. Our findings reveal that the hepatic glucose metabolism links nutrient state to whole-body energetics through the rhythmic regulation of Sirt2.

Keywords:  Acetylation; Glycolysis; Metabolism; PKM2; Sirt2

DOI:  https://doi.org/10.1186/s12967-024-05435-w
Biol Methods Protoc. 2024 ;9(1): bpae048

Machine learning of cellular metabolic rewiring.

Joao B Xavier.

Metabolic rewiring allows cells to adapt their metabolism in response to evolving environmental conditions. Traditional metabolomics techniques, whether targeted or untargeted, often struggle to interpret these adaptive shifts. Here, we introduce MetaboLiteLearner, a lightweight machine learning framework that harnesses the detailed fragmentation patterns from electron ionization (EI) collected in scan mode during gas chromatography/mass spectrometry to predict changes in the metabolite composition of metabolically adapted cells. When tested on breast cancer cells with different preferences to metastasize to specific organs, MetaboLiteLearner predicted the impact of metabolic rewiring on metabolites withheld from the training dataset using only the EI spectra, without metabolite identification or pre-existing knowledge of metabolic networks. Despite its simplicity, the model learned captured shared and unique metabolomic shifts between brain- and lung-homing metastatic lineages, suggesting cellular adaptations associated with metastasis to specific organs. Integrating machine learning and metabolomics paves the way for new insights into complex cellular adaptations.

DOI: https://doi.org/10.1093/biomethods/bpae048
Bio Protoc. 2024 Jul 05. 14(13): e5028

Analysis and Quantification of the Mitochondrial-ER Lipidome.

Alexis R Diaz-Vegas, Anthony S Don, James G Burchfield.

  Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress. Key features • Analysis and quantification of lipids in mitochondria-ER fraction through liquid chromatography-tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics). • LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems. • LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.

Keywords:  Cardiolipin; Ceramides; Endoplasmic reticulum; Lipidomics; Mitochondria; Subcellular fractionation

DOI:  https://doi.org/10.21769/BioProtoc.5028
Cancer Res. 2024 Jul 15. 84(14): 2297-2312

Metabolic Reprogramming Is an Initial Step in Pancreatic Carcinogenesis That Can Be Targeted to Inhibit Acinar-to-Ductal Metaplasia.

Thorsten Neuß, Min-Chun Chen, Nils Wirges, Sinem Usluer, Rupert Oellinger, Svenja Lier, Michael Dudek, Tobias Madl, Martin Jastroch, Katja Steiger, Werner Schmitz, Henrik Einwächter, Roland M Schmid.

Metabolic reprogramming is a hallmark of cancer and is crucial for cancer progression, making it an attractive therapeutic target. Understanding the role of metabolic reprogramming in cancer initiation could help identify prevention strategies. To address this, we investigated metabolism during acinar-to-ductal metaplasia (ADM), the first step of pancreatic carcinogenesis. Glycolytic markers were elevated in ADM lesions compared with normal tissue from human samples. Comprehensive metabolic assessment in three mouse models with pancreas-specific activation of KRAS, PI3K, or MEK1 using Seahorse measurements, nuclear magnetic resonance metabolome analysis, mass spectrometry, isotope tracing, and RNA sequencing analysis revealed a switch from oxidative phosphorylation to glycolysis in ADM. Blocking the metabolic switch attenuated ADM formation. Furthermore, mitochondrial metabolism was required for de novo synthesis of serine and glutathione (GSH) but not for ATP production. MYC mediated the increase in GSH intermediates in ADM, and inhibition of GSH synthesis suppressed ADM development. This study thus identifies metabolic changes and vulnerabilities in the early stages of pancreatic carcinogenesis. Significance: Metabolic reprogramming from oxidative phosphorylation to glycolysis mediated by MYC plays a crucial role in the development of pancreatic cancer, revealing a mechanism driving tumorigenesis and potential therapeutic targets. See related commentary by Storz, p. 2225.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-2213
Cell Death Dis. 2024 Jul 18. 15(7): 513

Targeting valine catabolism to inhibit metabolic reprogramming in prostate cancer.

Charles L Bidgood, Lisa K Philp, Anja Rockstroh, Melanie Lehman, Colleen C Nelson, Martin C Sadowski, Jennifer H Gunter.

Metabolic reprogramming and energetic rewiring are hallmarks of cancer that fuel disease progression and facilitate therapy evasion. The remodelling of oxidative phosphorylation and enhanced lipogenesis have previously been characterised as key metabolic features of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours, as well as upregulation of the enzymes associated with branched-chain amino acid (BCAA) catabolism. In this study, we hypothesised that the degradation of the BCAAs, particularly valine, may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool, as well as the maintenance of intracellular lipid metabolism. Through the suppression of BCAA availability, we report significantly reduced lipid content, strongly indicating that BCAAs are important lipogenic fuels in PCa. This work also uncovered a novel compensatory mechanism, whereby fatty acid uptake is increased in response to extracellular valine deprivation. Inhibition of valine degradation via suppression of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in a selective reduction of malignant prostate cell proliferation, decreased intracellular succinate and impaired cellular respiration. In combination with a comprehensive multi-omic investigation that incorporates next-generation sequencing, metabolomics, and high-content quantitative single-cell imaging, our work highlights a novel therapeutic target for selective inhibition of metabolic reprogramming in PCa.

DOI: https://doi.org/10.1038/s41419-024-06893-2
bioRxiv. 2024 Jul 12. pii: 2024.07.09.602707. [Epub ahead of print]

Amino acids trigger MDC-dependent mitochondrial remodeling by altering mitochondrial function.

Nidhi Raghuram, Adam Hughes.

Cells utilize numerous pathways to maintain mitochondrial homeostasis, including a recently identified mechanism that adjusts the content of the outer mitochondrial membrane (OMM) through formation of OMM-derived multilamellar domains called mitochondrial-derived compartments, or MDCs. MDCs are triggered by perturbations in mitochondrial lipid and protein content, as well as increases in intracellular amino acids. Here, we sought to understand how amino acids trigger MDCs. We show that amino acid-activation of MDCs is dependent on the functional state of mitochondria. While amino acid excess triggers MDC formation when cells are grown on fermentable carbon sources, stimulating mitochondrial biogenesis blocks MDC formation. Moreover, amino acid elevation depletes TCA cycle metabolites in yeast, and preventing consumption of TCA cycle intermediates for amino acid catabolism suppresses MDC formation. Finally, we show that directly impairing the TCA cycle is sufficient to trigger MDC formation in the absence of amino acid stress. These results demonstrate that amino acids stimulate MDC formation by perturbing mitochondrial metabolism.

DOI: https://doi.org/10.1101/2024.07.09.602707
Cold Spring Harb Perspect Med. 2024 Jul 15. pii: a041551. [Epub ahead of print]

Imaging Tumor Metabolism.

Thomas Ruan, Kayvan R Keshari.

Molecular imaging-the mapping of molecular and cellular processes in vivo-has the unique capability to interrogate cancer metabolism in its spatial contexts. This work describes the usage of the two most developed modalities for imaging metabolism in vivo: positron emission tomography (PET) and magnetic resonance (MR). These techniques can be used to probe glycolysis, glutamine metabolism, anabolic metabolism, redox state, hypoxia, and extracellular acidification. This review aims to provide an overview of the strengths and limitations of currently available molecular imaging strategies.

DOI: https://doi.org/10.1101/cshperspect.a041551
Proc Natl Acad Sci U S A. 2024 Jul 23. 121(30): e2319782121

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Daniel Maxim Iascone, Xue Zhang, Patricia Brafford, Clementina Mesaros, Yogev Sela, Samuel Hofbauer, Shirley L Zhang, Sukanya Madhwal, Kieona Cook, Pavel Pivarshev, Ben Z Stanger, Stewart Anderson, Chi V Dang, Amita Sehgal.

  Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Keywords:  adenocarcinoma; cancer; circadian rhythms; luciferase; metabolism

DOI:  https://doi.org/10.1073/pnas.2319782121
Sci Rep. 2024 Jul 18. 14(1): 16626

Metabolism changes caused by glucose in normal and cancer human brain cell lines by Raman imaging and chemometric methods.

Monika Kopec, Karolina Beton-Mysur, Jakub Surmacki, Halina Abramczyk.

  Glucose is the main source of energy for the human brain. This paper presents a non-invasive technique to study metabolic changes caused by glucose in human brain cell lines. In this paper we present the spectroscopic characterization of human normal brain (NHA; astrocytes) and human cancer brain (CRL-1718; astrocytoma and U-87 MG; glioblastoma) control cell lines and cell lines upon supplementation with glucose. Based on Raman techniques we have identified biomarkers that can monitor metabolic changes in lipid droplets, mitochondria and nucleus caused by glucose. We have studied the vibrations at 750 cm-1, 1444 cm-1, 1584 cm-1 and 1656 cm-1 as a function of malignancy grade. We have compared the concentration of cytochrome, lipids and proteins in the grade of cancer aggressiveness in normal and cancer human brain cell lines. Chemometric analysis has shown that control normal, control cancer brain cell lines and normal and cancer cell lines after supplementation with glucose can be distinguished based on their unique vibrational properties. PLSDA (Partial Least Squares Discriminant Analysis) and ANOVA tests have confirmed the main role of cytochromes, proteins and lipids in differentiation of control human brain cells and cells upon supplementation with glucose. We have shown that Raman techniques combined with chemometric analysis provide additional insight to monitor the biology of astrocytes, astrocytoma and glioblastoma after glucose supplementation.

Keywords:  Biomarkers; Brain cancer; Glucose; PLS-DA; Raman imaging; Raman spectroscopy

DOI:  https://doi.org/10.1038/s41598-024-67718-y
Nat Commun. 2024 Jul 16. 15(1): 5980

Metabolic imaging across scales reveals distinct prostate cancer phenotypes.

Nikita Sushentsev, Gregory Hamm, Lucy Flint, Daniel Birtles, Aleksandr Zakirov, Jack Richings, Stephanie Ling, Jennifer Y Tan, Mary A McLean, Vinay Ayyappan, Ines Horvat Menih, Cara Brodie, Jodi L Miller, Ian G Mills, Vincent J Gnanapragasam, Anne Y Warren, Simon T Barry, Richard J A Goodwin, Tristan Barrett, Ferdia A Gallagher.

Hyperpolarised magnetic resonance imaging (HP-13C-MRI) has shown promise as a clinical tool for detecting and characterising prostate cancer. Here we use a range of spatially resolved histological techniques to identify the biological mechanisms underpinning differential [1-13C]lactate labelling between benign and malignant prostate, as well as in tumours containing cribriform and non-cribriform Gleason pattern 4 disease. Here we show that elevated hyperpolarised [1-13C]lactate signal in prostate cancer compared to the benign prostate is primarily driven by increased tumour epithelial cell density and vascularity, rather than differences in epithelial lactate concentration between tumour and normal. We also demonstrate that some tumours of the cribriform subtype may lack [1-13C]lactate labelling, which is explained by lower epithelial lactate dehydrogenase expression, higher mitochondrial pyruvate carrier density, and increased lipid abundance compared to lactate-rich non-cribriform lesions. These findings highlight the potential of combining spatial metabolic imaging tools across scales to identify clinically significant metabolic phenotypes in prostate cancer.

DOI: https://doi.org/10.1038/s41467-024-50362-5
bioRxiv. 2024 Jul 02. pii: 2024.07.02.601567. [Epub ahead of print]

Role of vaccinia virus growth factor in stimulating the mTORC1-CAD axis of the de novo pyrimidine pathway under different nutritional cues.

Lara Dsouza, Anil Pant, Blake Pope, Zhilong Yang.

Vaccinia virus (VACV), the prototype poxvirus, actively reprograms host cell metabolism upon infection. However, the nature and molecular mechanisms remain largely elusive. Given the diverse nutritional exposures of cells in different physiological contexts, it is essential to understand how VACV may alter various metabolic pathways in different nutritional conditions. In this study, we established the importance of de novo pyrimidine biosynthesis in VACV infection. We elucidated the significance of vaccinia growth factor (VGF), a viral early protein and a homolog of cellular epidermal growth factor, in enabling VACV to phosphorylate the key enzyme CAD of the de novo pyrimidine pathway at serine 1859, a site known to positively regulate CAD activity. While nutrient-poor conditions typically inhibit mTORC1 activation, VACV activates CAD via mTORC1-S6K1 signaling axis, in conditions where glutamine and asparagine are absent. However, unlike its cellular homolog, epidermal growth factor (EGF), VGF peptide alone in the absence of VACV infection has minimal ability to activate CAD, suggestive of the involvement of other viral factor(s) and differential functions to EGF acquired during poxvirus evolution. Our research provides a foundation for understanding the regulation of a significant metabolic pathway, namely, de novo pyrimidine synthesis during VACV infection, shedding new light on viral regulation under distinct nutritional environments. This study not only has the potential to contribute to the advancement of antiviral treatments but also improve the development of VACV as an oncolytic agent and vaccine vector.
Importance: Our research provides new insights into how VACV alters the mTORC1-CAD signaling axis under different nutritional cues. The identification of how VACV regulates a major enzyme, CAD, within the de novo pyrimidine synthesis pathway, establishes a molecular mechanism for determining how VACV reshapes this essential pathway, necessary for facilitating efficient VACV replication. We further emphasize that, despite nutrient-poor conditions, which typically inhibit mTORC1 activation, VACV can stimulate mTORC1. We identify its early growth factor, VGF, as an important factor for this stimulation of mTORC1 and its downstream effector CAD, revealing a new mechanism for how VACV sustains mTORC1-CAD axis activation under these nutrient deficient conditions. This work provides fresh insights into the molecular mechanisms of mTORC1-CAD regulation, which has the potential to be utilized to enhance VACV as an oncolytic tool, vaccine vector and aid in the development of antiviral drugs.

DOI: https://doi.org/10.1101/2024.07.02.601567
Nat Rev Mol Cell Biol. 2024 Jul 18.

Regulation of and challenges in targeting NAD+ metabolism.

Marie E Migaud, Mathias Ziegler, Joseph A Baur.

Nicotinamide adenine dinucleotide, in its oxidized (NAD+) and reduced (NADH) forms, is a reduction-oxidation (redox) co-factor and substrate for signalling enzymes that have essential roles in metabolism. The recognition that NAD+ levels fall in response to stress and can be readily replenished through supplementation has fostered great interest in the potential benefits of increasing or restoring NAD+ levels in humans to prevent or delay diseases and degenerative processes. However, much about the biology of NAD+ and related molecules remains poorly understood. In this Review, we discuss the current knowledge of NAD+ metabolism, including limitations of, assumptions about and unappreciated factors that might influence the success or contribute to risks of NAD+ supplementation. We highlight several ongoing controversies in the field, and discuss the role of the microbiome in modulating the availability of NAD+ precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), the presence of multiple cellular compartments that have distinct pools of NAD+ and NADH, and non-canonical NAD+ and NADH degradation pathways. We conclude that a substantial investment in understanding the fundamental biology of NAD+, its detection and its metabolites in specific cells and cellular compartments is needed to support current translational efforts to safely boost NAD+ levels in humans.

DOI: https://doi.org/10.1038/s41580-024-00752-w
Nat Commun. 2024 Jul 17. 15(1): 6002

Acetyl-CoA synthetase activity is enzymatically regulated by lysine acetylation using acetyl-CoA or acetyl-phosphate as donor molecule.

Chuan Qin, Leonie G Graf, Kilian Striska, Markus Janetzky, Norman Geist, Robin Specht, Sabrina Schulze, Gottfried J Palm, Britta Girbardt, Babett Dörre, Leona Berndt, Stefan Kemnitz, Mark Doerr, Uwe T Bornscheuer, Mihaela Delcea, Michael Lammers.

The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5'-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.

DOI: https://doi.org/10.1038/s41467-024-49952-0
Cell Rep. 2024 Jul 11. pii: S2211-1247(24)00817-9. [Epub ahead of print]43(7): 114488

Therapeutic targeting of immunometabolism reveals a critical reliance on hexokinase 2 dosage for microglial activation and Alzheimer's progression.

Juan F Codocedo, Claudia Mera-Reina, Peter Bor-Chian Lin, Paul B Fallen, Shweta S Puntambekar, Brad T Casali, Nur Jury-Garfe, Pablo Martinez, Cristian A Lasagna-Reeves, Gary E Landreth.

  Neuroinflammation is a prominent feature of Alzheimer's disease (AD). Activated microglia undergo a reprogramming of cellular metabolism necessary to power their cellular activities during disease. Thus, selective targeting of microglial immunometabolism might be of therapeutic benefit for treating AD. In the AD brain, the levels of microglial hexokinase 2 (HK2), an enzyme that supports inflammatory responses by promoting glycolysis, are significantly increased. In addition, HK2 displays non-metabolic activities that extend its inflammatory role beyond glycolysis. The antagonism of HK2 affects microglial phenotypes and disease progression in a gene-dose-dependent manner. HK2 complete loss fails to improve pathology by exacerbating inflammation, while its haploinsufficiency reduces pathology in 5xFAD mice. We propose that the partial antagonism of HK2 is effective in slowing disease progression by modulating NF-κB signaling through its cytosolic target, IKBα. The complete loss of HK2 affects additional inflammatory mechanisms related to mitochondrial dysfunction.

Keywords:  Alzheimer’s disease; CP: Metabolism; CP: Neuroscience; NF-κB; amyloid; hexokinase 2; inflammation; lonidamine; microglia; mitochondria

DOI:  https://doi.org/10.1016/j.celrep.2024.114488
J Mol Biol. 2024 Jul 13. pii: S0022-2836(24)00317-6. [Epub ahead of print] 168708

Structural basis of nucleotide selectivity in pyruvate kinase.

Atsushi Taguchi, Ryosuke Nakashima, Kunihiko Nishino.

  Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

Keywords:  ADP; GDP; Glycolysis; X-ray crystallography; prokaryotic metabolism

DOI:  https://doi.org/10.1016/j.jmb.2024.168708
Proc Natl Acad Sci U S A. 2024 Jul 23. 121(30): e2313609121

Mfn2-dependent fusion pathway of PE-enriched micron-sized vesicles.

Daniel A Peñalva, Ajay K Monnappa, Paolo Natale, Iván López-Montero.

  Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.

Keywords:  giant unilamellar vesicles; membrane fusion; mitochondrial dynamics; mitofusin 2

DOI:  https://doi.org/10.1073/pnas.2313609121
bioRxiv. 2024 Jul 04. pii: 2024.07.01.601572. [Epub ahead of print]

The metabolic benefits of thermogenic stimulation are preserved in aging.

Duraipandy Natarajan, Bhuvana Plakkot, Kritika Tiwari, Shoba Ekambaram, Weidong Wang, Michael Rudolph, Mahmoud A Mohammad, Shaji K Chacko, Madhan Subramanian, Stefano Tarantini, Andriy Yabluchanskiy, Zoltan Ungvari, Anna Csiszar, Priya Balasubramanian.

Adipose thermogenesis has been actively investigated as a therapeutic target for improving metabolic dysfunction in obesity. However, its applicability to middle-aged and older populations, which bear the highest obesity prevalence in the US (approximately 40%), remains uncertain due to age-related decline in thermogenic responses. In this study, we investigated the effects of chronic thermogenic stimulation using the β3-adrenergic (AR) agonist CL316,243 (CL) on systemic metabolism and adipose function in aged (18-month-old) C57BL/6JN mice. Sustained β3-AR treatment resulted in reduced fat mass, increased energy expenditure, increased fatty acid oxidation and mitochondrial activity in adipose depots, improved glucose homeostasis, and a favorable adipokine profile. At the cellular level, CL treatment increased uncoupling protein 1 (UCP1)-dependent thermogenesis in brown adipose tissue (BAT). However, in white adipose tissue (WAT) depots, CL treatment increased glycerol and lipid de novo lipogenesis (DNL) and turnover suggesting the activation of the futile substrate cycle of lipolysis and reesterification in a UCP1-independent manner. Increased lipid turnover was also associated with the simultaneous upregulation of proteins involved in glycerol metabolism, fatty acid oxidation, and reesterification in WAT. Further, a dose-dependent impact of CL treatment on inflammation was observed, particularly in subcutaneous WAT, suggesting a potential mismatch between fatty acid supply and oxidation. These findings indicate that chronic β3-AR stimulation activates distinct cellular mechanisms that increase energy expenditure in BAT and WAT to improve systemic metabolism in aged mice. Our study provides foundational evidence for targeting adipose thermogenesis to improve age-related metabolic dysfunction.

DOI: https://doi.org/10.1101/2024.07.01.601572