bims-toxgon 2025-03-02 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2025–03–02
nineteen papers selected by
Lakesh Kumar, BITS Pilani

Toxoplasma gondii Type I TR and ROP16 Synergistically Downregulate IL-12 to Inhibit Host Reactive Oxygen Species Production.
Toxoplasma gondii (GRA10): A Closer Glance Into Immunogenic and Biochemical Characteristics Using In Silico Approach.
Toxoplasma gondii ROP18 induces maternal-fetal dysfunction by downregulating CD73 expression on decidual macrophages.
Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil.
Activity Evaluation and Mode of Action of ICA Against Toxoplasma gondii In Vitro.
Design, Synthesis, and Structure-Activity Relationship of 2-(Piperazin-1-yl)quinazolin-4(3H)-one Derivatives as Active Agents against Toxoplasma gondii.
Activity of Carbazole, Aminoguanidine and Diamine Anti-infectives against Toxoplasma gondii.
Visualization of Subcellular mTOR Complex 1 Activity with a FRET-Based Sensor (TORCAR).
[Functions and mechanisms of Zn2+-dependent histone deacetylase in plant responses to abiotic stress].
Advances in the Development of Mitochondrial Pyruvate Carrier Inhibitors for Therapeutic Applications.
FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK.
Trypanosoma cruzi cell cycle progression exhibits minimal variation in histone PTMs with unique histone H4 acetylation pattern.
Monitoring Cellular Energy Balance in Single Cells Using Fluorescent Biosensors for AMPK.
NAD+ Metabolism Reprogramming Drives SIRT1-Dependent Deacetylation Inducing PD-L1 Nuclear Localization in Cervical Cancer.
How does the tubulin code facilitate directed cell migration?
Determination of Nutrient Ligand-Sensor Binding Affinity.
A Genetically Encoded Fluorescent Biosensor for Intracellular Measurement of Malonyl-CoA.
Measuring Cellular Adenine Nucleotides by Liquid Chromatography-Coupled Mass Spectrometry.
Exploring the differential localization of protein kinase A isoforms in Candida albicans.

Pathogens. 2025 Feb 08. pii: 171. [Epub ahead of print]14(2):

Toxoplasma gondii Type I TR and ROP16 Synergistically Downregulate IL-12 to Inhibit Host Reactive Oxygen Species Production.

Xiaoling Geng, Ruifang Li, Jingying Du, Manyu Zhang, Wei Jiang, Qing Sun, Rongsheng Mi, Shuang Qin, Quan Wang.

  Toxoplasma gondii is an obligate intracellular opportunistic protozoan parasite. T. gondii invasion disturbs the balance between reactive oxygen species (ROS) production and antioxidant capacity in the host, triggering the oxidative stress response. Thioredoxin reductase (TR) of T. gondii helps to escape ROS-induced damage in the host, whereas T. gondii rhoptry protein 16 (ROP16) downregulates host innate immunity to suppress excessive inflammation and inhibit ROS production. However, whether TR and ROP16 synergistically promote resistance to ROS-induced damage remains unclear. Here, we used the CRISPR/Cas9 technology to successfully obtain a double TR and ROP16 gene knockout T. gondii strain. The double deletion of TR and ROP16 in T. gondii weakened its growth ability in vitro and decreased its virulence in vivo. Moreover, the double deletion of TR and ROP16 resulted in a lower antioxidant capacity, higher degree of lipid oxidation, and elevated ROS levels in the parasite and host cells. Interestingly, the deletion of the TR and ROP16 genes in T. gondii synergistically increased IL-12 levels, which triggered host cells to produce more ROS to resist T. gondii infection. These results show that TR and ROP16 in T. gondii play a synergistic role, facilitating resistance to ROS damage incurred by host immune cells through different pathways.

Keywords:  CRISPR/Cas9; Toxoplasma gondii; immune escape; oxidative stress; rhoptry protein 16; thioredoxin reductase

DOI:  https://doi.org/10.3390/pathogens14020171
Parasite Immunol. 2025 Feb;47(2): e70004

Toxoplasma gondii (GRA10): A Closer Glance Into Immunogenic and Biochemical Characteristics Using In Silico Approach.

Mahdi Khadem Mohammadi, Ali Dalir Ghaffari.

  Toxoplasma gondii, an obligate, intracellular, protozoan parasite, is known to infect a wide range of warm-blooded animals, including livestock, marine mammals and humans leading to the development of toxoplasmosis. The dense granule antigens (GRAs) have garnered crucial role in parasite survival, virulence and the formation of the parasitophorous vacuole. The study focusing on the GRA10 protein of T. gondii aims to elucidate its features further to support its potential inclusion in future vaccine formulations. The present study provided an exhaustive elucidation of the key characteristics of the GRA10 protein, encompassing its presence of a transmembrane domain, physico-chemical properties, subcellular localization, potential epitopes recognised by B-cells and T-cells, secondary and tertiary structures, as well as other significant attributes of this protein. The results indicated that the GRA10 protein possesses a total of 192 possible post-translational modification sites, with no transmembrane domains being detected within its structure. In terms of secondary structure, the GRA10 protein is composed of 27.74% alpha-helix, 13.53% extended strand and 58.72% random coil elements. Additionally, various potential B- and T-cell epitopes were pinpointed for the GRA10 protein, suggesting its immunogenic properties. The assessment of antigenicity and allergenicity further confirmed that GRA10 is immunogenic but non-allergenic, making it a promising candidate for further study. Furthermore, the induction of IFN-γ and IL-4 highlighted the ability of related MHC-II molecules to interact with GRA10, indicating its potential role in immune responses. These findings shed light on the multifaceted nature of the GRA10 protein and its significance in immunological processes. The study presented crucial fundamental and theoretical information regarding GRA10 in order to facilitate the creation of a potent vaccine against both acute and chronic toxoplasmosis, warranting additional in vivo examinations.

Keywords:   Toxoplasma gondii ; GRA10; bioinformatics; vaccines

DOI:  https://doi.org/10.1111/pim.70004
Parasit Vectors. 2025 Feb 24. 18(1): 72

Toxoplasma gondii ROP18 induces maternal-fetal dysfunction by downregulating CD73 expression on decidual macrophages.

Jingjing Guo, Xiaohui Wang, Lei Wei, Shuai Li, Junwei Wang, Yan Zhang, Ruohan Yang, Han Zhang, Aiqun Xu, Yuzhu Jiang, Xuemei Hu.

   BACKGROUND: Decidual macrophages (dMφ) are pivotal in maintaining maternal-fetal immune tolerance during normal pregnancy by expressing a range of immune-suppressive molecules, including CD73. It has been demonstrated that Toxoplasma gondii (T. gondii) infection during pregnancy can impair dMφ function, potentially leading to adverse pregnancy outcomes, through downregulation of these inhibitory molecules. T. gondii rhoptry protein 18 (TgROP18), a key virulence factor of T. gondii, is associated with the incapacitation of the host's innate and adaptive immune responses to protect the parasite from elimination. However, the role of TgROP18 in modulating CD73 expression on dMφ and the underlying mechanisms remain to be elucidated.
METHODS: Wild-type (WT) and CD73-deficient (CD73-/-) pregnant mice were subjected to intraperitoneal injection of T. gondii RH or RH-Δrop18 on gestational day (Gd) 8, and subsequently euthanized on Gd 14. Pregnancy outcomes were then evaluated, and the expression levels of CD73, arginase 1 (Arg-1), and interleukin 10 (IL-10) were quantified by flow cytometry. Mononuclear cells isolated from the human aborted decidual tissues were also infected with T. gondii RH or RH-Δrop18 for the analysis of CD73 expression with flow cytometry. Additionally, infected human dMφ were used to assess the expression levels of CD73, Arg-1, IL-10, and their associated signaling molecules by western blot analysis. Furthermore, chromatin immunoprecipitation (ChIP) assays were performed to validate the involved signaling pathways.
RESULTS: Compared with the T. gondii RH-infected group, milder adverse pregnancy outcomes and attenuated expression levels of CD73 on dMφ were observed in T. gondii RH-Δrop18-infected pregnant mice and human decidual tissues. Lysine-specific histone demethylase1 (LSD1) and snail family transcriptional repressor 1 (SNAIL1) were found to be involved in the downregulation of CD73 expression on dMφ following T. gondii infection. Subsequently, reduced expression of CD73 contribute to the downregulation of Arg-1 and IL-10 expression through adenosine A2a receptor (A2AR) / protein kinase A (PKA) / phosphorylated cAMP-response element binding protein (p-CREB) / CCAAT enhancer binding protein B (C/EBPβ) pathway.
CONCLUSIONS: TgROP18 can significantly reduce CD73 expression on dMφ through LSD1/SNAIL1 pathway, subsequently leading to the decreased expression levels of Arg-1 and IL-10 via adenosine/A2AR/PKA/p-CREB/C/EBPβ pathway, which ultimately contributes to maternal-fetal tolerance dysfunction of dMφ.

Keywords:   Toxoplasma gondii ; Abnormal pregnancy outcome; CD73; Decidual macrophage; ROP18

DOI:  https://doi.org/10.1186/s13071-025-06713-2
Microorganisms. 2025 Jan 29. pii: 301. [Epub ahead of print]13(2):

Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil.

Júlia Gatti Ladeia Costa, Érica Santos Martins-Duarte, Lorena Velozo Pinto, Ramon Araujo de Castro Baraviera, Wagner Martins Fontes do Rego, Ricardo Wagner de Almeida Vitor.

  Toxoplasma gondii is an obligate intracellular protozoan parasite distributed worldwide that infects a wide range of warm-blooded animals, including humans. Recent studies sought to clarify the relationship between the alleles GRA15, ROP5, ROP16, ROP17, and ROP18 and the virulence of T. gondii isolates in mice. This work aims to analyze the variability of genes that express T. gondii virulence proteins of 103 strains. Most strains were virulent for mice (76/103-73.79%); within these, 30 were 100% lethal, and 46 caused a cumulative mortality range from 20% to 93%. For the GRA15 marker, most strains presenting allele 2 were non-lethal. For the ROP17 marker, allele 4 was associated with mortality, compared to allele 1. For the ROP18 marker, alleles 1 and 4 were associated with mortality, compared to alleles 2 and 3. A combined analysis of alleles showed low cumulative mortality when the strains presented alleles 3 and 1 for ROP18 and ROP16, respectively. On the other hand, allele 4 of ROP17 was a determinant for virulence when associated with ROP18 allele 3 and ROP16 allele 1. Our analysis shows that ROP18 is the primary determinant of the virulence of atypical strains in mice. Additionally, ROP17 genotyping should not be overlooked, as it has proven critical to enhance this prediction.

Keywords:  PCR-RFLP; cumulative mortality; genotyping; rhoptry proteins; toxoplasmosis

DOI:  https://doi.org/10.3390/microorganisms13020301
Biomolecules. 2025 Feb 01. pii: 202. [Epub ahead of print]15(2):

Activity Evaluation and Mode of Action of ICA Against Toxoplasma gondii In Vitro.

Yanhua Qiu, Weiwei Wang, Qing Wang, Jing Xu, Guonian Dai, Yubin Bai, Jiyu Zhang.

  Toxoplasmosis is a significant zoonotic parasitic disease. Currently, there is no effective vaccine available to prevent human infection, and treatment primarily relies on chemotherapy. However, the lack of specific therapeutic agents and the limitations of existing drugs highlight the urgent need for novel, safe, and effective anti-Toxoplasma gondii (T. gondii) medications. In this study, we evaluated the toxicity of ICA (N-(pyridin-2-yl)-4-(pyridine-2-yl)thiazol-2-amine) to host cells and assessed its inhibitory and anti-proliferative effects on T. gondii tachyzoites. We further investigated the impact of ICA on the ultrastructure of T. gondii using transmission electron microscopy (TEM). Additionally, we measured alterations in mitochondrial membrane potential, superoxide levels, and ATP levels in T. gondii to assess the effect of ICA on mitochondrial function. Our findings demonstrated that ICA exhibits a safe and effective inhibitory effect on T. gondii, with a selectivity index (SI) of 258.25. Notably, ICA demonstrated a more potent anti-proliferative effect than pyrimethamine (PYR). Ultrastructural observations revealed that ICA induces mitochondrial swelling and membrane rupture in T. gondii. Further investigations confirmed that ICA leads to mitochondrial dysfunction in T. gondii. In conclusion, our results suggest that ICA possesses the potential to serve as a lead compound for the development of novel anti-T. gondii therapies.

Keywords:  ICA; Toxoplasma gondii; mitochondria; ultrastructure

DOI:  https://doi.org/10.3390/biom15020202
J Agric Food Chem. 2025 Feb 26.

Design, Synthesis, and Structure-Activity Relationship of 2-(Piperazin-1-yl)quinazolin-4(3H)-one Derivatives as Active Agents against Toxoplasma gondii.

Yu Deng, Yuan-Di Yu, Chao Song, Guo-Yang Xu, Yue Xu, Chang-Ju Ye.

  A novel series of quinazolin-4(3H)-one derivatives were synthesized using a hybridization strategy that combined the quinazolin-4(3H)-one scaffold, the diarylether fragment, and the piperazine ring. The in vitro activity evaluation of these compounds against Toxoplasma gondii demonstrated that most of this series of compounds showed moderate to good effectiveness, with IC50 values ranging from 5.94 to 102.2 μM. Among the synthesized derivatives, compounds 11 and 18 emerged as the most potent inhibitors, significantly reducing the replication rate of T. gondii with IC50 values of 6.33 and 5.94 μM, as well as demonstrated low cytotoxicity with CC50 values of 285 and 59.2 μM, respectively. The structure-activity relationship investigation indicates that the substituent at the N-3 position of the quinazolin-4(3H)-one is important for anti-T. gondii activity while the replacements at the phenyl moiety of the quinazolin-4(3H)-one and at the diarylether fragment cannot improve activity. The invasion and proliferation assay demonstrated that compound 11 could inhibit both parasite invasion and replication ability. Further investigation of the in vitro efficacy revealed irreversible action of compound 11 against T. gondii. In vivo investigations conducted within a murine model of acute infection revealed that compounds 11 and 18 exhibited a remarkable capacity to significantly diminish the parasitic load in comparison to the control group while also extending the survival duration of the subjects. These results underscore the potential of compound 11 as a candidate for further exploration in the development of antitoxoplasmosis therapies.

Keywords:  Toxoplasma gondii; antiparasitic; diarylether; quinazolinone

DOI:  https://doi.org/10.1021/acs.jafc.4c06788
bioRxiv. 2025 Feb 16. pii: 2025.02.15.638445. [Epub ahead of print]

Activity of Carbazole, Aminoguanidine and Diamine Anti-infectives against Toxoplasma gondii.

Davinder Singh, Melissa A Sleda, Akanksha Pandey, Satish R Malwal, Yiyuan Chen, Ruijie Zhou, Feyisola Adewole, Katie Sadowska, Oluseye K Onajole, Silvia N J Moreno, Eric Oldfield.

We report the observation that carbazole anti-infectives developed as antibacterial and antifungal drug leads have activity against the tachyzoite-stage growth of the Apicomplexan parasite Toxoplasma gondii with IC 50 values as low as 2 μM. We show that a phenylthiazole aminoguanidine with antibacterial as well as antifungal activity inhibits growth with an IC 50 value of 2.1 μM. We also tested a series of 18 analogs of the diamine SQ109, a tuberculosis drug candidate which likewise has both antibacterial and antifungal activity, finding activity as low as 2.3 μM. We tested all compounds for their activity in collapsing the ΔpH component of the promotive force, the results indicating that all compounds acted, at least in part, as protonophore uncouplers. Finally, we also investigated the correlation between the activity of all compounds against the yeast Saccharomyces cerevisiae and the bacterium Mycobacterium smegmatis , finding significant correlations between the collapse of the proton motive force and anti- fungal/antibacterial activity.

DOI: https://doi.org/10.1101/2025.02.15.638445
Methods Mol Biol. 2025 ;2882 139-162

Visualization of Subcellular mTOR Complex 1 Activity with a FRET-Based Sensor (TORCAR).

Ayse Z Sahan, Sohum Metha, Jin Zhang.

  The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing complex that integrates inputs from several pathways to promote cell growth and proliferation. mTORC1 localizes to many cellular compartments, including the nucleus, lysosomes, and plasma membrane. However, little is known about the spatial regulation of mTORC1 and the specific functions of mTORC1 at these locations. To address these questions, we previously developed a Förster resonance energy transfer (FRET)-based mTORC1 activity reporter (TORCAR) to visualize the dynamic changes in mTORC1 activity within live cells. Here, we describe a detailed protocol for using subcellularly targeted TORCAR constructs to investigate subcellular mTORC1 activities via live-cell fluorescence microscopy.

Keywords:  Biosensor; Compartmentalized signaling; Fluorescence; Location-specific

DOI:  https://doi.org/10.1007/978-1-0716-4284-9_7
Sheng Wu Gong Cheng Xue Bao. 2025 Feb 25. 41(2): 491-509

[Functions and mechanisms of Zn2+-dependent histone deacetylase in plant responses to abiotic stress].

Ming Wei, Meng Zhao, Xinrui Wu, Guoqiang Wu.

  The HDAs (a subfamily of histone deacetylases), a class of Zn2+-dependent histone deacetylases, are highly homologous to the reduced potassium dependency 3 (RPD3) in yeast. HDAs extensively regulate chromosome stability, gene transcription, and protein activity by catalyzing the removal of acetyl group from histone and non-histone lysine residues. HDA-mediated deacetylation is essential for plant growth, development, and responses to abiotic stress. We review the research progress in HDAs regarding the discovery, structures, classification, deacetylation process, and roles in regulating plant responses to abiotic stress. Furthermore, this paper prospects the future research on HDAs, aiming to provide theoretical support for the research on epigenetic regulation mediated by HDAs.

Keywords:  Zn2+-dependent histone deacetylases; abiotic stress; deacetylation; epigenetics

DOI:  https://doi.org/10.13345/j.cjb.240570
Biomolecules. 2025 Feb 03. pii: 223. [Epub ahead of print]15(2):

Advances in the Development of Mitochondrial Pyruvate Carrier Inhibitors for Therapeutic Applications.

Henry Politte, Lingaiah Maram, Bahaa Elgendy.

  The mitochondrial pyruvate carrier (MPC) is a transmembrane protein complex critical for cellular energy metabolism, enabling the transport of pyruvate from the cytosol into the mitochondria, where it fuels the citric acid cycle. By regulating this essential entry point of carbon into mitochondrial metabolism, MPC is pivotal for maintaining cellular energy balance and metabolic flexibility. Dysregulation of MPC activity has been implicated in several metabolic disorders, including type 2 diabetes, obesity, and cancer, underscoring its potential as a therapeutic target. This review provides an overview of the MPC complex, examining its structural components, regulatory mechanisms, and biological functions. We explore the current understanding of transcriptional, translational, and post-translational modifications that modulate MPC function and highlight the clinical relevance of MPC dysfunction in metabolic and neurodegenerative diseases. Progress in the development of MPC-targeting therapeutics is discussed, with a focus on challenges in designing selective and potent inhibitors. Emphasis is placed on modern approaches for identifying novel inhibitors, particularly virtual screening and computational strategies. This review establishes a foundation for further research into the medicinal chemistry of MPC inhibitors, promoting advances in structure-based drug design to develop therapeutics for metabolic and neurodegenerative diseases.

Keywords:  MPC (mitochondrial pyruvate carrier); MPC inhibitors; drug design; energy metabolism; medicinal chemistry; metabolic disorders (e.g., diabetes, obesity, and cancer); neurodegenerative disorders; pyruvate transport; therapeutic target; virtual screening

DOI:  https://doi.org/10.3390/biom15020223
Methods Mol Biol. 2025 ;2882 15-45

FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK.

Roland Abi Nahed, Martin Pelosse, Francesco Aulicino, Florine Cottaz, Imre Berger, Uwe Schlattner.

  AMP-activated protein kinase (AMPK) has evolved to detect a critical increase in cellular AMP/ATP and ADP/ATP concentration ratios as a signal for limiting energy supply. Such energy stress then leads to AMPK activation and downstream events that maintain cellular energy homeostasis. AMPK activation by AMP, ADP, or pharmacological activators involves a conformational switch within the AMPK heterotrimeric complex. We have engineered an AMPK-based sensor, AMPfret, which translates the activating conformational switch into a fluorescence signal, based on increased fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. Here we describe how this sensor can be used to analyze direct AMPK activation by small molecules in vitro using a fluorimeter, or to estimate changes in the energy state of cells using standard fluorescence or confocal microscopy.

Keywords:  ADP; AMP; AMP-activated protein kinase; ATP; Cellular energy state; Fluorescence resonance energy transfer; Genetically encoded sensors; Large cargo vectors

DOI:  https://doi.org/10.1007/978-1-0716-4284-9_2
J Proteomics. 2025 Feb 24. pii: S1874-3919(25)00040-5. [Epub ahead of print] 105413

Trypanosoma cruzi cell cycle progression exhibits minimal variation in histone PTMs with unique histone H4 acetylation pattern.

A P J Menezes, A M Silber, M C Elias, J P C Cunha.

  Histones are crucial proteins in eukaryotic cells that undergo extensive posttranslational modifications (PTMs) such as methylation, acetylation, and phosphorylation, which are associated to chromatin structure, gene expression, DNA damage/repair and cell cycle. In Trypanosoma cruzi, the primary sequence of histones differs from that of other eukaryotes. Despite this, they display a vast range of PTMs, though their modulation throughout the cell cycle remains largely unexplored. In this study, we investigated the dynamic modulation of histone PTMs across G1/S, S, and G2/M phases of T. cruzi cell cycle using hydroxyurea- synchronized parasites. We applied a workflow that included histone derivatization, trypsin digestion followed by a high-resolution mass spectrometry and data independent analysis. Quantitative analysis of 141 histone peptide isoforms revealed that there are only minor variations in histone PTM levels throughout the cell cycle. The H3K76 trimethylation remained predominant throughout all phases, with an increase in monomethylation during G2/M. Additionally, hyperacetylation of the N-terminal region of histone H4 was observed, particularly at lysine residues 2, 5, and 10, suggesting their importance in cell cycle progression. Striking, acetylation of histone H4 at K2 and K5 increases during the S-phase, mirroring the H4K5acK12ac pattern observed in mammals, which are related to histone nuclear import and chromatin deposition. Overall, the results suggest that the T. cruzi cell cycle maintains stable global levels of histone PTMs, relying on variations in only a few specific PTMs. Further investigations are warranted to elucidate the functional significance of these PTMs and their impact on cell cycle regulation and chromatin dynamics in T. cruzi. SIGNIFICANCE: Histone posttranslational modifications (PTMs) are key regulators of chromatin architecture and cellular processes such as gene expression and cell cycle control. In Trypanosoma cruzi, the etiological agent of Chagas disease, histones have a distinct primary structure compared to other eukaryotes, yet they display a wide variety of PTMs. This study provides a comprehensive analysis of histone PTM dynamics across the G1/S, S, and G2/M phases of the T. cruzi cell cycle, revealing that global histone PTM levels remain largely stable, with variations in a few specific marks. Notably, the study highlights the increased acetylation of histone H4 at lysines 2 and 5 during the S-phase, contrasting with the well-conserved acetylation at lysines 5 and 12 observed in mammals involved in nuclear import and chromatin assembly. These findings underscore the evolutionary divergence and functional specificity of histone modifications and provide a foundation for further investigations into their roles in parasite biology, with potential implications for understanding chromatin dynamics and identifying novel therapeutic targets.

Keywords:  Cell cycle; Histone PTMs; Trypanosoma cruzi

DOI:  https://doi.org/10.1016/j.jprot.2025.105413
Methods Mol Biol. 2025 ;2882 47-79

Monitoring Cellular Energy Balance in Single Cells Using Fluorescent Biosensors for AMPK.

Nicholaus DeCuzzi, Nont Kosaisawe, Michael Pargett, Markhus Cabel, John G Albeck.

  5'-Adenosine monophosphate-activated protein kinase (AMPK) senses cellular metabolic status and reflects the balance between ATP production and ATP usage. This balance varies from cell to cell and changes over time, creating a need for methods that can capture cellular heterogeneity and temporal dynamics. Fluorescent biosensors for AMPK activity offer a unique approach to measure metabolic status nondestructively in single cells in real time. In this chapter, we provide a brief rationale for using live-cell biosensors to measure AMPK activity, survey the current AMPK biosensors, and discuss considerations for using this approach. We provide methodology for introducing AMPK biosensors into a cell line of choice, setting up experiments for live-cell fluorescent microscopy of AMPK activity, and calibrating the biosensors using immunoblot data.

Keywords:  AMPKAR; Biosensors; Fluorescent protein reporters; Forster resonance energy transfer (FRET); Live-cell microscopy; Metabolic signaling; Single cell

DOI:  https://doi.org/10.1007/978-1-0716-4284-9_3
Adv Sci (Weinh). 2025 Feb 23. e2412109

NAD+ Metabolism Reprogramming Drives SIRT1-Dependent Deacetylation Inducing PD-L1 Nuclear Localization in Cervical Cancer.

Xinyi Lu, Pingping Jin, Qianyun Tang, Min Zhou, Hanjie Xu, Chen Su, Lei Wang, Feng Xu, Min Zhao, Yongxiang Yin, Jinqiu Zhang, Zhen Jia, Xinrui Peng, Jie Zhou, Lu Wang, Yan Chen, Min Wang, Min Yang, Daozhen Chen, Yu Chen.

  Cervical cancer (CC) is a major health threat to women, with immunotherapies targeting the programmed death receptor 1/programmed death ligand 1(PD-1/PD-L1) axis showing promise but encountering resistance in a significant patient population. This resistance has driven a critical quest to uncover the underlying mechanisms. This study uncovers a novel metabolic axis involving the nicotinamide adenine dinucleotide (NAD+) salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) and the deacetylase Sirtuin 1 (SIRT1), which regulates PD-L1 expression and nuclear localization in CC. This axis may be a key factor contributing to the resistance observed in immunotherapy. This study reveals that PD-L1 overexpression in cancers is regulated by both transcriptional and post-transcriptional processes. Acetyl-proteomic analysis pinpoints SIRT1 as a central regulator in the deacetylation of histone H3 at lysines 27, which may influence PD-L1 subcellular distribution. This finding reveals the epigenetic control of immune checkpoint proteins by metabolic pathways, offering a new perspective on the regulation of PD-L1. The identification of the NAMPT/SIRT1 metabolic axis as a critical factor suggests that targeting this axis may enhance therapeutic responses.

Keywords:  NAD+ metabolism; PD‐L1, SIRT1; acetyl‐proteomics; cervical cancer; immunotherapy resistance

DOI:  https://doi.org/10.1002/advs.202412109
Biochem Soc Trans. 2025 Feb 21. pii: BST20240841. [Epub ahead of print]53(1):

How does the tubulin code facilitate directed cell migration?

Miguel Marques Simoes-da-Silva, Marin Barisic.

  Besides being a component of the cytoskeleton that provides structural integrity to the cell, microtubules also serve as tracks for intracellular transport. As the building units of the mitotic spindle, microtubules distribute chromosomes during cell division. By distributing organelles, vesicles, and proteins, they play a pivotal role in diverse cellular processes, including cell migration, during which they reorganize to facilitate cell polarization. Structurally, microtubules are built up of α/β-tubulin dimers, which consist of various tubulin isotypes that undergo multiple post-translational modifications (PTMs). These PTMs allow microtubules to differentiate into functional subsets, influencing the associated processes. This text explores the current understanding of the roles of tubulin PTMs in cell migration, particularly detyrosination and acetylation, and their implications in human diseases.

Keywords:  Cell migration; Microtubules; Tubulin PTMs; Tubulin acetylation; Tubulin code; Tubulin detyrosination

DOI:  https://doi.org/10.1042/BST20240841
Methods Mol Biol. 2025 ;2882 163-178

Determination of Nutrient Ligand-Sensor Binding Affinity.

Xin Gu.

  Cells contain dedicated mechanisms to sense nutrient levels in the environment to regulate their growth by balancing anabolism and catabolism [1, 2]. The mechanistic Target of Rapamycin Complex 1 (mTORC1), a multi-protein kinase complex, serves as an essential growth regulator that integrates various upstream inputs including growth factors and nutrients like amino acids [1, 2] Nutrient sensors upstream of mTORC1 directly bind cognate nutrient ligands to convey their availability and thereby regulate mTORC1 signaling [1, 2]. A reliable method is needed to quantitatively determine the binding affinity (Kd) of the nutrient sensor to its ligand. In parallel, quantitative metabolomic analysis can reveal metabolite levels in fed and starved cells; which represent the physiological range of the nutrient of interest. Whether or not the binding affinity is within the physiological range serves as an indicator to determine the physiological relevance of the sensing mechanism. This chapter describes a generalizable protocol that allows reproducible determination of nutrient ligand-nutrient sensor binding affinity. Here, the S-adenosylmethionine (nutrient ligand)-SAMTOR (nutrient sensor) pair is used as an example [3]. Nutrient sensor purification, radioactive nutrient ligand incubation, and eventual scintillation counting are included, along with a description of the mathematical equation that is used to calculate the binding affinity.

Keywords:  Affinity beads; Competitive binding assays; Nutrient sensors; Nutrients; Protein purification; Radioactive ligands; Scintillation; mTORC1

DOI:  https://doi.org/10.1007/978-1-0716-4284-9_8
ACS Bio Med Chem Au. 2025 Feb 19. 5(1): 184-193

A Genetically Encoded Fluorescent Biosensor for Intracellular Measurement of Malonyl-CoA.

Brodie L Ranzau, Tiffany D Robinson, Jack M Scully, Edmund D Kapelczak, Teagan S Dean, Tara TeSlaa, Danielle L Schmitt.

Malonyl-CoA is the essential building block of fatty acids and regulates cell function through protein malonylation and allosteric regulation of signaling networks. Accordingly, the production and use of malonyl-CoA is finely tuned by the cellular energy status. Most studies of malonyl-CoA dynamics rely on bulk approaches that take only a snapshot of the average metabolic state of a population of cells, missing out on heterogeneous differences in malonyl-CoA and fatty acid biosynthesis that could be occurring among a cell population. To overcome this limitation, we have developed a genetically encoded fluorescent protein-based biosensor for malonyl-CoA that can be used to capture malonyl-CoA dynamics in single cells. This biosensor, termed Malibu (malonyl-CoA intracellular biosensor to understand dynamics), exhibits an excitation-ratiometric change in response to malonyl-CoA binding. We first used Malibu to monitor malonyl-CoA dynamics during inhibition of fatty acid biosynthesis using cerulenin in Escherichia coli, observing an increase in Malibu response in a time- and dose-dependent manner. In HeLa cells, we used Malibu to monitor the impact of fatty acid biosynthesis inhibition on malonyl-CoA dynamics in single cells, finding that two inhibitors of fatty acid biosynthesis, cerulenin and orlistat, which inhibit different steps of fatty acid biosynthesis, increase malonyl-CoA levels. Altogether, we have developed a new genetically encoded biosensor for malonyl-CoA, which can be used to study malonyl-CoA dynamics in single cells, providing an unparalleled view into fatty acid biosynthesis.

DOI: https://doi.org/10.1021/acsbiomedchemau.4c00103
Methods Mol Biol. 2025 ;2882 3-14

Measuring Cellular Adenine Nucleotides by Liquid Chromatography-Coupled Mass Spectrometry.

Ashley J Ovens, Dingyi Yu, Toby A Dite, Bruce E Kemp, Jonathan S Oakhill.

  Adenine nucleotides (AXPs, also referred to as adenosines or adenylates) are a group of organic molecules including adenosine 5'- mono-, di-, and tri-phosphate (AMP, ADP, and ATP, respectively) that, combined, resembles an electrochemical storage cell to facilitate cellular energy storage and transfer. ATP, generated from ADP by photosynthesis, anaerobic respiration, and oxidative phosphorylation, powers many energy-requiring processes in the cell through hydrolysis of its terminal (γ) phosphate, whereas ADP is equilibrated with AMP and ATP by the adenylate kinase reaction. AXPs are major signaling molecules that regulate a wide range of anabolic and catabolic enzymes including AMP-activated protein kinase (AMPK), phosphofructokinase, and pyruvate dehydrogenase.Methods to determine concentrations of AXPs from cells and biological samples have historically relied on high-performance liquid chromatography (HPLC)/capillary electrophoresis techniques to measure [ATP] and [ADP]. However, due to its low basal concentrations, these techniques lack sufficient sensitivity to directly measure [AMP], which must be extrapolated using assumptions of adenylate kinase equilibrium that neglect AMP degradation and synthesis pathways. Here, we describe a detailed protocol to accurately measure [AXP] from cells by liquid chromatography-coupled mass spectrometry (LC/MS), applicable to a wide range of fields including our specific interest in AMPK-dependent metabolic regulation.

Keywords:  AMPK; Adenine nucleotides; Energy; Enzyme regulation; Mass spectrometry; Metabolism

DOI:  https://doi.org/10.1007/978-1-0716-4284-9_1
mSphere. 2025 Feb 25. e0103724

Exploring the differential localization of protein kinase A isoforms in Candida albicans.

Saif Hossain, Zhongle Liu, Nicole Robbins, Leah E Cowen.

  The cAMP-dependent protein kinase A (PKA) plays important roles in a wide range of biological processes in eukaryotic organisms. In the fungal pathogen Candida albicans, PKA is a critical regulator of morphological transitions, which are a key virulence trait. PKA is composed of two catalytic isoforms, Tpk1 and Tpk2, which are often thought to act together in a complex with the regulatory subunit Bcy1. Although Tpk1 and Tpk2 have some redundant functions, they also have distinct cellular functions for which the mechanistic underpinnings remain largely elusive. Here, we constructed functional GFP-tagged fusion proteins for Tpk1, Tpk2, and Bcy1 to explore the localization of PKA isoforms. We observed that the PKA holoenzyme is mainly found in the cytoplasm, as Bcy1 is always excluded from the nucleus. Under glucose-replete conditions, both Tpk1 and Tpk2 translocate into the nucleus from the cytosol. In the presence of glycerol, Tpk1 resides in the cytosol, whereas Tpk2 and Bcy1 become enriched on the vacuolar membrane. As the C-terminal domains of Tpk are highly homologous, we investigated the localization and function of hybrid Tpk proteins with exchanged N-terminal domains. We found the catalytic C-terminus of Tpk1 is required for morphogenesis in solid medium, whereas the C-terminus of Tpk2 is critical for filamentation in liquid. Interestingly, the N-terminus of Tpk2 drives its localization to the vacuolar membrane. Our work highlights environmentally contingent localization patterns for the PKA subunits and suggests that the nuclear localization of Tpk is not sufficient to induce the filamentation program in a leading fungal pathogen of humans.IMPORTANCEFungal pathogens have a devastating impact on human health worldwide. They infect billions of people and kill more than 2.5 million per year. Candida albicans is a leading human fungal pathogen responsible for causing life-threatening systemic disease in immunocompromised individuals. A key virulence trait in C. albicans is the ability to switch between yeast and filamentous forms. The conserved protein kinase A (PKA) regulates diverse functions in the cell, including growth and filamentation. Although PKA has been studied in C. albicans for decades, the subcellular localization of PKA has not been thoroughly investigated. Here, we constructed functional GFP-tagged PKA subunits to explore their localization. We identified differential localization patterns for the PKA subunits that are carbon-source dependent and report that these proteins localize into foci in response to diverse environmental stresses. These findings further our understanding of a critical regulator of growth and virulence in C. albicans.

Keywords:  Candida albicans; filamentation; fungal pathogen; protein kinase A; protein localization

DOI:  https://doi.org/10.1128/msphere.01037-24