bims-toxgon 2026-02-22 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2026–02–22
twenty-two papers selected by
Lakesh Kumar, BITS Pilani

Toxoplasma gondii GRA3 activates interferon-stimulated genes and STAT6 by the cGAS-STING pathway to promote parasite proliferation.
In vitro growth of Toxoplasma gondii tachyzoites on different host cell lines selects for changes in efficiency of invasion and parasite surface antigen gene expression.
Long-term feeder cell-free cat intestinal organoid cultures to study Toxoplasma gondii's sexual development.
The HosA histone deacetylase regulates stress resistance, host cell interactions, and virulence in Aspergillus fumigatus.
Dual function of mitochondrial complex III in Plasmodium falciparum.
Structural insights into the Urm1-Uba4 pathway and its biological roles.
Covalent capture and genetic code expansion enables chemoproteomic profiling and functional characterization of lysine acetoacetylation.
AMPylation: key roles in biological regulation across prokaryotes and eukaryotes.
Restoring NAD⁺-Sirtuins Signaling: A Novel Approach to Combat Male Reproductive Aging.
Nutrient Deprivation Promotes Bladder Cancer Metastasis through Beclin-1 Deacetylation-Mediated Autophagy Activation.
Mass spectrometry reveals the evolutionary conservation of phycobiliprotein complexes.
Advances in LC-MS/MS methods and sample preparation strategies for therapeutic drug monitoring of immunosuppressive drugs.
Structural Characterization of Glycolytic Enzymes from Trypanosoma cruzi.
Quantitative analysis of H-Ras localization using confocal microscopy.
Alkyne lipids as tracers of lipid metabolism.
Metabolic labeling of glycerophospholipids.
Epigenetic regulation of serine biosynthesis by PHF8 during neurogenesis.
Core principles of autophagy initiation mechanisms.
Subcellular localization as a driver of protein function.
Heme Synthesis Inhibition Induces the Intrinsic Apoptosis Pathway in Leukemia with OSGIN1 Upregulation.
cPLA2α targeting to exosomes connects nuclear deformation to LTB4-signaling during neutrophil chemotaxis.
Structural insights into Phospholipase Cε activity and regulation.

PLoS Negl Trop Dis. 2026 Feb 19. 20(2): e0014035

Toxoplasma gondii GRA3 activates interferon-stimulated genes and STAT6 by the cGAS-STING pathway to promote parasite proliferation.

Minmin Wu, Peihao Wang, Ru Wang, Mengting Zhan, Jie Wang, Haijian Cai, Lixia Zha, Nan Zhou, Qingli Luo, Lijian Chen, Jian Du.

Toxoplasma gondii is an opportunistic protozoan parasite that can establish latent infections in humans, causing toxoplasmosis in immunocompromised individuals. Type I interferons (IFN-I), particularly IFN-β, are critical for controlling Toxoplasma gondii infection, but the parasite has evolved various strategies to manipulate the host immune response. Interferon regulatory factor 3 (IRF3) is a key transcription factor that regulates the expression of antiviral genes, including IFN-I and ISGs. Unlike IFN-β, IRF3-activated ISG56 can enhance T. gondii proliferation. Furthermore, STAT6 activation has also been reported to promote the proliferation of Toxoplasma gondii. In this study, we found that GRA3 is highly expressed in the less virulent ME49 strain. Furthermore, we discovered that GRA3 interacted with STING to activate the cGAS/STING pathway. This interaction promotes STING oligomerization and the nuclear translocation of phosphorylated-IRF3, which in turn enhances IFN-β production. GRA3 in ME49 tachyzoites promoted both IRF3-mediated ISG56 expression and STAT6 phosphorylation, thereby enhancing the proliferation of these less virulent parasites. Interestingly, GRA3 enhances parasite proliferation via a mechanism mediated by ISG56 and STAT6, rather than by IFN-β. This study highlights how less virulent strains modulate host immunity to promote T. gondii survival and replication, establish latent infections, and ultimately achieve widespread dissemination in humans.

DOI: https://doi.org/10.1371/journal.pntd.0014035
FEMS Microbes. 2026 ;7 xtag006

In vitro growth of Toxoplasma gondii tachyzoites on different host cell lines selects for changes in efficiency of invasion and parasite surface antigen gene expression.

Adit Naor, John Boothroyd.

  Toxoplasma gondii is remarkable for its intermediate host range, which includes most warm-blooded animals so far tested. Being such a generalist poses challenges for how the parasite can be optimized for growth in cell types that might be radically different in their metabolism and other aspects, including host defenses and microenvironments. To explore how the parasite might adapt to finding itself in a different host, we started with a Type II line of T. gondii (ME49) that had been grown for at least the past 20 years exclusively in vivo. This line was then used to repeatedly infect two cell lines in vitro, human foreskin fibroblasts and Madin-Darby bovine kidney cells. After at least 70 such passages in one or other host cell type, the resulting lines were compared with respect to growth differences, secretion of an important adhesin (MIC2), and transcriptome. The results showed that passage on these two host cell lines results in profound and reproducible differences in parasite phenotype, including attachment/invasion, MIC2 secretion, and gene expression. The transcriptomic differences were especially pronounced for parasite surface antigen genes. The implications of these results for how T. gondii deals with its breadth of possible intermediate hosts are discussed.

Keywords:  adaptation; host range; in-lab evolution; invasion; micronemes; surface antigens

DOI:  https://doi.org/10.1093/femsmc/xtag006
Commun Biol. 2026 Feb 14.

Long-term feeder cell-free cat intestinal organoid cultures to study Toxoplasma gondii's sexual development.

David Warschkau, Tobias Hoffmann, Michael Laue, Antonia Müller, Chandra Ramakrishnan, Giulia Rigamonti, Fabrizia Veronesi, Elvio Lepri, Mohamed Ali Hakimi, Christian Klotz, Frank Seeber.

Toxoplasma gondii is a protozoan parasite able to infect and survive in diverse host environments. However, its sexual reproduction, culminating in infectious oocysts, occurs exclusively in feline intestines. Recent studies identified the transcription factors AP2XII-1 and AP2XI-2 as crucial for pre-sexual development. Their depletion enabled merozoite formation in human fibroblasts, but progression to sexual stages appeared to require additional cues. Host-specific factors governing this process are suspected but remain elusive. Here, we describe a robust continuous feline intestinal organoid culture system without feeder cells to investigate whether the feline cellular and metabolic environment promotes sexual development of in vitro-generated merozoites. Using ultrastructural and transcriptional analyses, we found elevated levels of sexual stage-specific transcripts. While advanced sexual stage formation could not yet be observed, our feline intestinal organoid model provides a controlled and reproducible experimental system to systematically uncover the feline host factors and molecular mechanisms of T. gondii's sexual development.

DOI: https://doi.org/10.1038/s42003-026-09710-y
Microbiol Spectr. 2026 Feb 19. e0403625

The HosA histone deacetylase regulates stress resistance, host cell interactions, and virulence in Aspergillus fumigatus.

Hong Liu, Pamela Lee, Alice Vo, Sanjoy Paul, Quynh T Phan, Jianfeng Lin, Vincent M Bruno, Mark A Stamnes, W Scott Moye-Rowley, Scott G Filler.

  The capacity of Aspergillus fumigatus to cause invasive pulmonary aspergillosis depends on its ability to adapt to dynamic and stressful microenvironments within the host. Epigenetic regulation, including histone deacetylation, plays a critical role in fungal adaptation to stress. Here, we investigated the role of the class I histone deacetylase HosA in A. fumigatus stress resistance, host cell interactions, and virulence. A ΔhosA mutant had increased susceptibility to intracellular oxidant stress induced by menadione. It also had impaired capacity to invade and damage two pulmonary epithelial cell lines in vitro. In a corticosteroid-immunosuppressed mouse model of invasive aspergillosis, mice infected with the ΔhosA mutant survived significantly longer than those infected with the wild-type strain, despite having similar pulmonary fungal burden. The ΔhosA mutant also induced a weaker inflammatory response than the wild-type strain. Transcriptomic analysis revealed that HosA regulates genes involved in secondary metabolite biosynthesis and energy metabolism, functioning as both an activator and repressor of distinct gene sets. Collectively, these results indicate that HosA is a key epigenetic regulator that governs A. fumigatus interactions with host cells and virulence during invasive pulmonary aspergillosis.IMPORTANCEEpigenetic modifications in Aspergillus fumigatus can be induced by environmental changes and stresses such as those induced by interaction with host cells. HosA, a class I histone deacetylase, has been shown to play a key role in regulating secondary metabolism in several Aspergillus species, but its function in A. fumigatus was previously unknown. We found that deletion of hosA increased susceptibility to intracellular, but not extracellular, oxidative stress. The ΔhosA mutant also exhibited significantly reduced pulmonary epithelial cell invasion and host cell damage, as well as attenuated virulence in immunosuppressed mice. Together, these findings indicate that HosA functions as a key epigenetic regulator that governs stress resistance, secondary metabolism, and fungal-host interactions. Defining the functions of HosA could provide critical insight into the epigenetic mechanisms that control fungal pathogenicity and may reveal a potential therapeutic target for invasive aspergillosis.

Keywords:  Aspergillus fumigatus; histone deacetylase; virulence regulation

DOI:  https://doi.org/10.1128/spectrum.04036-25
PLoS One. 2026 ;21(2): e0334727

Dual function of mitochondrial complex III in Plasmodium falciparum.

River S Rell, Anurag Shukla, Joanne M Morrisey, Ijeoma C Okoye, Michael W Mather, Akhil B Vaidya.

Complex III of the malaria parasite mitochondrial electron transport chain (mtETC) has been validated as an attractive target for currently used antimalarials. We previously showed that the main function of mtETC in blood stage Plasmodium falciparum is to regenerate ubiquinone, which serves as an obligatory co-substrate of dihydroorotate dehydrogenase (DHOD), an essential mitochondrial enzyme for pyrimidine biosynthesis. P. falciparum can be rendered resistant to all mtETC inhibitors by provision of a bypass mediated by cytosolic yeast DHOD, a fumarate-reducing enzyme. Malaria parasite mitochondrial DNA (mtDNA) encodes only 3 proteins, each a component of mtETC. However, attempts to eliminate mtDNA in transgenic parasites expressing yDHOD have been unsuccessful, suggesting the possibility that essential function(s) other than the canonical redox reactions of the mtETC also require mtDNA maintenance. Here we have tested the hypothesis that Complex III serves the dual functions of processing imported mitochondrial proteins, as well as ubiquinone regeneration. We have generated transgenic lines that conditionally express mitochondrial processing peptidase a (MPPα), which is also a component of Complex III. Using these parasites, we have determined that MPPα is essential even when the need for mitochondrial electron transport is bypassed. MPPα knockdown also resulted in hypersensitivity of the parasites to proguanil, a drug that synergizes with mtETC inhibitors such as atovaquone. Pulldown with MPPα followed by proteomics revealed the association of multiple mitochondrially targeted proteins, in addition to all components of Complex III. These results are consistent with the suggestion that Complex III in P. falciparum serves both mtETC and protein processing functions in mitochondrial physiology.

DOI: https://doi.org/10.1371/journal.pone.0334727
Essays Biochem. 2025 Dec 22. pii: EBC20253041. [Epub ahead of print]69(4):

Structural insights into the Urm1-Uba4 pathway and its biological roles.

Dominika Kwasna, Keerthiraju E Ravichandran, Anna Biela, Sebastian Glatt.

  Ubiquitin-related modifier 1 (Urm1) is a unique and evolutionarily conserved member of the ubiquitin-like protein (UBL) family that represents a molecular link between ancestral sulfur carrier proteins (SCPs) and canonical eukaryotic UBLs. Urm1 is required for the thiolation of tRNAs and a non-canonical post-translational modification, called 'urmylation'. Activation of Urm1 by its E1-like enzyme, ubiquitin-like protein activator 4 (Uba4), involves the sequential adenylation, thioesterification, and thiocarboxylation of Urm1's C-terminus. Thereby, Urm1 can provide sulfur for the tRNA modification reaction or catalyze its conjugation to target proteins through a mechanism that is independent of E2-conjugating enzymes and E3 ligases. Recent structural studies have resolved several key intermediates of the fungal Uba4-Urm1 system, shedding light onto its two distinct subdomains and their dynamical interplay. Notably, Urm1 also interacts with several additional up- or downstream partners of the two pathways. Foremost, urmylation couples an UBL-conjugation reaction with the persulfidation of a cysteine residue in the target proteins. This protective oxidative post-translational modification underscores Urm1's central role in redox regulation and cellular stress responses. Here, we aim to summarize the most recent mechanistic insights and structural advances in the eukaryotic Urm1-Uba4 pathway.

Keywords:  TRNA; Urm1; persulfidation; thiolation; ubiquitin signaling; ubiquitins

DOI:  https://doi.org/10.1042/EBC20253041
Sci Adv. 2026 Feb 20. 12(8): eaeb5106

Covalent capture and genetic code expansion enables chemoproteomic profiling and functional characterization of lysine acetoacetylation.

Xiaohan Song, Yuhan Lu, Xinlong Guo, Yanan Zheng, He Huang.

Lysine acetoacetylation (Kacac) driven by metabolite acetoacetic acid represents a molecular mechanism by which ketone bodies regulate cellular functions beyond energy provision. However, comprehensive characterization of Kacac has been hindered by technical limitations in detection and functional validation. Here, we report an integrated platform for systematic Kacac investigation. Exploiting the unique reactive ketone carbonyl moiety, we developed Aca-Bio, a hydroxylamine-based probe enabling specific enrichment of Kacac peptides through ketone-targeted covalent labeling and pH-controlled reversible enrichment. Application to mouse liver identified 260 Kacac sites across 125 proteins, revealing notable enrichment in metabolic pathways. Concurrently, we established a genetic code expansion system enabling site-specific Kacac incorporation. Using this approach, we demonstrated that K310acac in HMGCS2 substantially attenuates catalytic activity through impaired substrate binding. This dual-platform approach establishes a comprehensive framework for global profiling and site-specific functional characterization of Kacac, thereby facilitating systematic exploration of its physiological roles and pathological implications.

DOI: https://doi.org/10.1126/sciadv.aeb5106
Front Cell Infect Microbiol. 2026 ;16 1763599

AMPylation: key roles in biological regulation across prokaryotes and eukaryotes.

Xinyi Wang, Junyong Yang, Zhaotai Zang, Yanan Wang, Zihan Shao, Bingqing Li, Haihong Jia.

  AMPylation, as a crucial post-translational modification, is widely present in both prokaryotes and eukaryotes, playing a key role in regulating biological functions. The regulation of biological functions by AMPylation is a complex and diverse process. In prokaryotes, AMPylation plays a role in processes such as self-metabolic regulation, gene expression control, and maintenance of cellular redox homeostasis. Eukaryotes utilize AMPylation to regulate endoplasmic reticulum stress, participate in disease progression, and modulate immune responses. During interactions between prokaryotes and eukaryotes, bacteria can influence host cytoskeletal function, anti-apoptotic processes, and vesicular transport through AMPylation, thereby enhancing their survival within the host. Currently, AMPylation has been applied in numerous directions, such as detecting modifications, constructing disease models, and studying protein functions. This article highlights the diverse roles of AMPylation in regulating biological functions and reviewed the application progress in various fields, aiming to provide theoretical foundations for understanding their mechanisms in pathogen control and eukaryotic disease prevention.

Keywords:  AMPylation; application; biological function; prokaryotes and eukaryotes; regulatory mechanisms

DOI:  https://doi.org/10.3389/fcimb.2026.1763599
World J Mens Health. 2026 Feb 06.

Restoring NAD⁺-Sirtuins Signaling: A Novel Approach to Combat Male Reproductive Aging.

Lu Zhou, Xiucheng Lan, Meijing Wang, Xiaoya Li, Xin Zeng, Sixue Peng, Jingyi Zhang, Junjun Li, Liang Dong, Xujun Yu, Fang Yang.

  Male reproductive aging manifests as progressive declines in testosterone synthesis, spermatogenic efficiency, and fertility, driven by multi-system dysfunction. This review synthesizes evidence establishing the nicotinamide adenine dinucleotide (NAD⁺)-Sirtuins axis as a central regulator of testicular homeostasis and its age-related dysregulation. Mechanistically, NAD⁺ depletion impairs energy metabolism (glycolysis/TCA cycle), antioxidant defenses, and steroidogenesis, while reduced Sirtuins activity (SIRT1/SIRT3/SIRT6) disrupts spermatogonial stem cell maintenance, blood-testis barrier (BTB) integrity, epigenetic programming, and immune privilege. Key pathological features include: (1) Structural compromise (lysosomal dysfunction in Sertoli cells, BTB disruption); (2) Metabolic-oxidative stress (lipid accumulation, mitochondrial failure, reactive oxygen species overproduction); (3) Chronic inflammation (NLRP3 inflammasome activation, T-cell infiltration); (4) Epigenetic dysregulation (aberrant histone acetylation, ncRNA imbalances); and (5) hormonal synthesis disorder. Interventions targeting this axis-NAD⁺ precursors (NMN/NR), Sirtuin activators (resveratrol/quercetin), and lifestyle strategies (intermittent fasting/exercise)-demonstrate efficacy in preclinical models: NMN restores sperm quality (+40% normality) and testosterone synthesis; resveratrol reduces sperm DNA fragmentation by 45% via SIRT3 activation. Clinical data suggest improved physiological parameters (e.g., muscle function, biological age) with NMN supplementation. Critical knowledge gaps persist regarding tissue-specific Sirtuin functions, long-term safety of NAD⁺ boosters, and individualized intervention protocols. Future research should prioritize testicular-targeted delivery systems, NAD⁺-based biomarkers, and randomized trials. Restoring NAD⁺-Sirtuins signaling represents a promising therapeutic strategy against age-related male infertility.

Keywords:  Aging, male; NAD; Sirtuins; Testosterone/biosynthesis; Therapeutics

DOI:  https://doi.org/10.5534/wjmh.250248
Cancer Lett. 2026 Feb 16. pii: S0304-3835(26)00100-X. [Epub ahead of print] 218337

Nutrient Deprivation Promotes Bladder Cancer Metastasis through Beclin-1 Deacetylation-Mediated Autophagy Activation.

Yan Sun, Hang Tong, Guozhi Zhao, Linfeng Wu, Xiaoyu Zhang, Tinghao Li, Junlong Zhu, Kunyao Zhu, Hubin Yin, Xinyuan Li, Weiyang He.

  Deprivation of nutrients in the tumor microenvironment drives malignant progression, yet the molecular mechanisms linking metabolic stress to metastasis in bladder cancer remain incompletely understood. Here, we report that nutrient-deprivation stress promotes metastasis by orchestrating a post-translational modification cascade centered on Beclin-1. Clinical analysis revealed that acetylation of Beclin-1 at lysine residues K430 and K437 was significantly reduced in muscle-invasive bladder cancer (MIBC) compared with non-muscle-invasive bladder cancer (NMIBC), a molecular signature inversely correlated with elevated phospho-eIF2α, a marker of cellular starvation. Mechanistically, nutrient deprivation dynamically regulates the expression of the deacetylase SIRT1 and acetyltransferase p300, shifting the balance toward Beclin-1 deacetylation. This deacetylation event serves a dual function: it enhances Beclin-1 protein stability by shielding it from TRIM21-mediated K48-linked ubiquitination and proteasomal degradation, and it promotes autophagosome formation by strengthening its interaction with pro-autophagic partners VPS34, ATG14, and UVRAG while weakening its binding to the inhibitor Rubicon. Consequently, this leads to sustained autophagy activation and epithelial-mesenchymal transition. Genetic and pharmacological interventions further confirmed the central role of this axis, demonstrating that SIRT1 activation by resveratrol promoted metastasis, whereas p300 activation by CTB suppressed it. Crucially, these effects were abrogated in cells expressing acetylation-mimetic Beclin-1 mutants, suggesting a direct causal link. Our study unveils the SIRT1/p300-Beclin-1-TRIM21 axis as a key nutrient-sensing pathway that promotes bladder cancer metastasis through crosstalk between acetylation and ubiquitination. These findings identify new therapeutic vulnerabilities in advanced bladder cancer.

Keywords:  Bladder cancer; acetylation; autophagy; nutrient deprivation; ubiquitination

DOI:  https://doi.org/10.1016/j.canlet.2026.218337
Nat Commun. 2026 Feb 16.

Mass spectrometry reveals the evolutionary conservation of phycobiliprotein complexes.

Jaspreet K Sound, Giorgio Bianchini, Thrupthi A Ashok, Cecilia Rad-Menéndez, David H Green, Patricia Sánchez-Baracaldo, Aneika C Leney.

Cyanobacteria are a highly taxonomically and ecologically diverse group of oxygenic phototrophs that have colonized many different environments on our planet. Despite their differences, almost all cyanobacteria rely on highly efficient light-harvesting protein complexes, termed phycobilisomes, for effective photosynthesis. Phycobilisomes, along with the phycobiliproteins that make them up, have maintained their function throughout evolutionary history while also diversifying to optimize energy capture and transfer in different conditions. Here, we use a combination of evolutionary proteomics, phylogenomics, and structural bioinformatics to probe how phycobiliproteins have maintained their function while adapting to different habitats. Using high-resolution native mass spectrometry, we show that the two most abundant phycobiliprotein complexes, phycocyanin and allophycocyanin, are highly dynamic. Moreover, upon mixing phycobiliproteins from cyanobacterial strains representing diverse environments and evolutionary lineages, heterologous phycobiliprotein complexes rapidly form, comprising building blocks from different cyanobacterial strains. Bioinformatics and structural prediction methods allow us to identify critical residues involved in these interactions. We thus demonstrate that key structural features within the phycobiliprotein components have remained conserved over three billion years of cyanobacterial evolution, ensuring effective photosynthesis across a wide variety of natural environments.

DOI: https://doi.org/10.1038/s41467-026-69558-y
J Chromatogr A. 2026 Feb 11. pii: S0021-9673(26)00128-7. [Epub ahead of print]1773 466798

Advances in LC-MS/MS methods and sample preparation strategies for therapeutic drug monitoring of immunosuppressive drugs.

Selen Al, Demet Dincel, Aykut Kul, Olcay Sagirli.

  Immunosuppressive drugs play a critical role in preventing organ rejection following transplantation and require close monitoring due to their narrow therapeutic ranges and interindividual pharmacokinetic variability. This review provides an overview of the pharmacokinetic properties, mechanisms of action, and clinical applications of drugs such as cyclosporine A, tacrolimus, sirolimus, everolimus, mycophenolate mofetil, and azathioprine. Within this clinical context, therapeutic drug monitoring (TDM) represents a cornerstone of individualized immunosuppressive therapy, directly linking drug exposure to efficacy and safety outcomes. Analytical approaches commonly used in the therapeutic monitoring of these agents-high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS)-are described, with particular emphasis on LC-MS/MS as the primary analytical platform due to its superior selectivity, sensitivity, and multi-analyte capability. Recent advancements in sample preparation techniques are discussed in relation to their role in minimizing matrix effects and meeting the stringent analytical requirements of TDM. Tabulated data are presented on instrumentation, sample matrices, sample preparation techniques, calibration ranges, lower limits of quantification (LLOQ), analysis times, mobile phase compositions, and chromatographic columns for each analytical method. By critically integrating analytical methodology with clinical TDM needs, this review is intended to provide researchers and clinicians with a useful reference that supports the advancement of more effective and dependable strategies for therapeutic drug monitoring in clinical settings.

Keywords:  Analytical methods; Immunosuppressive drugs; LC-MS/MS; Sample preparation; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.chroma.2026.466798
Mol Biochem Parasitol. 2026 Feb 17. pii: S0166-6851(26)00015-0. [Epub ahead of print] 111736

Structural Characterization of Glycolytic Enzymes from Trypanosoma cruzi.

Kenneth Austin, Vincent A Obachi, Florence L Muzenda, Marakiya T Moetlediwa, Chelsea Agyei, Timothy Craig, Jan Abendroth, Tom Edwards, Mary Nguyen, Ngoc Tran, Bart Staker, Sandhya Subramanian, Peter Myler, Tawanda Zininga, Krishna K Govender, Graham Chakafana.

  Trypanosoma cruzi, the etiological agent of Chagas disease, depends on glycolysis for ATP production, rendering its glycolytic enzymes attractive targets for therapeutic development. Here, we report the high-resolution crystal structures of two essential glycolytic enzymes, glucose-6-phosphate isomerase (Tc PGI, 1.8Å) and enolase (Tc enolase, 2.4Å) and provide structural and computational analyses to support structure-based drug design. Tc PGI adopts a dimeric αβα sandwich fold and features a parasite-specific 53-residue N-terminal extension and a unique C-terminal hook region which both distinguish it from its human ortholog. Tc enolase exhibits the conserved (α/β) 8 TIM barrel fold but harbors minor distinct structural deviations, including an extended α17 helix and a structured α1 region, which differentiate it from human isoforms. Both enzymes exhibited high thermal stability, consistent with adaptation to the parasite's complex life cycle. Structure-based virtual screening using a scaffold with known multi-target potential identified distinct high-affinity inhibitors for each enzyme. Molecular dynamics simulations further confirmed stable enzyme-inhibitor interactions and favorable binding energetics. Collectively, these findings reveal structural signatures unique to T. cruzi glycolytic enzymes and lay the groundwork for the development of antiparasitic therapeutics.

Keywords:  Glycolysis; Structure; Trypanosoma cruzi; X-ray crystallography

DOI:  https://doi.org/10.1016/j.molbiopara.2026.111736
Methods Enzymol. 2026 ;pii: S0076-6879(25)00490-2. [Epub ahead of print]726 269-287

Quantitative analysis of H-Ras localization using confocal microscopy.

Anjana P Sundaresan, Mary E Brown, Mark D Distefano.

  Protein prenylation is an essential post-translational modification, wherein an isoprenoid group is irreversibly attached to the C-terminus of approximately 2 % of the mammalian proteome. This modification facilitates membrane association and functional regulation of diverse signaling proteins, including Ras GTPases. Ras proteins are members of a large superfamily of small GTPases, and therefore disruptions in prenylation impact their localization and importantly contribute to pathologies including cancer and metabolic diseases. This chapter presents a robust, semi-automated workflow for quantifying the subcellular distribution of GFP-H-Ras in adherent epithelial cells, providing a reproducible platform for comparative studies of prenylation and isoprenoid analog effects. The procedure combines high-resolution fluorescence microscopy with image analysis to calculate raw integrated density as a metric of localization. This pipeline is scalable for high-content imaging datasets and adaptable to a variety of prenylated protein systems for mechanistic studies of protein membrane targeting.

Keywords:  Cellular localization; GFP-H-Ras; Imaging; MDCK cells; Post-translational modification; Prenylation; Quantitative imaging

DOI:  https://doi.org/10.1016/bs.mie.2025.11.009
Methods Enzymol. 2026 ;pii: S0076-6879(25)00486-0. [Epub ahead of print]726 85-104

Alkyne lipids as tracers of lipid metabolism.

Christoph Thiele.

  The high complexity of cellular lipidomes and of the underlying metabolic pathways requires powerful labeling and detection systems for systematic lipid tracing experiments. Alkyne fatty acids are tracers with favorable biological properties very similar to unlabeled natural counterparts. We have developed a labeling and detection system based on alkyne lipid tracers and specialized reporter molecules that confer high specificity and sensitivity to labeled metabolites. Tracers are added to living cells and metabolites are extracted in pulse-chase setups to achieve time resolution. Copper(I)-dependent click reaction between extracted lipids and the C171 or C175 reporter molecules is followed by mass spectrometry analysis. The reporter carries a positive charge leading to improved ionization and increased sensitivity. Uniform and predictable neutral loss-type fragmentation in tandem mass spectrometry leads to reliable identification and quantification of labeled metabolites. Parallel multi-labeling with several precursors, combined with multiplexed analysis enables efficient high-content tracing. This chapter introduces the basic concepts and a step-by-step protocol with detailed explanation of key procedures to obtain optimal results.

Keywords:  Click reaction; Lipid metabolism; Mass spectrometry; Reporter molecule

DOI:  https://doi.org/10.1016/bs.mie.2025.11.005
Methods Enzymol. 2026 ;pii: S0076-6879(25)00491-4. [Epub ahead of print]726 289-319

Metabolic labeling of glycerophospholipids.

Robert J Maraski, Christelle F Ancajas, Jinchao Lou, Michael D Best.

  The approach of metabolic labeling provides an invaluable tool for elucidating previously unknown and poorly understood metabolic processes within cells. By introducing clickable versions of substrates into cells, the products of these biomolecule mimics can be conveniently tracked via post-derivatization of the clickable tag with a variety of reporter groups. Here, we will describe lipid metabolic labeling as an invaluable approach for interrogating lipid metabolic pathways, which can yield crucial information regarding complex lipid biosynthesis and trafficking networks that can open new therapeutic targets involving downstream natural products. In this chapter, we present detailed experimental procedures for the development of clickable serine probes for the labeling of phosphatidylserine (PS) and other lipids, including probe design and synthesis as well as analysis of biological incorporation via confocal microscopy, thin-layer chromatography (TLC), and liquid chromatography mass spectrometry (LCMS). This strategy provides a powerful approach for interrogating lipid biosynthetic pathways centered around PS.

Keywords:  Click chemistry; Fluorescence microscopy; Lipids; Membranes; Metabolic labeling; Phosphatidylserine; Phospholipids

DOI:  https://doi.org/10.1016/bs.mie.2025.11.010
EMBO Rep. 2026 Feb 19.

Epigenetic regulation of serine biosynthesis by PHF8 during neurogenesis.

Marta H Artes, Simona Iacobucci, María J Barallobre, Paula Carballeira, Marta Garcia-Cajide, Alejandro Pérez-Venteo, Natalia Padilla, Bárbara S Viegas, Aitana Díaz-Vásquez, A Silvina Nacht, Guillermo P Vicent, Maria L Arbonés, Xavier de la Cruz, Marta Nieto, Neus Agell, Caroline Mauvezin, Marian A Martínez-Balbás.

  Progenitor proliferation during neurodevelopment requires tight coordination of epigenetic regulation and metabolism. However, the crosstalk between these processes remains poorly understood. To investigate this, we examine in neural stem cells the role of PHF8, a histone demethylase whose mutations are linked to Siderius-Hamel syndrome, a rare neurodevelopmental disorder. Through an integrated multi-omics approach - combining transcriptomics, epigenomics, and metabolomics - we identify PHF8 as a key driver of the serine biosynthesis pathway, safeguarding the intracellular serine pool essential for neural progenitor proliferation. PHF8 fine-tunes chromatin accessibility at promoters of metabolic genes, ensuring their activation during development. Loss of PHF8 disrupts amino acid metabolism, blocks autophagy, and hinders vesicle formation. Ultimately PHF8 depletion leads to replication defects, DNA damage, and proliferation arrest. In vivo, PHF8 deficiency in mouse embryos halts neurogenesis, progenitor expansion, and neuron generation in the developing brain. These findings identify PHF8 as a key molecular link between chromatin regulation, metabolic control, and neural development, offering new insights into the epigenetic basis of neurodevelopmental and metabolic disorders.

Keywords:  Gene Transcription; Neural Stem Cells; Neurogenesis; PHF8; Serine Biosynthesis Histone Demethylation

DOI:  https://doi.org/10.1038/s44319-026-00713-8
Nat Struct Mol Biol. 2026 Feb 20.

Core principles of autophagy initiation mechanisms.

Tetsuya Kotani, Hitoshi Nakatogawa.

Autophagy is a conserved intracellular degradation system essential for maintaining cellular homeostasis and adapting to a variety of environmental or metabolic cues. Different types of autophagy are induced in response to various physiological signals through distinct mechanisms. In this Review, we highlight recent advances in understanding the molecular mechanisms that induce autophagic degradation of cytoplasmic material in bulk upon nutrient or energy deprivation, and those that trigger the selective autophagic removal of specific cellular components for their quality or quantity control. We discuss mechanistic principles shared across different types of autophagy, such as phase-separation-mediated assembly and activation of related factors, and the coordination between cargo recognition and membrane biogenesis, delineating how diverse mechanisms converge on core principles to ensure context-specific control of autophagy initiation.

DOI: https://doi.org/10.1038/s41594-026-01752-4
Nat Rev Mol Cell Biol. 2026 Feb 18.

Subcellular localization as a driver of protein function.

Alina Sigaeva, Charlotte Hutchings, Anthony Cesnik, Kathryn S Lilley, Emma Lundberg.

Biological functions depend on the spatiotemporal distribution of proteins within cells. Key cellular activities such as signal transduction, metabolism, cell cycle and cell death are driven by the interactions of proteins that are localized in multiple cellular compartments. Such multilocalization can even allow protein with identical sequences to display multifunctionality, a phenomenon known as moonlighting. Despite its biological importance, the relationship between protein localization and function remains underexplored. In this Review, we discuss the known mechanisms of protein localization (including RNA transport, role of proteoforms and molecular interactions) and how subcellular localization controls protein function. Proper regulation of protein localization is crucial for specialized cell and tissue functions, including cell differentiation, polarization and the epithelial-mesenchymal transition. Protein mislocalization can also have important roles in pathological processes, such as in cancer, neurodegeneration and autoimmunity. We end with a discussion of current technological and conceptual challenges in the field of subcellular proteomics and spatial biology. Addressing these challenges will allow us to link the dynamic nature of protein localization and function across biological scales and contexts, with great impact on fundamental cell biology and clinical applications.

DOI: https://doi.org/10.1038/s41580-026-00947-3
Exp Hematol. 2026 Feb 13. pii: S0301-472X(26)00034-2. [Epub ahead of print] 105401

Heme Synthesis Inhibition Induces the Intrinsic Apoptosis Pathway in Leukemia with OSGIN1 Upregulation.

Yan Yan, Hiroki Kato, Sayaka Sano, Eijiro Furukawa, Satoshi Ichikawa, Koichi Onodera, Yasushi Onishi, Noriko Fukuhara, Hisayuki Yokoyama, Tohru Fujiwara, Hideo Harigae.

  Acute myeloid leukemia (AML) is one of the hematological malignancies with a poor outcome. AML has a unique metabolic status, and identifying its metabolic vulnerabilities is warranted. Recent genome-wide screenings suggest that heme synthesis might be such a vulnerability. Heme is required not only for hemoglobin synthesis but also for the proper function of hemoproteins. Cytochromes are such hemoproteins and are necessary for mitochondrial respiration. Therefore, heme synthesis inhibition can diminish AML by altering mitochondrial status and function. However, still little is known about the importance of heme synthesis in leukemia cells. To reveal the roles of heme synthesis in leukemia, we treated human leukemia cell lines with heme synthesis inhibitors, succinylacetone (SA) or N-methyl Protoporphyrin IX (NMPP). Heme synthesis inhibition induced cell growth inhibition and cell death in a concentration-dependent manner. Therefore, heme synthesis is required for leukemia cell proliferation and survival. Increased pro-apoptotic factors (cleaved caspase 3 and cleaved PARP) and decreased anti-apoptotic factor (XIAP) were observed following heme synthesis inhibition. Cytochrome c and Smac were released into the cytoplasm by heme synthesis inhibition, suggesting that heme synthesis inhibition led to mitochondrial outer membrane permeabilization and activation of the intrinsic pathway of apoptosis. Comprehensive transcriptomic analysis revealed that heme synthesis inhibition induced OSGIN1 expression, leading to the release of cytochrome c and Smac from mitochondria into the cytoplasm. Therefore, heme synthesis inhibition induced leukemia apoptosis by activating the intrinsic apoptosis pathway.

Keywords:  OSGIN1; heme; intrinsic apoptosis pathway; leukemia

DOI:  https://doi.org/10.1016/j.exphem.2026.105401
Sci Adv. 2026 Feb 20. 12(8): eaea2784

cPLA2α targeting to exosomes connects nuclear deformation to LTB4-signaling during neutrophil chemotaxis.

Subhash B Arya, Fatima Jordan-Javed, Kristen Loesel, Yehyun Choi, Samuel P Collie, Lauren E Hein, Brendon M Baker, Euisik Yoon, Carole A Parent.

Efficient neutrophil chemotaxis requires the integration of mechanical forces and lipid-mediated signaling. While the signaling lipid leukotriene B4 (LTB4) reinforces cellular polarity, how mechanical cues regulate its production remains unclear. We now show that cytosolic phospholipase A2α (cPLA2α), which is essential for the synthesis of LTB4, functions as a nuclear curvosensor. cPLA2α responds to nuclear squeezing by localizing to ceramide-rich inner nuclear membrane microdomains and incorporating onto the exofacial surface of nuclear envelope-derived exosomes. This unique topology enables localized LTB4 synthesis, which synchronizes calcium spikes, promotes myosin light chain II phosphorylation, and sustains polarity and directional persistence after constriction. In neutrophils passing through tight spaces, cPLA2α activity drives the chemotactic response to nuclear squeezing by promoting exosomal LTB4 production and persistence after constriction. These findings uncover a cPLA2α-dependent mechanochemical axis linking nuclear architecture to chemotactic efficiency and offer alternative strategies to modulate inflammatory responses.

DOI: https://doi.org/10.1126/sciadv.aea2784
J Biol Chem. 2026 Feb 16. pii: S0021-9258(26)00159-6. [Epub ahead of print] 111289

Structural insights into Phospholipase Cε activity and regulation.

Ketaki A Mahurkar, Stephanie L Barrios, Faith D McCauley, Angeline M Lyon.

  Phospholipase Cε (PLCε) is a complex, multifunctional enzyme that responds to and integrates signals from G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through the direct binding of G proteins. These activators translocate PLCε to the cytoplasmic leaflets of the plasma and perinuclear membranes where the lipase hydrolyzes phosphatidylinositol (PI) lipids to produce inositol polyphosphates (IPs) and diacylglycerol (DAG). These second messengers increase intracellular Ca2+ and/or activate protein kinase C, respectively, stimulating numerous pathways. Recent studies have broadened our understanding of this enzyme, revealing roles for PLCε in Ca2+-induced Ca2+-release (CICR) processes in the kidneys and pancreas, as well as cancer. These are complemented by structural studies that provide more complete insights into its basal and G protein-bound conformations. Here, we summarize and discuss these advances in understanding the regulation and roles of PLCε in normal and pathological contexts.

Keywords:  G protein; PIP(2); heterotrimeric G protein; phosphatidylinositol; phosphatidylinositol signaling; phospholipase C; protein structure; second messenger; small GTPase

DOI:  https://doi.org/10.1016/j.jbc.2026.111289