bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2025–09–07
43 papers selected by
Lakesh Kumar, BITS Pilani



  1. Int J Parasitol. 2025 Aug 28. pii: S0020-7519(25)00161-4. [Epub ahead of print]
      The protozoan parasite Toxoplasma gondii relies on antioxidant proteins and systems to protect against the host's immune responses and to neutralize free radicals produced by its own metabolism. In this study, we identified and characterized a new thioredoxin protein, TgTrx1, which is mainly found in the cytoplasm of T. gondii tachyzoites and contains a conserved -CXXC- catalytic motif. Using CRISPR-Cas9 gene editing, we disrupted the TgTrx1 gene to generate a knockout strain (RHΔtrx1) and studied the effect of gene loss on various aspects of the infection process. RHΔtrx1 parasites showed a marked reduction in their ability to invade host cells, secrete micronemes, replicate intracellularly, egress from host cells, and tolerate oxidative stress. They also displayed abnormal mitochondrial morphology and asynchronous cell division. Transcriptomic analysis revealed significant changes in the expression of genes involved in oxidative stress response and bradyzoite differentiation. Mice injected intraperitoneally with 106 RHΔtrx1 tachyzoites showed no clinical symptoms. However, the immunity induced by these attenuated tachyzoites conferred only partial protection against subsequent acute and chronic T. gondii infections. This limited protective effect is likely related to the parasite's impaired replication, which may lead to rapid clearance by the host immune system and insufficient antigenic stimulation to elicit a fully protective immune response. These findings establish TgTrx1 as a multifunctional redox protein important for T. gondii survival, redox balance, synchronous cell division, and virulence.
    Keywords:  Oxidative stress; Parasite fitness; Thioredoxin; Toxoplasma gondii; Virulence
    DOI:  https://doi.org/10.1016/j.ijpara.2025.08.015
  2. mSphere. 2025 Aug 29. e0002625
      Toxoplasma gondii is a protozoan parasite that causes persistent infection in warm-blooded vertebrates by undergoing differentiation from a replicative stage (tachyzoites) to a latent encysted stage (bradyzoites). Stage differentiation is critical for transmission and pathogenesis and relies on gene regulation driven by a network of transcription and epigenetic factors. We previously found in non-cystogenic type I RH strain parasites that the lysine acetyltransferase (KAT), GCN5a, is dispensable in tachyzoites but required to upregulate stress-response genes, suggesting a link with bradyzoite conversion. To address this possibility, we generated endogenously tagged GCN5a parasites and a genetic knockout in cystogenic type II Pru strain. We show that GCN5a protein, but not mRNA, increases during differentiation and complexes with unique protein partners, most of which contain AP2 domains. Pru strain tachyzoites lacking GCN5a augment bradyzoite-specific gene expression in the absence of stress. Loss of GCN5a slowed tachyzoite replication and heightened sensitivity to bradyzoite conversion but resulted in smaller cyst sizes compared to wild type. Using CUT&Tag, we delineated the chromosomal occupancy of GCN5a relative to the essential KAT, GCN5b. While GCN5b localizes to coding regions, GCN5a surprisingly localizes exclusively to telomeres. These findings suggest that the loss of GCN5a leads to telomere dysfunction, which slows replication and promotes the transition to latency.IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in warm-blooded hosts, including humans, because it converts into latent tissue cysts. Switching from its replicating form into dormant cysts is a tightly regulated process that involves epigenetic factors such as lysine acetyltransferases GCN5a and GCN5b. This study is the first to examine the role of GCN5a in a cyst-forming Toxoplasma strain. We found that GCN5a protein, but not mRNA, increases during cyst development. Additionally, parasites lacking GCN5a replicate more slowly and are quicker to form cysts when stressed. We show that GCN5a and GCN5b work in different multi-protein complexes and localize to different areas of the genome; while GCN5b targets promoters of gene coding regions, GCN5a is exclusively found at telomeric regions. Our findings suggest a novel role for GCN5a in telomere biology that, when depleted, produces a fitness defect that favors development of latent stages.
    Keywords:  Toxoplasma; apicomplexa; chromatin; epigenetics; gene expression; parasite; protozoa
    DOI:  https://doi.org/10.1128/msphere.00026-25
  3. Curr Opin Microbiol. 2025 Aug 30. pii: S1369-5274(25)00084-0. [Epub ahead of print]88 102662
      Toxoplasma gondii, a widespread apicomplexan parasite, navigates a complex life cycle in which it switches back and forth between tachyzoites and bradyzoites to balance acute infection and persistence while also undergoing an often-overlooked one-way commitment to the sexual stage. Emerging evidence suggests that Toxoplasma's developmental trajectories are orchestrated by an interconnected network of transcriptional switches and chromatin remodeling mechanisms, acting in concert to ensure robust stage transitions. This review outlines an updated epigenetic framework for stage transitions and highlights the functional synergy between an Imitation SWItch family chromatin remodeler and the MORC/HDAC3 repressor complex, which together modulate chromatin accessibility to maintain or shift developmental states. We also spotlight recent findings, showing that simultaneous depletion of AP2XII-1 and AP2XI-2 transcription factors reactivates the presexual program in vitro, eliminating the need for cat hosts and allowing ethical, scalable exploration of sexual stages. Strikingly, the knockdown of these transcription factors also reveals a previously unrecognized hybrid zoite co-expressing bradyzoite and merozoite markers, suggesting a transitional state that defies classical stage definition. This hybrid form, as well as more recent data on bradyzoite development, support a new model in which T. gondii development unfolds along a continuum of epigenetically primed states rather than a rigid sequence of binary fate decisions. These findings provide a powerful conceptual and experimental framework for dissecting parasite transmission and persistence and have far-reaching implications for the biology of other parasites in the phylum.
    DOI:  https://doi.org/10.1016/j.mib.2025.102662
  4. bioRxiv. 2025 Aug 27. pii: 2025.08.27.672535. [Epub ahead of print]
       Background: A hallmark of the eukaryotic cell is the regulated transport between the nucleus and cytoplasm, which is mediated by a multi-subunit protein assembly called the nuclear pore complex (NPC). While its overall architecture has been preserved across eukaryotes, variations in NPC structure appear to have tuned its function in different organisms. Outside of a handful of model systems, the NPC has not been comprehensively studied. This is particularly true of species that are not closely related to well-studied models, such as apicomplexan parasites. Indeed, the evolutionary divergence of Apicomplexa has complicated facile prediction of these proteins in these organisms. Because of this, the NPC components remain largely unidentified, and therefore NPC cellular function in Apicomplexa is poorly understood.
    Principal Findings: Here we identified, experimentally validated, and functionally characterized protein components of the NPC in the apicomplexan parasite Toxoplasma gondii . By combining proximity biotinylation with careful bioinformatic analysis we identified 15 previously uncharacterized proteins that localize to the Toxoplasma NPC. We demonstrated 7 of these proteins are essential to parasite replication. Importantly, we defined components of the mRNA export machinery, as well as subunits required for the stability and/or assembly of specific NPC subcomplexes. Consistent with the evolutionary distance between Toxoplasma and well-studied models, the majority of our newly validated NPC components show no clear homology to NPC proteins in yeast, animals, or plants. Moreover, we demonstrated that the Toxoplasma mRNA export machinery has a distinct composition from other well-established systems. Intriguingly, several well-defined domains the TREX-2 complex that are essential in other systems are missing from the Toxoplasma genome. In contrast, others, such as Centrin-3, have been conserved in Toxoplasma , but are not required for mRNA export in the parasite.
    Conclusion: Our work highlights the distinct composition of multiple subcomplexes of the Toxoplasma NPC and paves the way for future studies to provide high-resolution structural information on the parasite's unusual NPC architecture.
    DOI:  https://doi.org/10.1101/2025.08.27.672535
  5. bioRxiv. 2025 Aug 29. pii: 2025.08.26.672290. [Epub ahead of print]
      Chromera velia is a photosynthetic, free-living alga that is closely related to the apicomplexans, a phylum of intracellular parasites responsible for many devastating diseases, including malaria, cryptosporidiosis, and toxoplasmosis. With molecular and cellular landmarks that are clearly related to but distinguishable from those found in apicomplexan parasites, Chromera provides a fantastic opportunity to investigate the evolutionary origin of the structures and processes needed for intracellular parasitism. However, tools for defining localization and functions of gene products do not exist for Chromera , which creates a major bottleneck for exploring its biology. Here we report two major advances in exploring the cell biology of this free-living relative of a large group of intracellular parasites: 1) successful cell transformation and 2) the implementation of expansion microscopy. The initial analysis enabled by these tools generated new insights into subcellular organization in different life stages of Chromera. These new developments boost the potential of Chromera as a model system for understanding the evolution of parasitism in apicomplexans.
    DOI:  https://doi.org/10.1101/2025.08.26.672290
  6. J Pediatric Infect Dis Soc. 2025 Aug 27. pii: piaf060. [Epub ahead of print]14(8):
      Toxoplasma gondii is an apicomplexan parasite with an enormous global reach, infecting over a billion people worldwide. An opportunist in humans, T. gondii causes severe disease only in a select few scenarios but is otherwise relatively benign. Through mechanisms that are unclear T. gondii has a propensity to persist in neuronal tissues including the brain and retina, and it is in these sites that it can cause the most severe disease. Disease occurs in those with suppressed immune function, including HIV/AIDS and organ transplant patients. However, infection can also lead to recurrent ocular disease in otherwise healthy individuals, causing temporary vision loss and in the most severe cases, blindness. The propensity for this organism to reside and cause disease in tissues of the central nervous system is of great interest, and here we explore what is known about the neurovirulent outcomes of T. gondii infection.
    Keywords:  Toxoplasma gondii; host–parasite interactions; neurovirulence; ocular toxoplasmosis; toxoplasmic encephalitis
    DOI:  https://doi.org/10.1093/jpids/piaf060
  7. Parasites Hosts Dis. 2025 Aug;63(3): 201-214
      Toxoplasma gondii is a neurotropic apicomplexan protozoan estimated to affect approximately 30% of the global population. In this review, we aimed to examine scientific evidence on the potential role of T. gondii infection in the development of autism spectrum disorder (ASD), a heterogeneous neurodevelopmental disorder. This review summarizes the current literature exploring the possible association between T. gondii and ASD. Findings indicate that toxoplasmosis may contribute to host alterations, including the induction of humoral and cellular immune responses, production of various cytokines, and changes in neurotransmitter levels (e.g., serotonin, dopamine, acetylcholine, gamma-aminobutyric acid, and glutamate), as well as the activation of enzymes such as indoleamine 2,3-dioxygenase, which may influence the pathophysiology of ASD. In conclusion, this review suggests that T. gondii infection could act as a potential risk factor for ASD. However, further intensive studies are necessary to clarify the role of this parasite in the etiology and progression of ASD. This review is anticipated to stimulate further studies aimed at understanding and potentially reducing the burden of neurodevelopmental disorders worldwide.
    Keywords:  Toxoplasma gondii; autism; autism spectrum disorder; immune response; neurotransmitter; toxoplasmosis
    DOI:  https://doi.org/10.3347/PHD.24066
  8. PLoS Pathog. 2025 Sep 03. 21(9): e1013475
      Toxoplasma gondii is a significant pathogen in both humans and animals, with disease progression driven by the rapid proliferation of its tachyzoite stage. In this study, we identify the PP2A-2 holoenzyme as a key regulator of daughter cell emergence during parasite division. This holoenzyme, likely composed of the regulatory subunit TgPR48 (PP2A-B2), the catalytic subunit PP2A-C2, and the scaffolding subunit PP2A-A2, is essential for proper cytokinesis. Disruption of any single component severely impairs daughter cell separation and emergence. Phosphoproteomic analysis following PP2A-C2 depletion revealed numerous differentially phosphorylated proteins. Among these, DCS1 and DCS2 were prioritized as potential effectors. While phosphomimetic and non-phosphorylatable mutations in DCS1 and DCS2 did not significantly impair their function, depletion of either protein disrupted TgPR48 localization. Interestingly, TgPR48 overexpression partially rescued the phenotypes associated with DCS2 loss, but not DCS1, indicating divergence in their downstream pathways and implicating additional, yet unidentified, substrates. These findings establish PP2A-2-mediated dephosphorylation as a central mechanism in regulating tachyzoite cytokinesis and highlight a promising regulatory axis for therapeutic intervention against T. gondii.
    DOI:  https://doi.org/10.1371/journal.ppat.1013475
  9. ACS Infect Dis. 2025 Aug 28.
      We investigated the antiparasitic activity of several antimicrobial drug leads against Toxoplasma gondii tachyzoites and, in one case, bradyzoites. Carbazole and phenylthiazole aminoguanidine anti-infectives, originally developed as antibacterial and antifungal agents, showed potent activity, with IC50 values as low as 2 μM. This potency was comparable to that observed with the tuberculosis drug candidate SQ109 and a series of its analogs. Notably, SQ109 also significantly reduced the viability of in vivo-derived bradyzoites. All compounds acted, at least in part, as protonophore uncouplers by collapsing the ΔpH component of the proton motive force. Furthermore, SQ109 and the tetrahydrocarbazole (THCz) compounds disrupted the mitochondrial membrane potential in T. gondii tachyzoites. While SQ109 is known to activate macrophages to an M1 phenotype, we observed no significant difference in its activity against T. gondii grown in fibroblasts versus macrophages, likely due to the parasite's residence within the protective parasitophorous vacuole. We also examined correlations between compound activity against the yeast Saccharomyces cerevisiae, and the bacterium Mycobacterium smegmatis, finding significant correlations between the collapse of the proton motive force and antiproliferative activity. Taken together, our findings underscore the potential of these antimicrobial agents as promising leads for the development of new antiparasitic therapies against T. gondii.
    Keywords:  SQ109; antiparasitic; carbazole; protonophore; toxoplasmosis
    DOI:  https://doi.org/10.1021/acsinfecdis.5c00609
  10. Acta Trop. 2025 Aug 29. pii: S0001-706X(25)00282-7. [Epub ahead of print] 107812
      Toxoplasma gondii (T. gondii) infection is a globally prevalent and potentially severe disease, particularly in infants and immunocompromised individuals. Despite its widespread impact, no licensed vaccine is currently available for human use. The T. gondii antigens MIC8, AMA1, and RON4 have been identified as candidates capable of inducing immunity against toxoplasmosis. In this study, we generated recombinant vaccinia viruses (rVV) expressing MIC8 (MIC8-rVV), AMA1 (AMA1-rVV), or RON4 (RON4-rVV), and evaluated the vaccine efficacy of each rVV in mice. Mice were intranasally immunized twice with rVV and subsequently challenged with T. gondii ME49 strain. All three rVV vaccines induced T. gondii-specific immunoglobulin (Ig)G and IgA responses, as well as significant activation of CD8⁺ T cells and total B cells. All immunized mice 100% survived a lethal challenge, whereas the naïve control group did not. Among the three vaccines, MIC8-rVV elicited the strongest CD8⁺ T cell and B cell responses, resulting in the highest reduction in brain cyst counts following T. gondii ME49 strain challenge. These findings demonstrate that rVV-based delivery of MIC8, AMA1, and RON4 antigens can confer protective immunity against T. gondii ME49 strain infection, with MIC8 emerging as a particularly promising vaccine candidate.
    Keywords:  AMA1; MIC8; RON4; T. gondii ME49 strain; recombinant vaccinia virus
    DOI:  https://doi.org/10.1016/j.actatropica.2025.107812
  11. EXCLI J. 2025 ;24 749-773
      This immunoinformatics-based study utilized a suite of online predictive tools to characterize the structural and immunogenic properties of Toxoplasma gondii rhoptry neck proteins (TgRONs). Full-length amino acid sequences of TgRON2, TgRON4, TgRON4L1, TgRON5, TgRON8, TgRON9, TgRON10, and TgRON13 were retrieved from ToxoDB and subjected to comprehensive analysis. Except for TgRON4L1, all proteins were predicted to be possess antigenic potential, with none identified as allergenic. Solubility predictions indicated that TgRON9 and TgRON10 are the most likely to be expressed as soluble antigens. Aliphatic index values, ranging from 51.17 to 84.63, suggest acceptable thermostability, while negative GRAVY scores across all proteins indicate favorable hydrophilicity. Additionally, multiple post-translational modification sites were identified, underscoring the functional complexity of these antigens. Initial 3D structure modeling showed that 60.21-92.41 % of residues fell within favored regions on Ramachandran plots, with refinement increasing this to 92.27-98.58 %, reflecting substantial improvements in structural quality. Several potential T-cell (CTL and HTL) and B-cell epitopes were predicted for all candidate proteins. Immune simulation models further suggested that these antigens could elicit robust humoral and cellular immune responses when delivered in a three-dose regimen at four-week intervals. These findings offer valuable preliminary insights and support the further investigation of TgRONs, particularly TgRON9 and TgRON10, as promising targets for experimental validation in the development of vaccines against T. gondii infection. See also the graphical abstract(Fig. 1).
    Keywords:  Toxoplasma gondii; immunoinformatics; in silico analysis; rhoptry neck protein; vaccine
    DOI:  https://doi.org/10.17179/excli2025-8304
  12. Vet Med Sci. 2025 Sep;11(5): e70595
      Toxoplasma gondii is the causative agent of toxoplasmosis, an obligate intracellular parasite of warm-blooded animals; the definitive host is cats and felines. Transmission of this parasite in herbivorous intermediate hosts occurs through contaminated water and forage by the oocyst stage of the parasite, and in cats and humans, it occurs through eating contaminated meat and milk by the cystic stage, tachyzoite and oocyst of the parasite. Some people consume the milk of various animals, including cows, sheep, goats, camels and donkeys. Such kinds of milk are supposed to be a good source of protein and vital minerals. Nonetheless, they are frequently ingested uncooked or not heated with high temperatures to destroy dangerous microbes. Hence, the potential viability of T. gondii tachyzoites in the milk of these animals under various temperatures needs to be investigated, as this could be a significant risk indicator of human infections. For this purpose, purchase 1000.0 mL of cow's milk from milk supply centres and then divide it into different parts, and after adding T. gondii tachyzoites of the RH strain (107 × 5) to each milk sample, they are subjected to rapid pasteurization temperatures (75°C for 15 s), slow pasteurizations (60°C for 15 min), temperatures of 25°C and 35°C for 3, 6 and 12 h and refrigerator temperatures (4°C for 6, 12 and 24 h), and after centrifugation, the resulting precipitate was injected three times into three mice. One mouse without parasite tachyzoite injection was considered a negative control, and three mice injected with parasite tachyzoites were considered as positive controls. Our results showed that in the first trial, T. gondii tachyzoites treated at 75°C, 60°C, 35°C, 25°C and 4°C survived after 15 s and caused the death of mice. However, in the second trial, at 75°C, the parasite tachyzoites were completely eliminated after both 15 min and 3 h, and all treated mice survived. In contrast, in the third trial, at 4°C, 25°C and 35°C, after 15 min, 3, 6, 12 and 24 h, the parasite survived and caused the death of mice. Statistical analysis showed that the mortality of mice treated at 75°C and 60°C during the treatment periods was significant. This study showed that milk possibly contaminated with parasitic tachyzoites can survive in rapid and slow pasteurization and refrigerator temperatures, suggesting heating milk at 60°C for more than 15 min is recommended.
    Keywords:  East Azerbaijan; Toxoplasma gondii; cow; heat; milk
    DOI:  https://doi.org/10.1002/vms3.70595
  13. mBio. 2025 Aug 28. e0215925
      Toxoplasma gondii, a widespread human parasite, persists in hosts through complex molecular interactions. Protein-protein interactions (PPIs) underpin essential biological processes, including parasite-host interactions and cellular invasion. Herein, we utilized advanced crosslinking mass spectrometry (XL-MS) techniques to map a T. gondii tachyzoite cytosolic extract interactome. By integrating MS-cleavable and non-cleavable analysis, we identified a total of 196 unique PPIs at medium confidence (false discovery rate [FDR] < 5%) and 171 at high confidence (FDR < 1%), revealing both known and novel interactions within critical cellular complexes such as the ribosome, proteasome, and dense granule proteins. Structural validation confirmed spatial proximity of crosslinked residues, while comparative analyzes against existing data sets (hyperLOPIT, ToxoNET, and STRING) corroborated the biological relevance of identified interactions. Furthermore, we introduced a machine learning approach leveraging biological annotations and experimental data to significantly enhance the detection and validation of PPIs. Our findings not only provide a refined view of T. gondii's molecular architecture but also highlight the utility of XL-MS coupled with computational tools in dissecting complex parasite proteomes. The XL-MS interactome map provides a new valuable resource for understanding parasite biology and developing targeted therapeutic strategies.IMPORTANCEOur work presents a novel application of crosslinking mass spectrometry (XL-MS) integrated with machine learning to systematically characterize the cytosolic protein-protein interactions in Toxoplasma gondii-a pathogen of significant clinical and epidemiological interest. This study addresses an important gap in microbial proteomics by leveraging advanced XL-MS techniques to capture transient and novel interactions, which are often challenging to detect with conventional methods. By combining both MS-cleavable and non-cleavable strategies with a robust machine learning approach, we were able to significantly enhance the identification of genuine protein interactions. The methodology described not only improves the depth and accuracy of interactome analysis but also offers a framework that can be applied to other complex microbial systems. We believe that the insights gained from our study will be of great interest to the microbiology community, particularly researchers focusing on host-pathogen interactions and the molecular mechanisms underlying parasitic infections.
    Keywords:  GRA; Toxoplasma gondii; XL-MS; interactome; machine learning; mass spectrometry; protein crosslinking agents; protein-protein interactions; proteomics; ribosome
    DOI:  https://doi.org/10.1128/mbio.02159-25
  14. Annu Rev Microbiol. 2025 Aug 28.
      Apicomplexan and trypanosomatid parasites cause important human diseases, including malaria, toxoplasmosis, Chagas disease, and human leishmaniasis. The mammalian-infective stages of these parasites colonize nutrient-rich, intracellular niches in a range of different host cells. These niches include specialized vacuoles (Plasmodium spp., Toxoplasma gondii), the mature lysosome of phagocytic cells (Leishmania), and the cytoplasm of nucleated host cells (Trypanosoma cruzi). Here, we review the different growth and metabolic strategies utilized by each of these protists to survive in these niches. Although all stages utilize sugars as preferred carbon sources, different species or developmental stages vary markedly in their dependence on aerobic fermentation versus respiratory metabolism and their co-utilization of other carbon sources. Stage-specific differences in glycolytic and mitochondrial respiratory capacity may be a hardwired feature of each stage and reflect the trade-off of achieving high growth rates at the expense of host range adaptability and establishing long-lived persistent infections.
    DOI:  https://doi.org/10.1146/annurev-micro-032421-120925
  15. Glycobiology. 2025 Sep 01. pii: cwaf051. [Epub ahead of print]
      Toxoplasma gondii is a highly successful intracellular mammalian and avian pathogen that must adapt to a wide range of intracellular and extracellular environments. A mechanism that may support this is the modification of hydroxyamino acid rich sequences of nucleocytoplasmic proteins with O-fucose. O-fucosylation of possibly hundreds of proteins is mediated by a single highly conserved nucleocytoplasmic enzyme. Deletion of the SPY O-fucosyltransferase gene is tolerated but inhibits parasite proliferation in fibroblasts and their accumulation in mouse brains. A prior ectopic expression study suggested that O-fucose is required to detect proteins considered essential. To distinguish whether the SPY requirement was specific to the method or for protein expression per se, GPN1, an RNA polymerase chaperone, was epitope-tagged at its endogenous locus in both normal and SPYΔ strains. GPN1 was shown to be substantially and quantitatively O-fucosylated and exhibited a modest 24% reduction in level in SPYΔ cells. Proteomic analysis of its interactome indicated that fucosylation did not affect its association with RNA polymerase subunits. GPN1 was mostly cytoplasmic based on super-resolution immunofluorescence microscopy, and this localization was not affected by O-Fuc. A fusion of its O-fucosylated serine-rich domain to yellow fluorescent protein behaved similarly. In comparison, the abundance of a Zn-finger containing protein also depended on SPY, whereas the abundance and localization of ERK7 were not affected nor were levels of two other proteins. Thus O-fucose directly but modestly promotes the accumulation of select targets, but it does not enforce their localization in nuclear assemblies that are highlighted by immunofluorescence studies.
    Keywords:  GPN-loop GTPase; O-fucose; nucleocytoplasmic glycosylation; spindly; toxoplasma gondii
    DOI:  https://doi.org/10.1093/glycob/cwaf051
  16. Vaccines (Basel). 2025 Aug 15. pii: 862. [Epub ahead of print]13(8):
      Background:Toxoplasma gondii (T. gondii) infection causes serious diseases in immunocompromised patients and causes congenital toxoplasmosis in infants. T. gondii microneme protein 8 (MIC8) and apical membrane antigen 1 (AMA1) are essential proteins involved in parasitic invasion. Methods: In this study, we generated virus-like particles (VLPs) and recombinant vaccinia virus (rVV) containing MIC8 or AMA1 proteins. Vaccine efficacy was evaluated in mice (BALB/c) upon challenge infection with T. gondii ME49. Results: Intramuscular immunization with heterologous vaccines (rVV + VLPs; rVV for prime and VLPs for boost) elicited T. gondii-specific IgG antibody responses in mice. Four weeks after the boost, all mice were orally challenged with T. gondii ME49, and protective immunity was assessed. The responses of antibody-secreting cells for IgG2a and IgG2b and those of memory B cells and CD4+ and CD8+ T cells were higher in the rVV + VLP group than in the VLP + VLP group. The rVV + VLP group exhibited a significant reduction in cyst count in the brain. Conclusions: These findings indicate that heterologous vaccination with vaccinia viruses and VLPs improves vaccine efficacy.
    Keywords:  AMA1; MIC8; T. gondii; VLPs; rVV; vaccine
    DOI:  https://doi.org/10.3390/vaccines13080862
  17. Adv Exp Med Biol. 2025 ;1478 421-443
      Hyperacetylation of proteins represents a stress to aged organisms. Increased consumption and loss of NAD+ homeostasis underlie a major mechanism for the disturbed acetylation/deacetylation balance during aging. Nicotinamide adenine dinucleotide (NAD) is a versatile chemical compound serving as a coenzyme in metabolic pathways and as a substrate to support the enzymatic functions of sirtuins (SIRTs), poly (ADP-ribose) polymerase-1 (PARP-1), and cyclic ADP ribose hydrolase (CD38). Under normal physiological conditions, NAD+ consumption is matched by its synthesis primarily via the salvage pathway catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). However, aging and muscular contraction enhance NAD+ utilization, whereas NAD+ replenishment is limited by cellular sources of NAD+ precursors and/or enzyme expression. This chapter will briefly review NAD+ metabolic functions, its roles in regulating cell signaling, mechanisms of its degradation and biosynthesis, and major challenges to maintain its cellular level in skeletal muscle. The effects of aging, physical exercise, and dietary supplementation on NAD+ homeostasis will be highlighted based on recent literature.
    Keywords:  Acetylation; Aging; Exercise; NAD+; Sirtuin; Skeletal muscle
    DOI:  https://doi.org/10.1007/978-3-031-88361-3_17
  18. PLoS Negl Trop Dis. 2025 Sep;19(9): e0013428
      Toxoplasma gondii infection induces anxiety in hosts during the chronic stage; however, its role in pre-anxiety-like behaviors during the acute stage remains poorly understood. This study investigates the role of Bradyzoite Formation Deficient 2 (BFD2), a transcription factor essential for tachyzoite-to-bradyzoite differentiation, in inflammation, apoptosis, and behavioral changes during acute T. gondii infection. Using CRISPR/Cas9-mediated gene editing, we generated a Bfd2 knockout strain (ME49∆bfd2) and observed reduced parasite proliferation and plaque formation, indicating BFD2's role in promoting T. gondii survival. RNA sequencing analysis of infected BV2 cells revealed that Bfd2 deletion significantly downregulated inflammatory responses, with reduced expression of key inflammatory markers (interleukin 1 beta ((IL-1β), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α)) during acute infection. Next, we used western blotting, real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs) to verify that BFD2 improves the inflammation induced by acute stage T. gondii infection. In vivo studies confirmed that BFD2 exacerbates brain inflammation and neuronal apoptosis specifically during the acute stage, with no significant effects during the chronic stage. Behavior was assessed using the elevated plus maze test and open field test. Compared with the uninfected group and ME49∆bfd2 group, the ME49 group mice showed an increased percentage of distance in the open arms and time in the open arm. The results showed that the total distance traveled, distance in the center, and time in the center were significantly decreased in the ME49 group, and the total distance traveled (mm) had no significant changes in the ME49∆bfd2. These demonstrated that BFD2 contributes to pre-anxiety-like behaviors in mice during acute stage T. gondii infection. These findings highlight BFD2 as a critical regulator of acute-stage inflammation, neuronal damage, and behavioral alterations, providing insights to develop targeted interventions against T. gondii infection.
    DOI:  https://doi.org/10.1371/journal.pntd.0013428
  19. Dis Model Mech. 2025 Sep 05. pii: dmm.052197. [Epub ahead of print]
      Mitochondria are the regulators of energy production and play a vital role in modulating ageing and age-associated diseases. We investigated the role of sirtuins, a well-studied class of longevity-associated proteins (NAD+-dependent histone deacetylases), in mitochondrial biology and Parkinson's disease pathology. In particular, we endeavored to study the functional implications of mitochondrial sirtuin, sir-2.2 (ortholog of human SIRT4), in regulating neuroprotection employing Caenorhabditis elegans model. We observed that upon sir-2.2 knockdown, the alpha-synuclein aggregation was increased and expression of dopamine transporter, dat-1, was reduced. Also, the levels of marker proteins for innate immunity, oxidative stress, mitophagy, UPRmt, and autophagy, were decreased, suggesting an important function of sir-2.2 in maintaining mitochondrial homeostasis, regulating protein clearance and ameliorating the disease condition. Because of their crucial role in regulating oxidative stress and mitochondrial quality control, studying mitochondrial sirtuin will provide therapeutic insights into the metabolic regulation of ageing and neurodegeneration.
    Keywords:   Caenorhabditis elegans.; Sir-2.2 ; Mitochondria; Neurodegeneration; Parkinson's disease
    DOI:  https://doi.org/10.1242/dmm.052197
  20. Crit Rev Oncol Hematol. 2025 Sep 03. pii: S1040-8428(25)00308-7. [Epub ahead of print] 104920
      Colorectal cancer (CRC) is a predominant malignancy of the digestive tract globally, with primary treatment strategies including surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Recently, histone deacetylases (HDACs) and their inhibitors (HDACi) have emerged as promising therapeutic targets in CRC. As critical epigenetic regulators, HDACs influence gene expression and cellular processes, thereby affecting tumor initiation, progression, and immune evasion. Growing evidence suggests that HDAC inhibitors not only induce apoptosis and suppress cell proliferation but also potentiate the effectiveness of immune checkpoint inhibitors (ICIs) and counteract drug resistance. This review summarizes the classification and functional mechanisms of HDACs in CRC, evaluates the clinical advancement of HDAC inhibitors as monotherapies and combination therapies (e.g., with chemotherapy or ICIs), and highlights innovations in HDAC inhibitors, such as PROTAC-based degraders and nanomaterial-mediated delivery systems. These developments provide valuable insights for the precision treatment of CRC.
    Keywords:  Colorectal cancer; Epigenetic regulation; Histone deacetylase; Histone deacetylase inhibitors; Immune checkpoint inhibitors
    DOI:  https://doi.org/10.1016/j.critrevonc.2025.104920
  21. J Biol Chem. 2025 Aug 28. pii: S0021-9258(25)02488-3. [Epub ahead of print] 110636
      Ketone bodies are a key alternative energy source during carbohydrate deficiency. In addition to their metabolic function, they regulate essential cellular processes, including metabolism, signal transduction, and protein post-translational modifications (PTMs). However, the role of ketone body metabolism in tumorigenesis remains poorly understood. Here, we demonstrate that ketone body synthesis metabolism is activated in pancreatic cancer, while exogenous ketone supplementation does not affect PDAC cell proliferation. Moreover, we observe a significant upregulation of β-Hydroxybutyrate dehydrogenase (BDH1), a key enzyme in ketone body metabolism, in human pancreatic ductal adenocarcinoma (PDAC) tissues compared to adjacent normal pancreatic tissues. BDH1 promotes PDAC cell proliferation by maintaining mitochondrial acetylation levels through regulation of the intracellular NAD+/NADH ratio. These findings underscore the importance of ketone body metabolism in pancreatic cancer progression and highlight the regulatory role of BDH1 in maintaining cellular NAD+/NADH balance and mitochondrial acetylation.
    Keywords:  BDH1; Ketone body; NAD(+)/NADH; Pancreatic Cancer; mitochondrial acetylation
    DOI:  https://doi.org/10.1016/j.jbc.2025.110636
  22. Bioorg Med Chem. 2025 Aug 27. pii: S0968-0896(25)00313-X. [Epub ahead of print]130 118372
      Breast cancer remains the most common prevalent malignancy among women globally, constituting the primary cause of cancer-associated mortality. While therapeutic advancements have been achieved, persistent challenges in treatment resistance, disease recurrence, and distant metastasis continue to undermine clinical outcomes. Histone deacetylases (HDACs), a conserved family of epigenetic regulators, catalyze the removal of acetyl groups from histone substrates, thereby orchestrating chromatin remodeling and transcriptional regulation. Beyond their canonical epigenetic functions, these enzymes critically modulate diverse oncogenic progresses, including cell proliferation, differentiation, and metastasis, positioning them as promising therapeutic targets in oncology. Recent studies have demonstrated the therapeutic prospects of dual-target inhibitors. Current evidence suggests such combinatorial approaches not only enhance anti-neoplastic efficacy through multi-modal mechanisms but also circumvent the drug resistance frequently observed in single-target therapy. This therapeutic paradigm shift underscores the clinical potential of HDAC-based dual inhibitors for breast cancer management. In this review, we systematically analyze recent advancements in dual-target HDAC inhibitors (HDACis), integrating mechanistic insights, preclinical evidences, and translational implications to establish a foundational framework for future therapeutic development and clinical implementation.
    Keywords:  Antitumor; Dual-target; Epigenetic factor; Growth factor receptor; Histone deacetylase; Nuclear receptor
    DOI:  https://doi.org/10.1016/j.bmc.2025.118372
  23. bioRxiv. 2025 Aug 28. pii: 2025.08.22.671872. [Epub ahead of print]
      The interdependence of chromatin states and transcription factor (TF) binding in eukaryotic genomes is critical for the proper regulation of gene expression. In this study, we explore the connection between TFs and chromatin states in the human malaria parasite, Plasmodium falciparum , throughout its 48-hour asexual intraerythrocytic developmental cycle (IDC). Most P. falciparum genes are expressed in a periodic manner during the IDC, accompanied by dynamic shifts in histone modifications and chromatin accessibility. Leveraging genome-wide profiles of chromatin accessibility, histone modifications, and Heterochromatin Protein 1 (HP1) occupancy, we characterize chromatin state dynamics during the IDC. Our results indicate that several chromatin states remain stable throughout the lifecycle, while others are dynamic and are linked to gene activation or repression. We further characterize chromatin state dynamics at the genome-wide DNA binding sites for a selection of Plasmodium TFs, allowing us to group TFs according to their chromatin preferences. By correlating changes in chromatin accessibility, histone modifications, and TF binding, we provide a global overview of the chromatin state dynamics that coordinate P. falciparum asexual blood stage development.
    DOI:  https://doi.org/10.1101/2025.08.22.671872
  24. bioRxiv. 2025 Aug 23. pii: 2025.08.23.671938. [Epub ahead of print]
      Reactivation of toxoplasmosis is a significant health threat to chronically infected individuals, especially those who are or become immunocompromised. An estimated one-third of the world population is infected with Toxoplasma , placing millions at risk. The Toxoplasma cyst is the foundation of disease with its ingestion leading to infection and its reactivation, from slow replicating bradyzoites to fast replicating tachyzoites, leading to cell lysis in tissues such as the brain. There are no treatments that prevent or eliminate cysts in part due to our poor understanding of the mechanisms that underlie cyst formation and recrudescence. In this study, we aimed to understand the biology of bradyzoites prior to recrudescence and the developmental pathways they initiate. We have discovered ME49EW cysts from infected mice harbor multiple bradyzoite subtypes that can be identified by their expression of distinct proteins. Sorting of these subtypes revealed they initiate distinct developmental pathways in animals and in primary astrocyte cell cultures. Single bradyzoite RNA sequencing indicates 5 major bradyzoite subtypes occur within these cysts. We further show that a crucial subtype comprising the majority of bradyzoites in chronically infected mice is absent from conventional in vitro models of bradyzoite development. Altogether this work establishes new foundational principles of Toxoplasma cyst development and reactivation that operate during the intermediate life cycle of Toxoplasma .
    DOI:  https://doi.org/10.1101/2025.08.23.671938
  25. Nat Commun. 2025 Sep 02. 16(1): 8190
      SOX2 is a potent oncodriver for various squamous cancers, but the underlying mechanism is largely unknown. Here we uncover a role of SOX2 in promoting global histone acetylation in esophageal squamous cancer cells (ESCCs). Mechanistic studies reveal that SOX2 promotes global histone acetylation in an AKT-independent manner, and does so by promoting histone acetylation at both SOX2 binding and non-SOX2 binding sites, and accounts for the formation of about half of the super-enhancers. Combined metabolic and transcriptional analyses reveal two mechanisms by which SOX2 enhances global histone acetylation: promoting the expression of multiple histone acetyltransferases and reducing acetyl-CoA consuming fatty acid synthesis in part by repressing the expression of ACSL5. Finally, SOX2 expression correlates negatively with ACSL5 and positively with histone acetylation in clinical esophageal squamous tumors. Altogether, our study uncovers a role of SOX2 in reprogramming lipid metabolism and driving histone hyperacetylation and super-enhancer function, providing mechanistic insights of SOX2 acting as a potent oncodriver.
    DOI:  https://doi.org/10.1038/s41467-025-63591-z
  26. J Parasit Dis. 2025 Sep;49(3): 691-700
      Toxoplasma gondii is a protozoan parasite that infects more than a third of the world's population. The drugs used today to treat toxoplasmosis cause severe side effects in many people and have poor success in treating chronic infections. In the current study, oil extracted from tea leaf was loaded into solid lipid nanoparticles (SLNs) and its anti-Toxoplasma properties were analyzed. The double emulsification technique was employed to provide SLNs and their physical characteristics were assessed using transmission electron microscope (TEM) and dynamic light scattering (DLS). Cell toxicity and anti-intracellular Toxoplasma activity were investigated using a MTT assay. The anti-Toxoplasma activity of Tea tree oil (TTO)-SLNs was evaluated through trypan-blue staining. The TTO-SLNs were round with a mean particle size of 85.23 nm and clear and stable margins. An association was observed between the cell toxicity of TTO-SLNs with the concentration of the component (P-value = 0.009). The cytotoxic concentration (CC50) against Toxoplasma was > 10 mg/mL, while it was concentration-dependent (P-value < 0.0001). the viability of T. gondii- infected Vero cells were higher in lower concentrations of TTO-SLNs (P-value = 0.0174), while at least 80% of T. gondii- infected Vero cells remaining alive at concentrations greater than >1 mg/mL. Overall, our findings demonstrated high anti-T. gondii properties of TTO-SLNs, suggesting a promising role of SLNs in carrying TTO. In addition, our findings showed prolonged release of the TTO from SLNs capsulation of the can lead to, suggesting the potential of TTO-SLNs for applications in the chronic phase (cyst stages), which should be further investigated in animal models.
    Keywords:  Herbal medicine; Solid lipid nanoparticles; Tea tree oil (TTO); Toxoplasma gondii
    DOI:  https://doi.org/10.1007/s12639-025-01786-3
  27. Genetics. 2025 Sep 05. pii: iyaf185. [Epub ahead of print]
      Mitochondrial sirtuins regulate metabolism and are emerging drug targets for metabolic and age-related diseases such as cancer, diabetes, and neurodegeneration. Yet, the extent of their functions remain unclear. Here, we uncover a physiological role for the C. elegans mitochondrial sirtuins, sir-2.2 and sir-2.3, in lifespan regulation. Using genetic alleles with deletions that destroy catalytic activity, we demonstrate that sir-2.2 and sir-2.3 mutants live an average of 25% longer than controls when fed the normal lab diet of live E. coli OP50. While decreased consumption of food is a known mechanism for lifespan extension, we did not find evidence of reduced pharyngeal pumping. Interestingly, lifespan extension effected by loss of sir-2.2 or sir-2.3 is sensitive to the diet. The lifespan extension of the sir-2.2 mutants is eliminated and that of sir-2.3 mutants is attenuated when the animals are fed the E. coli strain HT115, which is typically used for RNAi experiments. We used growth ability of the food source and a virulent pathogenic strain to ask if differences in pathogenicity are related to the mechanisms for lifespan extension. sir-2.3 deletion results in lifespan extension in all conditions. However, removing the ability of the food source to grow eliminated the sir-2-mediated effect. We also examine the response of the mutants to oxidative stress, and our results suggest that a hormetic response contributes to lifespan extension in both mutants. Our data suggest that sir-2.2 and sir-2.3 use overlapping yet distinct mechanisms for regulating lifespan.
    Keywords:   C. elegans ; HT115; OP50; Pseudomonas; SIRT4; WormBase; hormesis; lifespan; oxidative stress; pathogenicity; sirtuin
    DOI:  https://doi.org/10.1093/genetics/iyaf185
  28. Bioorg Chem. 2025 Aug 29. pii: S0045-2068(25)00812-0. [Epub ahead of print]165 108932
      Histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic target in drug discovery. Aberrant expression of HDAC6 is associated with various diseases, including cancer, neurodegenerative disorders, and pathological autoimmune responses. Inhibition of HDAC6 has been extensively investigated for the treatment of multiple diseases, particularly cancer. A variety of HDAC6 inhibitors have been developed and are at different stages of drug development. Currently, diverse strategies targeting HDAC6 are being explored, such as isoform selective inhibitors, multi-targeted inhibitors and PROTACs. This review summarizes the functions of HDAC6, its roles in different disorders, and various types of modulators targeting HDAC6. These insights may provide valuable information for further design of therapeutic drugs by targeting HDAC6.
    Keywords:  Dual targeting; HDAC6; Inhibitor; PROTAC; Selectivity
    DOI:  https://doi.org/10.1016/j.bioorg.2025.108932
  29. Acta Pharmacol Sin. 2025 Sep 02.
      Progressive loss of vascular smooth muscle cells (VSMCs) is the pathophysiological basis for aortic aneurysm and dissection (AAD), a life-threatening disease, but the underlying mechanisms are largely unknown. Sirtuin 6 (SIRT6), a class III histone deacetylase, is critical for maintenance of VSMC homeostasis and prevention of vascular remodeling-related diseases. In this study, we investigated the role of VSMC SIRT6 in AAD and the molecular mechanism. We showed that the expression levels of SIRT6 were significantly reduced in VSMCs of the thoracic aorta in AAD patients. We constructed a VSMC-specific Sirt6 deficient mouse line and found that loss of Sirt6 in VSMCs dramatically accelerated angiotensin II (Ang II)-induced AAD formation and rupture, even without an Apoe-deficient background. In human aortic smooth muscle cells (HASMCs), knockdown of SIRT6 led to mitochondrial dysfunction and accelerated VSMC senescence. We revealed that SIRT6 bound to and deacetylated NRF2, a key transcription factor for mitochondrial biogenesis. However, Sirt6 deficiency inhibited NRF2 and reduced mRNAs encoding mitochondrial complex proteins. Notably, MDL-811, a newly developed small-molecule SIRT6 agonist, effectively reversed Ang II-induced mitochondrial dysfunction in HASMCs. In a BAPN-induced TAAD mouse model, administration of MDL-811 (20 mg/kg, i.p., every other day for 28 d) effectively mitigated AAD progression and reduced mortality. These results suggest that SIRT6 plays a protective role against AAD development, and targeting SIRT6 with small-molecule activators such as MDL-811 could represent a promising therapeutic strategy for AAD.
    Keywords:  MDL-811; Sirtuin 6; aortic aneurysm and dissection; mitochondrial dysfunction; nuclear factor erythroid 2-related factor 2; vascular smooth muscle cells
    DOI:  https://doi.org/10.1038/s41401-025-01628-1
  30. Chem Biodivers. 2025 Sep 02. e01492
      Unregulated epigenetic modifications, including histone acetylation/deacetylation mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), contribute to cancer progression. HDACs, often overexpressed in cancer, downregulate tumor suppressor genes, making them crucial targets for treatment. This work aimed to develop non-hydroxamate benzoic acid-based HDAC inhibitors (HDACi) with comparable effect to the currently four FDA-approved HDACi, which are known for their poor solubility, poor distribution, and significant side effects. All compounds were structurally verified using FTIR, 1HNMR, 13CNMR, and mass spectrometry. In silico analysis showed that compound A3bn (3-chloro-4-((2-(2-(4-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)amino)benzoic acid) has strong binding affinity towards HDAC2, HDAC6, and HDAC8 and exhibits molecular similarity to trichostatin and SAHA (HDACi). A3bn achieved IC50 values comparable to SAHA against MCF-7 (20.3 vs. 39.2 µM) and K562 (42.0 vs. 36.1 µM) cancer cells. Western blot analysis confirmed that A3bn inhibited H3 and H4 deacetylation. Additionally, A3bn induced the extrinsic apoptotic pathway via caspase 8 activation, leading to cell death. Its enhanced activity across HDAC isoforms may result from its hydrophilic linker, facilitating zinc coordination. In conclusion, A3bn demonstrated efficacy similar to FDA-approved HDACi and represents a promising candidate for further optimization. Future studies will focus on structural modifications to enhance potency and selectivity at lower concentrations.
    Keywords:  Schiff bases; apoptosis; inhibitors; oxadiazole; thioamides
    DOI:  https://doi.org/10.1002/cbdv.202501492
  31. Biochem Biophys Res Commun. 2025 Aug 29. pii: S0006-291X(25)01276-8. [Epub ahead of print]782 152561
      Sirtuin 6 (Sirt6) is a member of the Sirtuin family, exhibiting histone deacetylase and ADP-ribosyltransferase activity. This enzyme is involved in several pathways, such as epigenetic regulation and inflammation control. It is essential for preserving cardiac equilibrium and postponing the emergence of cardiovascular disorders. Recent findings reveal that Sirt6 affects glucose and lipid metabolism and regulates oxidative stress via the HIF-1α/NF-κB signaling pathway, thereby delaying cardiomyocyte senescence and diminishing DNA damage accumulation. Sirt6 mitigates oxidative damage in cardiomyocytes by deacetylation, suppresses cardiac fibrosis, and improves cardiomyocyte survival rates. Sirt6 exhibits anti-atherosclerotic properties by enhancing DNA repair in endothelial cells, reducing lipid accumulation in macrophages, and promoting cholesterol transport via ATP-Binding Cassette A1 (ABCA1). Sirt6 promotes the degradation of the critical autophagic component Charged Multivesicular Body Protein 2B (CHMP2B) through the FoxO1-Atrogin-1 pathway. This action supports the autophagic process and mitigates ischemia-reperfusion harm. The regulatory mechanisms of Sirt6 in ferroptosis remain controversial. This article explores the specific molecular mechanisms of Sirt6 in the heart and various cell death pathways, including apoptosis, autophagy, and pyroptosis, while also considering the potential for targeted therapeutic applications of Sirt6 in cardiovascular medicine.
    Keywords:  Apoptosis; Cardiovascular diseases; Energy metabolism; Inflammation; Oxidative stress; Pyroptosis; Sirt6
    DOI:  https://doi.org/10.1016/j.bbrc.2025.152561
  32. Bioorg Med Chem. 2025 Aug 26. pii: S0968-0896(25)00312-8. [Epub ahead of print]130 118371
      The molecular chaperone heat shock protein 90 (Hsp90) has an important role in maintaining proteostasis in Plasmodium parasites, the causative agents of malaria, and is of interest as a potential antimalarial drug target. Inhibitors targeting its well-characterized N-terminal ATP-binding site are lethal, but the development of high-affinity binders with selectivity for the Plasmodium over the human homolog has been challenging given the high conservation of this domain. A binding site in the less conserved Hsp90 C-terminus has been reported to interact with nucleotides and inhibitors in other eukaryotic systems, which could offer an alternative route for antimalarial design. Herein, we characterize the potential ligandability of the C-terminus in the Plasmodium falciparum chaperone PfHsp90 with in silico and in vitro methods. We conducted affinity experiments with both a lysine-reactive nucleotide analog and an ATP resin that support a specific interaction between ATP and a C-terminal truncation of PfHsp90. We further explored the nucleotide structural requirements for this interaction with limited proteolysis experiments, which suggest association with ATP, dATP, and ADP, but not AMP or GTP. Lastly, we employed computational analyses and mutagenesis studies to interrogate the molecular basis for the interaction. Our findings provide the foundation for future studies to assess and develop C-terminal Hsp90 inhibitors against Plasmodium parasites.
    Keywords:  Cryptic site; Heat shock protein 90; Hsp90; Nucleotide interactions; Plasmodium
    DOI:  https://doi.org/10.1016/j.bmc.2025.118371
  33. Mol Divers. 2025 Aug 30.
      SIRT6, a pivotal member of the NAD+-dependent deacetylase superfamily, regulates critical biological processes, including DNA repair, transcriptional regulation, and aging. The deacetylase activity of SIRT6 is allosterically coupled to NAD⁺ binding, enabling site-specific removal of acetyl moieties from lysine substrates. Despite its physiological significance, the structural mechanisms underlying the allosteric regulation mediated by its N-terminal domain (NTD) have remained elusive. In this study, we establish that the NTD of SIRT6 plays an indispensable role in preserving the catalytic geometry by maintaining the NAD+ pocket conformation and stabilizing substrate coordination. Molecular dynamics simulations revealed that truncation of the NTD induces an open-state NAD+ pocket configuration, accompanied by a reduction in NAD+ binding affinity and an increase in the catalytic distance between NAD+ and the acetylated lysine substrate. Consistently, enzymatic assays demonstrated a twofold decrease in deacetylation efficiency in NTD-truncated enzyme compared to wild-type SIRT6. These results provide novel mechanistic insights into the NTD-mediated allosteric network essential for SIRT6 catalysis, offering a structural framework for developing modulators targeting this regulatory domain.
    Keywords:  Allosteric regulation; Community network analysis; Enzyme catalysis; Molecular dynamics simulations; SIRT6
    DOI:  https://doi.org/10.1007/s11030-025-11340-1
  34. J Chem Inf Model. 2025 Sep 03.
      Investigating structural variability is essential for understanding protein biological functions. Although AlphaFold2 accurately predicts static structures, it fails to capture the full spectrum of functional states. Recent methods have used AlphaFold2 to generate diverse structural ensembles, but they offer limited interpretability and overlook the evolutionary signals underlying the predictions. In this work, we enhance the generation of conformational ensembles and identify sequence patterns that influence the alternative fold predictions for several protein families. Building on prior research that clustered multiple sequence alignments to predict fold-switching states, we introduce a refined clustering strategy that integrates protein language model representations with hierarchical clustering, overcoming limitations of density-based methods. Our strategy effectively identifies high-confidence alternative conformations and generates abundant sequence ensembles, providing a robust framework for applying direct coupling analysis (DCA). Through DCA, we uncover key coevolutionary signals within the clustered alignments, leveraging them to design mutations that stabilize specific conformations, which we validate using alchemical free energy calculations from molecular dynamics. Notably, our method extends beyond fold-switching, effectively capturing a variety of conformational changes.
    DOI:  https://doi.org/10.1021/acs.jcim.5c01090
  35. Methods Enzymol. 2025 ;pii: S0076-6879(25)00214-9. [Epub ahead of print]718 107-128
      The N-terminus of a protein has an important regulatory impact on its in vivo stability and half-life. Proteins destined to chloroplasts and mitochondria are synthesized as precursor proteins in the cytosol with an N-terminal peptide sequence that ensures their correct targeting. During their cytosolic passage, precursor proteins are exposed to the cytosolic protein degradation machinery, hence, their N-termini must comply with regulatory processes for proteolytic degradation in the cytosol. We present here a method to determine the identity and modification state of plastid precursor protein N-termini in the cytosol by combining protoplast protein import assays with targeted mass spectrometry by means of parallel reaction monitoring (PRM). This method requires a hypothesis on potential modifications at the protein N-terminus such as methionine removal or N-terminal acetylation, that is decoded into an inclusion mass list to guide mass spectrometric data acquisition to specific peptides. This type of approach largely eliminates the stochastic nature of MS acquisition allowing different modifications to be tested as alternative hypotheses. Using Skyline, a quantitative assessment of different N-terminal modifications can be performed. We have used this method to determine the modification state of a model precursor protein RNP29 in two different genotypic backgrounds, but our workflow is easily expandable to different precursors.
    Keywords:  N-terminal acetylation; PRM; Plastid precursor proteins; Protoplast import assay; Skyline data analysis
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.006
  36. Methods Mol Biol. 2025 ;2972 135-152
      The proteome-wide detection of different posttranslational modifications (PTMs) still poses a notable analytical challenge. Nevertheless, the identification of protein pyrophosphorylation sites using mass spectrometry (MS) was reported recently. The implementation of an enrichment workflow proved to be key to characterize this substoichiometric modification within complex cell lysate samples. Analysis of the enriched samples using data-dependent neutral-loss triggered electron transfer/higher-energy collision dissociation (DDNL EThcD) MS enabled the detection of endogenous protein pyrophosphorylation. In this chapter, the bottom-up pyrophosphoproteomics analysis, including an enrichment workflow, to identify protein pyrophosphorylation sites is described.
    Keywords:  Bottom-up proteomics; EThcD; Enrichment; Fractionation; Lambda protein phosphatase; Mass spectrometry sample preparation; Posttranslational modification; Pyrophosphorylation; SIMAC
    DOI:  https://doi.org/10.1007/978-1-0716-4799-8_11
  37. Biochimie. 2025 Sep 03. pii: S0300-9084(25)00196-8. [Epub ahead of print]
      Metabolism involves a wide range of pathways and chemical reactions catalysed by specialized enzymes whose activity is fundamental for living cells. In the past three decades, metabolic enzymes have emerged as critical regulators of gene expression, thus revealing unexpected functions beyond their canonical metabolic roles. In this Review, we discuss the evidences that these enzymes, with a particular focus on enzymes participating in the glucose metabolism, can directly bind RNA. This binding has been recurrently shown to be involved in the post-trasncriptional gene regulation, by influencing processes such as RNA stability, localization, translation, and degradation. Although the mechanisms underlying RNA-enzyme interactions and their regulation are still not fully elucidated, several reports suggest that some of these interactions can be influenced by substrates, metabolites, and cellular metabolic states. In contrast, direct and specific binding of RNAs was also shown to regulate the activity, stability, interaction and localization of the enzymes. The discovery of the non-canonical RNA-binding activity of metabolic enzymes not only expands our understanding of these seemingly well-characterized proteins, but also provides new perspectives on the integration of metabolic and gene regulatory networks, besides revealing potential therapeutic vulnerabilities.
    Keywords:  Metabolic enzymes; RNA-binding proteins; post-transcriptional regulation; riboregulation
    DOI:  https://doi.org/10.1016/j.biochi.2025.08.017
  38. Structure. 2025 Aug 20. pii: S0969-2126(25)00303-X. [Epub ahead of print]
      Post-translational modifications, particularly protein lysine demethylation, intricately regulate diverse cellular processes. Dysregulation of this modification often precipitates human pathologies by perturbing substrate protein functions, stability, and interactions. Lysine demethylases (KDMs), such as the KDM5 family, are crucial in removing methyl marks. In particular, KDM5C has gained prominence for its role in cancer biology and drug resistance. These enzymes, specializing in erasing lysine methylation marks-especially from histone H3 lysine 4 (H3K4)-directly influence gene transcription. This study pioneers the design of a peptide inhibitor of KDM5C demethylase activity. This novel inhibitor displays remarkable selectivity for KDM5C over other family members. Intriguingly, in vivo experiments demonstrate that this inhibitor significantly reduces tumor growth. These findings highlight the potential of targeting KDM5C inhibition as a strategy for colon cancer treatment. Moreover, these findings underscore the promise of peptide inhibitors as targeted therapies, emphasizing their potential in altering the trajectory of cancer therapeutics.
    Keywords:  RNF20 methylation; histone H3K4me3; lysine demethylation; peptide arrays; peptide inhibitor; post-translational modification; therapeutics; xenograft model
    DOI:  https://doi.org/10.1016/j.str.2025.08.001
  39. Biol Open. 2025 Sep 05. pii: bio.062199. [Epub ahead of print]
      Yeast mitochondrial malate dehydrogenase, Mdh1p, is known to form supramolecular complexes with other TCA cycle and mitochondrial dehydrogenase enzymes, including the aldehyde dehydrogenase, Ald4p. These complexes have been proposed to facilitate NADH channeling. Here, we demonstrate that in cells grown to saturation and stationary phases, the endogenous Mdh1p, expressed without its mitochondrial targeting signal (MTS), stays outside mitochondria, in both a diffuse cytoplasmic distribution as well as localized to distinct puncta. The puncta formed by MTS-lacking Mdh1p show no co-localization with the MTS-lacking Ald4p, suggesting that they do not co-assemble into a supramolecular complex in the cytoplasm. However, we found that the MTS-lacking Mdh1p does co-localize with its cytoplasmic counterpart, Mdh2p, in puncta. Interestingly, Mdh2p has recently been reported to form heterocomplexes with the peroxisomal Mdh3p and to be transported into peroxisomes to assist in the glyoxylate cycle. We also show that the MTS-lacking Mdh1p co-localizes with a fluorescent peroxisome marker, Pex3p. Our findings suggest that different malate dehydrogenases can enter peroxisomes, potentially as a means to make the glyoxylate pathway more efficient.
    Keywords:  Malate dehydrogenase; Mitochondria; Peroxisome; Supramolecular assembly; Yeast
    DOI:  https://doi.org/10.1242/bio.062199
  40. Methods Enzymol. 2025 ;pii: S0076-6879(25)00236-8. [Epub ahead of print]718 129-165
      While antibodies specifically recognizing many post-translational modifications have existed for a long time, antibodies towards the acetylated N-termini (Nt) of proteins are only just emerging. Here we further explored the potential of an antibody developed to selectively recognize Nt-acetylated Met1 of proteins (anti-Nt-Ac-Met). While partly confirming previous characterizations of this antibody, showing it to be most useful for proteins whose N-terminal sequence matches that of the N-terminal acetyltransferases NatC, NatE and NatF, we here show that this antibody may additionally be used for selective detection of Nt-acetylated Met-starting proteins of the NatB-type sequence category, i.e. MD, ME, MN, MQ. We here demonstrate that this includes the Parkinson's disease-involved protein α-synuclein, with its MDVF-starting N-terminus. Thus, this antibody could potentially be useful to detect α-synuclein Nt-Ac state in Parkinson's tissue. Further, we show an improved sensitivity and signal-to-noise detection by the anti-Nt-Ac-Met antibody in peptide dot blots by adding a fixation step to the nitrocellulose membrane after the deposition of peptides. Finally, we here tested a new methodological concept and found that Nt-Ac-specific detection by antibodies can be used to measure the output of in vitro Nt-Ac enzyme activity assays.
    Keywords:  Acetyl detection; Antibody characterization; Enzyme activity assay; N-terminal acetyltransferase; NAA60; NatB; NatC; PTM; α-synuclein
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.028
  41. Gerontology. 2025 Aug 26. 1-22
      Sirtuin 3 (SIRT3), a NAD⁺-dependent deacetylase localized in the mitochondrial matrix, has emerged as a central regulator of aging and age-related pathologies. This review synthesizes evidence demonstrating SIRT3's tripartite anti-senescence mechanisms: 1) Enhancement of mitophagy via p53 deacetylation-mediated mitochondrial quality control, 2) Reinforcement of antioxidant defenses through SOD2/IDH2 activation, and 3) Optimization of metabolic homeostasis by coordinating fatty acid β-oxidation and glucose metabolism. In neurodegenerative models, SIRT3 ameliorates proteotoxic stress by promoting ketogenesis and reducing amyloid-β/tau pathology. SIRT3 mediates cardiovascular protection through dual modulation of fibrotic signaling cascades and nitric oxide biosynthesis. Paradoxically, SIRT3 exhibits context-dependent roles in oncology, suppressing tumor metabolism via HIF1α destabilization while potentially enabling chemoresistance through ferroptosis regulation. Within metabolic disorders, SIRT3 preserves β-cell function by neutralizing oxidative stress and SASP-driven inflammation, significantly delaying diabetes progression. Current therapeutic strategies leverage SIRT3's pleiotropic functions through natural compounds: Gastrodin (mitochondrial membrane stabilization), cocoa polyphenols (FOXO3-mediated antioxidant enhancement), and baicalein (anti-fibrotic signaling blockade). These advances position SIRT3 as a critical interface between mitochondrial energetics and systemic aging, offering a unified framework for developing precision gerotherapeutics.
    DOI:  https://doi.org/10.1159/000547549
  42. Methods Enzymol. 2025 ;pii: S0076-6879(25)00224-1. [Epub ahead of print]718 87-105
      N-terminal acetyltransferases (NATs) mediate co- and post-translational N-terminal protein acetylation to mediate essential protein functions, and their altered activities are linked to several diseases. Because of this connection of NATs to normal and abnormal cellular function, it is important to have assays available to evaluate NAT activity in vitro. This chapter outlines three in vitro assays for studying NAT activity: a sensitive radioactive method, and a scalable fluorescence-based approach and luminescence-coupled assay compatible with high-throughput screening. Using human NatB as a model NAT, we compare assay performance, highlighting differences in sensitivity, substrate requirements, and inhibitor detection. These methods enable investigations and support the development of NAT-targeted therapeutics, thus highlighting their utility in basic research and drug discovery.
    Keywords:  Acetyl-CoA synthetase short-chain (ACSS); Fluorescence assay; High-throughput screening (HTS); In-vitro activity assays; Luminescence assay; N-terminal acetyltransferases (NATs); Radioactivity assay
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.016
  43. bioRxiv. 2025 Aug 23. pii: 2025.08.12.670003. [Epub ahead of print]
      The ability of cells to power energy-demanding processes depends on maintaining the ATP hydrolysis reaction a billion-fold away from equilibrium. Cells respond to changes in energy state by sensing changes in ATP, ADP, AMP, and inorganic phosphate. A key barrier to a better understanding of the maintenance of energy homeostasis is a lack of tools for direct manipulation of energy state in living cells. Here, we report the development of ATPGobble-a genetically encoded tool for controlling cellular ATP hydrolysis rate. We validated ATPGobble by showing that it doubles the energy demand, decreases [ATP]/[ADP] and [ATP]/[AMP] ratios, and activates AMPK activity in human cells. We then used ATPGobble to systematically characterize the proteome and phosphoproteome changes caused by direct manipulation of the energy state. Our results establish ATPGobble as a powerful approach for dissecting the regulatory roles of energy state in human cells, opening new opportunities to study how cellular energy state governs physiology, stress responses, and disease processes.
    DOI:  https://doi.org/10.1101/2025.08.12.670003