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19 pages, 7360 KB  
Article
Class 1 Sugar Beet Phytoglobin Shows Strong Affinity to Glyceraldehyde-3-Phosphate Dehydrogenase and DNA In Vitro
by Leonard Groth, Miho Oda and Leif Bülow
Int. J. Mol. Sci. 2025, 26(19), 9404; https://doi.org/10.3390/ijms26199404 - 26 Sep 2025
Abstract
Class 1 phytoglobins (Pgbs) are known for their multifunctional roles in plant stress responses, with recent studies suggesting broader interactions involving metabolic and transcriptional regulation. Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights in many roles in colocalized spaces during cellular stress that are strikingly suitable [...] Read more.
Class 1 phytoglobins (Pgbs) are known for their multifunctional roles in plant stress responses, with recent studies suggesting broader interactions involving metabolic and transcriptional regulation. Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights in many roles in colocalized spaces during cellular stress that are strikingly suitable for supporting Pgb function. This study investigates the molecular interactions of class 1 Pgb from sugar beet (Beta vulgaris), BvPgb 1.2, and an alanine-substituted mutant (C86A), focusing on their ability to bind GAPDH and DNA. Using dual-emission isothermal spectral shift (SpS) analysis, we report strong binding interactions with GAPDH, with dissociation constants (KD) of 260 ± 50 nM for the recombinant wild-type protein (rWT) and a significantly stronger affinity for C86A (120 ± 40 nM), suggesting that the cysteine residue limits the interaction. Remarkably strong DNA-binding affinities were also observed for both variants, displaying biphasic binding. This behavior is characteristic of hexacoordinated globins and reflects the presence of two distinct species: a fast-reacting open pentacoordinated form and a slow-reacting closed hexacoordinated form with high apparent affinity. Here, the KD in the open configuration was 120 ± 50 nm and 50 ± 20 nM for rWT and C86A, respectively. In the closed configuration, however, the cysteine appears to support the interaction, as the KD was measured at 100 ± 10 pM and 230 ± 60 pM for rWT and C86A, respectively. Protein–protein docking studies reinforced these findings, revealing electrostatically driven interactions between BvPgb 1.2 and GAPDH, characterized by a substantial buried surface area indicative of a stable, biologically relevant complex. Protein–DNA docking similarly confirmed energetically favorable binding near the heme pocket without obstructing ligand accessibility. Together, these findings indicate a potential regulatory role for BvPgb 1.2 through its interaction with GAPDH and DNA. Full article
(This article belongs to the Section Biochemistry)
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21 pages, 2430 KB  
Article
ACE-Dependent Alzheimer’s Disease: Circulating ACE Phenotypes in Heterozygous Carriers of Rare ACE Variants
by Iaroslav V. Mironenko, Olga V. Kryukova, Anastasiia A. Buianova, Alexey V. Churov, Mikhail S. Arbatsky, Alyona A. Kubrikova, Yunna S. Petrusenko, Zhanna A. Repinskaia, Anna O. Shmitko, Galit A. Ilyina, Olga A. Kost, Steven M. Dudek, Irina D. Strazhesko, Ruslan I. Isaev, Elen A. Mkhitaryan, Olga N. Tkacheva, Denis V. Rebrikov and Sergei M. Danilov
Int. J. Mol. Sci. 2025, 26(18), 9099; https://doi.org/10.3390/ijms26189099 - 18 Sep 2025
Viewed by 265
Abstract
Damaging mutations of the Angiotensin I-converting enzyme (ACE) that result in low ACE levels may increase the risk of developing late-onset Alzheimer’s disease (AD). We quantified blood ACE levels in EDTA-plasma from 147 subjects with 23 different heterozygous ACE mutations (and 70 [...] Read more.
Damaging mutations of the Angiotensin I-converting enzyme (ACE) that result in low ACE levels may increase the risk of developing late-onset Alzheimer’s disease (AD). We quantified blood ACE levels in EDTA-plasma from 147 subjects with 23 different heterozygous ACE mutations (and 70 controls) and estimated the effect of these mutations on ACE phenotype, using a set of monoclonal antibodies (mAbs) to ACE and two ACE substrates. We identified several mutations in both ACE domains (including the most frequent ACE mutation, Y215C), which led to decreased ACE levels in the blood, and thus could be considered as putative risk factors for late-onset AD. The precipitation of several ACE mutants (Q259R, A725P, C734Y) by specific mAbs changed significantly, and therefore, these mAbs could be markers of these mutations. Analysis of 50 of the most frequent ACE mutations demonstrates that more than 1.5% of the adult population may have mutations which lead to decreased ACE levels, and thus, the role of low ACE levels in the development of AD may be underappreciated. Intriguingly, statistical and cluster analyses of longevity patients revealed trends towards higher frequency of cognitive impairment among affected individuals with damaging ACE mutations. Systematic analysis of blood ACE levels in patients with various ACE mutations identifies individuals with low blood ACE levels who may be at increased risk for late-onset AD. Patients with transport-deficient ACE mutations theoretically could benefit from therapeutic treatment with a combination of chemical and pharmacological chaperones and proteasome inhibitors, as was demonstrated previously on a cell model of the transport-deficient ACE mutation Q1069R. Moreover, clinical association analysis suggests a trend linking damaging ACE mutations with increased risk of cognitive impairment. Full article
(This article belongs to the Special Issue Molecular Insight into Alzheimer’s Disease)
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11 pages, 1634 KB  
Article
Botulinum Toxin Complex Serotype B-Okra Exerts Systemic Toxicity via the Oral Route by Disrupting the Intestinal Epithelial Barrier
by Chiyono Morimoto, Sho Amatsu, Takuhiro Matsumura, Masahiko Zuka and Yukako Fujinaga
Toxins 2025, 17(9), 443; https://doi.org/10.3390/toxins17090443 - 4 Sep 2025
Viewed by 659
Abstract
Botulinum toxin (BoNT) causes flaccid paralysis by blocking the release of neurotransmitters. BoNTs associate with neurotoxin-associated proteins to form medium and large progenitor toxin complexes. The large progenitor toxin complex serotype A-62A (L-PTC/A-62A) specifically targets intestinal M cells for invasion, whereas large progenitor [...] Read more.
Botulinum toxin (BoNT) causes flaccid paralysis by blocking the release of neurotransmitters. BoNTs associate with neurotoxin-associated proteins to form medium and large progenitor toxin complexes. The large progenitor toxin complex serotype A-62A (L-PTC/A-62A) specifically targets intestinal M cells for invasion, whereas large progenitor toxin complex serotype B-Okra (L-PTC/B-Okra) is mainly taken up by enterocytes and exhibits higher toxicity via the oral route. Hemagglutinin (HA) is a neurotoxin-associated protein that promotes BoNT absorption from the intestine and has carbohydrate-binding and barrier-disrupting activities. In this study, we established an in vitro reconstitution and purification system for recombinant L-PTC/B-Okra and created a recombinant L-PTC/B-Okra mutant rL-PTC/B-KA with carbohydrate-binding activity but not barrier-disrupting activity. rL-PTC/B-KA showed significantly reduced oral toxicity. Our results demonstrate that the B-Okra toxin disrupts the epithelial barrier of enterocytes and exerts oral toxicity. Full article
(This article belongs to the Special Issue Toxin–Host Interaction of Clostridium Toxins: 2nd Edition)
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14 pages, 1197 KB  
Article
Optimization of (Dithioperoxo)thiolate-Based Antifungal Agents for Triazole-Resistant Aspergillus Fumigatus
by Surya Karuturi, Kaitlyn L. Jobe, Melinda E. Varney, Michael D. Hambuchen, A. R. M. Ruhul Amin and Timothy E. Long
Pathogens 2025, 14(9), 878; https://doi.org/10.3390/pathogens14090878 - 3 Sep 2025
Viewed by 519
Abstract
This investigation on novel antifungal agents featuring a thiol-reactive (dithioperoxo)thiolate chemical nucleus [-NC(S)S-SR] established that the optimal levels of fungal growth inhibition were achieved with thiomethyl-bound derivatives (R = Me). The most efficacious analogs had MIC50/MIC90 values of 2/2 µg/mL [...] Read more.
This investigation on novel antifungal agents featuring a thiol-reactive (dithioperoxo)thiolate chemical nucleus [-NC(S)S-SR] established that the optimal levels of fungal growth inhibition were achieved with thiomethyl-bound derivatives (R = Me). The most efficacious analogs had MIC50/MIC90 values of 2/2 µg/mL and an MIC range of 1 to 2 µg/mL for a ten-member panel of voriconazole-resistant A. fumigatus mutants. Pharmacodynamic studies revealed that the lead (dithioperoxo)thiolates impaired conidial germination and germling development more effectively than voriconazole for the triazole-resistant strain AR-1295. Moreover, glutathione and Cu2+ were shown to have antagonistic interactions, which was attributed to the thiol-reactive, pro-oxidant properties of the (dithioperoxo)thiolates and their metabolic conversion to chelating agents. Cytotoxicity studies further showed that the compounds were less toxic to human fetal kidney cells than squamous carcinoma cells. The collective findings of the investigation indicate that (dithioperoxo)thiolates are effective antifungal agents against A. fumigatus to merit additional research on their therapeutic potential. Full article
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20 pages, 10269 KB  
Article
An AI-Designed Antibody-Engineered Probiotic Therapy Targeting Urease to Combat Helicobacter pylori Infection in Mice
by Feiliang Zhong, Xintong Liu, Xuefang Wang, Mengyu Hou, Le Guo and Xuegang Luo
Microorganisms 2025, 13(9), 2043; https://doi.org/10.3390/microorganisms13092043 - 1 Sep 2025
Cited by 1 | Viewed by 658
Abstract
Helicobacter pylori (Hp), a Class I carcinogen infecting over 50% of the global population, is increasingly resistant to conventional antibiotics. This study presents an AI-engineered probiotic strategy targeting urease, a key Hp virulence factor. A humanized single-domain antibody (UreBAb), previously identified and selected [...] Read more.
Helicobacter pylori (Hp), a Class I carcinogen infecting over 50% of the global population, is increasingly resistant to conventional antibiotics. This study presents an AI-engineered probiotic strategy targeting urease, a key Hp virulence factor. A humanized single-domain antibody (UreBAb), previously identified and selected in our laboratory, was synthesized commercially and modeled using AlphaFold2, with structural validation conducted via SAVES 6.0. Molecular docking (PyMOL/ClusPro2) and binding energy analysis (InterProSurf) identified critical urease-active residues: K40, P41, K43, E82, F84, T86, K104, I107, K108, and R109. Machine learning-guided optimization using mCSA-AB, I-Mutant, and FoldX prioritized four mutational hotspots (K43, E82, I107, R109), leading to the generation of nine antibody variants. Among them, the I107W mutant exhibited the highest activity, achieving 65.6% urease inhibition—a 24.95% improvement over the wild-type antibody (p < 0.001). Engineered Escherichia coli Nissle 1917 (EcN) expressing the I107W antibody significantly reduced gastric HP colonization by 4.42 log10 CFU in the treatment group and 3.30 log10 CFU in the prevention group (p < 0.001 and p < 0.05, respectively), while also suppressing pro-inflammatory cytokine levels. Histopathological (H&E) analysis confirmed that the I107W antibody group showed significantly enhanced mucosal repair compared to wild-type probiotic-treated mice. Notably, 16S rRNA sequencing revealed that intestinal microbiota diversity and the abundance of core microbial species remained stable across different ethnic backgrounds. By integrating AI-guided antibody engineering with targeted probiotic delivery, this platform provides a transformative and microbiota-friendly strategy to combat antibiotic-resistant Hp infections. Full article
(This article belongs to the Section Medical Microbiology)
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10 pages, 1168 KB  
Article
Gas-Binding Studies of Class 1 Sugar Beet Phytoglobin and C86A Mutant Using Isothermal Spectral Shifts in High-Precision Microliter Assay
by Leonard Groth and Leif Bülow
Int. J. Mol. Sci. 2025, 26(17), 8240; https://doi.org/10.3390/ijms26178240 - 25 Aug 2025
Cited by 1 | Viewed by 615
Abstract
Phytoglobins (Pgbs) are plant hemoglobin-like proteins with key roles in nitric oxide (NO) scavenging, oxygen sensing, and hypoxic stress responses. Their typical hexacoordination results in unusually high affinities for gaseous ligands such as NO and carbon monoxide (CO), complicating measurement using conventional methods. [...] Read more.
Phytoglobins (Pgbs) are plant hemoglobin-like proteins with key roles in nitric oxide (NO) scavenging, oxygen sensing, and hypoxic stress responses. Their typical hexacoordination results in unusually high affinities for gaseous ligands such as NO and carbon monoxide (CO), complicating measurement using conventional methods. Standard assays often require large sample volumes and lack sensitivity for high-affinity, low-abundance proteins like hexacoordinated Pgbs. Here, we present a microscale capillary-based fluorescence assay for the high-precision measurement of protein–gas binding. Fluorophore-labeled proteins are loaded into gas-saturated capillaries and analyzed via dual-wavelength fluorescence to monitor isothermal spectral shifts upon ligand binding. Phosphate-buffered saline with Tween20 (PBS-T20) ensures gas stability and minimizes nonspecific adsorption. Using this approach, we characterized CO and NO binding to the recombinant wildtype (rWT) of Beta vulgaris Pgb 1.2 (BvPgb 1.2) and its C86A mutant. CO titrations revealed biphasic binding, with EC50 ~400 nM and ~700 μM (rWT) and ~500 nM and ~400 μM (C86A). NO binding showed KD values of ~1600 nM (rWT) and ~400 nM (C86A), implicating Cys86 in ligand affinity. This assay provides a robust, low-volume method for high-affinity protein–gas studies and shows biphasic dynamics in BvPgbs. Full article
(This article belongs to the Section Biochemistry)
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15 pages, 1072 KB  
Article
Iron Ions Increase the Thermal Stability In Vitro and Activity In Vivo of the 447R Mutant Form of Mouse Tryptophan Hydroxylase 2
by Polina D. Komleva, Ekatherina I. Terentieva, Arseniy E. Izyurov and Alexander V. Kulikov
Int. J. Mol. Sci. 2025, 26(17), 8188; https://doi.org/10.3390/ijms26178188 - 23 Aug 2025
Viewed by 547
Abstract
Tryptophan hydroxylase 2 (TPH2) hydroxylates L-tryptophan to L-5-hydroxytryptophan (5-HTP) the first and rate-limiting step of serotonin (5-HT) synthesis in the mammalian brain. Some mutations in the Tph2 gene reducing TPH2 activity are associated with hereditary depressive disorders. The P447R substitution in the mouse [...] Read more.
Tryptophan hydroxylase 2 (TPH2) hydroxylates L-tryptophan to L-5-hydroxytryptophan (5-HTP) the first and rate-limiting step of serotonin (5-HT) synthesis in the mammalian brain. Some mutations in the Tph2 gene reducing TPH2 activity are associated with hereditary depressive disorders. The P447R substitution in the mouse TPH2 molecule reduces its thermal stability in vitro and its activity in the brain. The effects of iron ions on thermal stability in vitro and the activity in the brain of the mutant TPH2 were investigated. In the in vitro experiment effects of 0.01, 0.05, and 0.2 mM of FeSO4 and FeCl3 on the enthalpy (ΔH) and Gibbs free energy (ΔG) of thermal denaturation of the mutant TPH2 extracted from the midbrain of Balb/c mice were assayed. All FeSO4 concentrations and 0.05 and 0.2 mM concentrations of FeCl3 increased these thermodynamic characteristics of the mutant TPH2. Repeated (for 7 days) intramuscular administration of Fe(III) hydroxide dextran complex (15 and 30 mg/kg/day) increased TPH2 activity in the hippocampus, but not in the midbrain in Balb/c mice. Repeated (for 7 days) intramuscular administration of Fe(III) hydroxide dextran complex (15 and 30 mg/kg/day) together with thiamine (8 mg/kg/day) and cyanocobalamin (0.8 mg/kg/day) increased TPH2 activity in the hippocampus, while 30 mg/kg of Fe(III) hydroxide dextran also increased the enzyme activity in the midbrain in Balb/c mice. These results are the first evidence for chaperone-like effects of iron ions on thermal stability in vitro and activity in the brain of the mutant TPH2. Full article
(This article belongs to the Special Issue Protein Stability Research: 2nd Edition)
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18 pages, 2301 KB  
Article
Engineered TtgR-Based Whole-Cell Biosensors for Quantitative and Selective Monitoring of Bioactive Compounds
by Kyeongseok Song, Haekang Ji, Jiwon Lee, Geupil Jang and Youngdae Yoon
Biosensors 2025, 15(8), 554; https://doi.org/10.3390/bios15080554 - 21 Aug 2025
Viewed by 612
Abstract
TtgR, a transcriptional repressor from Pseudomonas putida, plays a key role in regulating multidrug resistance by controlling the expression of genes in response to various ligands. Despite its broad specificity, TtgR represents a promising candidate for the development of transcription factor (TF)-based [...] Read more.
TtgR, a transcriptional repressor from Pseudomonas putida, plays a key role in regulating multidrug resistance by controlling the expression of genes in response to various ligands. Despite its broad specificity, TtgR represents a promising candidate for the development of transcription factor (TF)-based biosensors. In this study, we utilized TtgR and its native promoter region (PttgABC) as genetic components to construct TF-based biosensors in Escherichia coli. By coupling TtgR and PttgABC with egfp, we developed a biosensor responsive to diverse flavonoids. To enhance the selectivity and specificity of the biosensor, we genetically engineered a TtgR-binding pocket. Engineered TtgR variants exhibited altered sensing profiles, enabling the development of biosensors with tailored ligand responses. Computational structural analysis and ligand docking provided insights into the interaction mechanisms between TtgR variants and flavonoids. Notably, biosensors based on wild-type TtgR and its N110F mutant were capable of quantifying resveratrol and quercetin at 0.01 mM with >90% accuracy. Although the precise molecular mechanisms involved remain unclear and further optimization is needed, the biosensors developed herein demonstrate strong potential for applications in numerous fields. This study lays the foundation for future research that could extend the utility of TtgR-based biosensors to synthetic biology, metabolic engineering, and beyond. Full article
(This article belongs to the Special Issue Biosensing Applications for Cell Monitoring)
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15 pages, 1908 KB  
Article
Enhancement of Protein–Protein Interactions by Destabilizing Mutations Revealed by HDX-MS
by Yoshitomo Hamuro, Anthony Armstrong, Jeffrey Branson, Sheng-Jiun Wu, Richard Y.-C. Huang and Steven Jacobs
Biomolecules 2025, 15(8), 1201; https://doi.org/10.3390/biom15081201 - 20 Aug 2025
Viewed by 670
Abstract
Enhancing protein–protein interactions is a key therapeutic strategy to ensure effective protein function in terms of pharmacokinetics and pharmacodynamics and can be accomplished with methods like directed evolution or rationale design. Previously, two papers suggested the possible enhancement of protein–protein binding affinity via [...] Read more.
Enhancing protein–protein interactions is a key therapeutic strategy to ensure effective protein function in terms of pharmacokinetics and pharmacodynamics and can be accomplished with methods like directed evolution or rationale design. Previously, two papers suggested the possible enhancement of protein–protein binding affinity via destabilizing mutations. This paper reviews the results of the previous literature and adds new data to show the generality of the strategy that destabilizing the unbound protein without significantly changing the free energy of the complex can enhance protein–protein interactions for therapeutic benefit. The first example presented is that of a variant of human growth hormone (hGHv) containing 15 mutations that improve the binding to the hGH binding protein (hGHbp) by 400-fold while retaining full biological activity. The second example is that of the YTE mutations (M252Y/S354T/T256E) in the Fc region of a monoclonal antibody (mAb). The YTE mutations improve the binding of the mAb to FcRn at pH 6.0 10-fold, resulting in elongated serum half-life of the mAb. In both cases, (i) chemical titration or differential scanning calorimetry (DSC) showed the mutations destabilize the unbound mutant proteins, (ii) isothermal titration calorimetry (ITC) showed extremely favorable enthalpy (ΔH) and unfavorable entropy (ΔS) upon binding to their respective target molecule compared with the wildtype, and (iii) hydrogen/deuterium exchange–mass spectrometry (HDX-MS) revealed that these mutations increase the free energy of unbound mutant protein without significantly affecting the free energy of the bound state, resulting in an enhancement to the binding affinities. The third example presented is that of the JAWA mutations (T437R/K248E) also located in the Fc region of a mAb. The JAWA mutations facilitate antibody multimerization upon binding to cell surface antigens, allowing for enhanced agonism and effector functions. Both DSC and HDX-MS showed that the JAWA mutations destabilize the unbound Fc, although the complex was not characterized due to weak binding. Enhancement of protein–protein interactions through incorporation of mutations that increase the free energy of a protein’s unbound state represents an alternative route to decreasing the protein–protein complex free energy through optimization of the binding interface. Full article
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15 pages, 4969 KB  
Article
Duplicated Genes on Homologous Chromosomes Decipher the Dominant Epistasis of the Fiberless Mutant in Cotton
by Yu Le, Xingchen Xiong, Zhiyong Xu, Meilin Chen, Yuanxue Li, Chao Fu, Chunyuan You and Zhongxu Lin
Biology 2025, 14(8), 983; https://doi.org/10.3390/biology14080983 - 2 Aug 2025
Viewed by 393
Abstract
Cotton fiber initiation determines the fiber yield, yet the genetic basis underlying lint and fuzz initiation has still not been fully uncovered. Here, map-based cloning was carried out to identify the fiberless mutant genes derived from a cross between Gossypium hirsutum acc. WT [...] Read more.
Cotton fiber initiation determines the fiber yield, yet the genetic basis underlying lint and fuzz initiation has still not been fully uncovered. Here, map-based cloning was carried out to identify the fiberless mutant genes derived from a cross between Gossypium hirsutum acc. WT and a natural fiberless mutant, fblSHZ. The 12:3:1 segregation ratio in F2 populations (including 1848 and 3100 individuals that were developed in 2016 and 2018, respectively) revealed dominant epistasis, with the fuzz gene exerting dominance over the lint gene. Genetic linkage analysis revealed that GhMYB25like_A12 controls fuzz fiber initiation, while both GhMYB25like_A12 and GhMYB25like_D12 regulate lint fiber development. Sequencing analyses showed that the fblSHZ mutant exhibited a K104M mutation in the R2R3 domain of GhMYB25like_A12 and a transposable element insertion in GhMYB25like_D12, leading to fiberless seeds. Knockout of GhMYB25like_A12 produced fuzzless seeds, knockout of GhMYB25like_D12 led to no obvious change in seeds, and knockout of both (GhMYB25like_A12&D12) resulted in fiberless seeds. The 12:3:1 ratio reappeared in the F2 population developed from the GhMYB25like_A12&D12 mutated plants as female and Jin668 as the male, which further confirmed the genetic interaction observed in fblSHZ. RNA-seq analysis revealed that GhMYB25like regulates cotton fiber initiation through multiple pathways, especially fatty acid metabolism. This study elucidates the key genes and their genetic interaction mechanisms governing cotton fiber initiation, providing a theoretical foundation for genetic improvement of cotton fiber traits. Full article
(This article belongs to the Special Issue Cotton: Genomics, Biotechnology and Molecular Breeding)
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20 pages, 3054 KB  
Article
Development of COVID-19 Vaccine Candidates Using Attenuated Recombinant Vesicular Stomatitis Virus Vectors with M Protein Mutations
by Mengqi Chang, Hui Huang, Mingxi Yue, Yuetong Jiang, Siping Yan, Yiyi Chen, Wenrong Wu, Yibing Gao, Mujin Fang, Quan Yuan, Hualong Xiong and Tianying Zhang
Viruses 2025, 17(8), 1062; https://doi.org/10.3390/v17081062 - 30 Jul 2025
Viewed by 819
Abstract
Recombinant vesicular stomatitis virus (rVSV) is a promising viral vaccine vector for addressing the COVID-19 pandemic. Inducing mucosal immunity via the intranasal route is an ideal strategy for rVSV-based vaccines, but it requires extremely stringent safety standards. In this study, we constructed two [...] Read more.
Recombinant vesicular stomatitis virus (rVSV) is a promising viral vaccine vector for addressing the COVID-19 pandemic. Inducing mucosal immunity via the intranasal route is an ideal strategy for rVSV-based vaccines, but it requires extremely stringent safety standards. In this study, we constructed two rVSV variants with amino acid mutations in their M protein: rVSV-M2 with M33A/M51R mutations and rVSV-M4 with M33A/M51R/V221F/S226R mutations, and developed COVID-19 vaccines based on these attenuated vectors. By comparing viral replication capacity, intranasal immunization, intracranial injection, and blood cell counts, we demonstrated that the M protein mutation variants exhibit significant attenuation effects both in vitro and in vivo. Moreover, preliminary investigations into the mechanisms of virus attenuation revealed that these attenuated viruses can induce a stronger type I interferon response while reducing inflammation compared to the wild-type rVSV. We developed three candidate vaccines against SARS-CoV-2 using the wildtype VSV backbone with either wild-type M (rVSV-JN.1) and two M mutant variants (rVSV-M2-JN.1 and rVSV-M4-JN.1). Our results confirmed that rVSV-M2-JN.1 and rVSV-M4-JN.1 retain strong immunogenicity while enhancing safety in hamsters. In summary, the rVSV variants with M protein mutations represent promising candidate vectors for mucosal vaccines and warrant further investigation. Full article
(This article belongs to the Special Issue Structure-Based Antiviral Drugs and Vaccine Design)
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16 pages, 871 KB  
Article
Primary HSV-2 Infection in an Immunocompromised Patient Reveals High Diversity of Drug-Resistance Mutations in the Viral DNA Polymerase
by Hanna Helena Schalkwijk, Sarah Gillemot, Emilie Frobert, Florence Morfin, Sophie Ducastelle, Anne Conrad, Pierre Fiten, Ghislain Opdenakker, Robert Snoeck and Graciela Andrei
Viruses 2025, 17(7), 962; https://doi.org/10.3390/v17070962 - 9 Jul 2025
Viewed by 1074
Abstract
Herpes simplex virus 2 (HSV-2) remains a significant cause of morbidity and mortality in immunocompromised individuals, despite the availability of effective antivirals. Infections caused by drug-resistant isolates are an emerging concern among these patients. Understanding evolutionary aspects of HSV-2 resistance is crucial for [...] Read more.
Herpes simplex virus 2 (HSV-2) remains a significant cause of morbidity and mortality in immunocompromised individuals, despite the availability of effective antivirals. Infections caused by drug-resistant isolates are an emerging concern among these patients. Understanding evolutionary aspects of HSV-2 resistance is crucial for designing improved therapeutic strategies. Here, we characterized 11 HSV-2 isolates recovered from various body sites of a single immunocompromised patient suffering from a primary HSV-2 infection unresponsive to acyclovir and foscarnet. The isolates were analyzed phenotypically and genotypically (Sanger sequencing of viral thymidine kinase and DNA polymerase genes). Viral clone isolations, deep sequencing, viral growth kinetics, and dual infection competition assays were performed retrospectively to assess viral heterogeneity and fitness. Sanger sequencing identified mixed populations of DNA polymerase mutant variants. Viral clones were plaque-purified and genotyped, revealing 17 DNA polymerase mutations (K533E, A606V, C625R, R628C, A724V, S725G, S729N, I731F, Q732R, M789T/K, Y823C, V842M, R847C, F923L, T934A, and R964H) associated with acyclovir and foscarnet resistance. Deep-sequencing of the DNA polymerase detected drug-resistant variants ranging between 1 and 95%, although the first two isolates had a wild-type DNA polymerase. Some mutants showed reduced fitness, evidenced by (i) the frequency of variants identified by deep-sequencing not correlating with the proportion of mutants found by plaque-purification, (ii) loss of the variants upon passaging in cell culture, or (iii) reduced frequencies in competition assays. This study reveals the rapid evolution of heterogeneous drug-resistant HSV-2 populations under antiviral therapy, highlighting the need for alternative treatment options and resistance surveillance, especially in severe infections. Full article
(This article belongs to the Special Issue Mechanisms of Herpesvirus Resistance)
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17 pages, 2623 KB  
Article
Conformational Remodeling and Allosteric Regulation Underlying EGFR Mutant-Induced Activation: A Multi-Scale Analysis Using MD, MSMs, and NRI
by Hui Duan, De-Rui Zhao, Meng-Ting Liu, Li-Quan Yang and Peng Sang
Int. J. Mol. Sci. 2025, 26(13), 6226; https://doi.org/10.3390/ijms26136226 - 27 Jun 2025
Viewed by 528
Abstract
Activating mutations in the epidermal growth factor receptor (EGFR) are key oncogenic drivers across multiple cancers, yet the structural mechanisms by which these mutations promote persistent receptor activation remain elusive. Here, we investigate how three clinically relevant mutations—T790M, L858R, and the T790M_L858R double [...] Read more.
Activating mutations in the epidermal growth factor receptor (EGFR) are key oncogenic drivers across multiple cancers, yet the structural mechanisms by which these mutations promote persistent receptor activation remain elusive. Here, we investigate how three clinically relevant mutations—T790M, L858R, and the T790M_L858R double mutant—reshape EGFR’s conformational ensemble and regulatory network architecture. Using multiscale molecular simulations and kinetic modeling, we show that these mutations, particularly in combination, enhance flexibility in the αC-helix and A-loop, favoring activation-competent states. Markov state modeling reveals a shift in equilibrium toward active macrostates and accelerated transitions between metastable conformations. To resolve the underlying coordination mechanism, we apply neural relational inference to reconstruct time-dependent interaction networks, uncovering the mutation-induced rewiring of allosteric pathways linking distant regulatory regions. This coupling of conformational redistribution with network remodeling provides a mechanistic rationale for sustained EGFR activation and suggests new opportunities for targeting dynamically organized allosteric circuits in therapeutic design. Full article
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15 pages, 1401 KB  
Communication
Evaluating the Effectiveness of Tyrosine Kinase Inhibitors on EGFR Mutations In Vitro
by Hanshuang Shao and Alan Wells
Int. J. Mol. Sci. 2025, 26(13), 6157; https://doi.org/10.3390/ijms26136157 - 26 Jun 2025
Viewed by 566
Abstract
Abnormal expressions and genetic mutations of EGFR are broadly involved in the progression of many human solid tumors, which has led to the development of small molecule inhibitors (TKIs). However, patients’ tumors usually develop resistance to targeted therapeutic TKIs after a period of [...] Read more.
Abnormal expressions and genetic mutations of EGFR are broadly involved in the progression of many human solid tumors, which has led to the development of small molecule inhibitors (TKIs). However, patients’ tumors usually develop resistance to targeted therapeutic TKIs after a period of treatment, mostly due to secondary mutations in EGFR. To date, three major and prevalent point mutations in EGFR, including L858R, T790M, and C797S, impact the use of TKIs in non-small cell lung cancer patients. Although at least four generations of TKIs have been designed and developed by targeting these mutations, how each mono, dual, or triple variant responds to clinical TKIs remains largely undeciphered. To fill this gap, we constructed a series of EGFR mutants and assessed their responses to clinical TKIs in vitro. The first-generation TKI, erlotinib, completely blocked the autophosphorylation of WT, L858R, C797S, and C797S/L858R, but only partially, if at all, in EGFR containing the T790M mutation alone or in combination. The third generation, osimertinib, completely abolished the autophosphorylation of WT, T790M, L858R, and T790M/L858R. It also significantly inhibited C797S and C790S/L858R, but had no effect on T790M/C797S or T790M/C797S/L858R. EAI045, as the fourth-generation TKI, almost completely inhibited WT and all mutants in complete growth media, but EGF-mediated phosphorylation of WT, C797S, and C797S/L858R were only partially inhibited in quiescence media, while the other mutants were fully inhibited. Furthermore, the abolishment of the enhanced tolerance to Dox in cells transiently expressing T790M/L858R and T790M/C797S/L858R by EAI045 suggests that their enhanced autophosphorylation is involved in their resistant ability. These findings provide some insights into how patients carrying typical mutations should be correctly and efficiently treated and why patients present side effects (because of non-specific inhibitory effects on cells without EGFR mutations). Full article
(This article belongs to the Special Issue Editorial Board Members’ Collection Series: "Enzyme Inhibition")
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31 pages, 12256 KB  
Article
Inter-Relationship Between Melanoma Vemurafenib Tolerance Thresholds and Metabolic Pathway Choice
by Pratima Nangia-Makker, Madison Ahrens, Neeraja Purandare, Siddhesh Aras, Jing Li, Katherine Gurdziel, Hyejeong Jang, Seongho Kim and Malathy P Shekhar
Cells 2025, 14(12), 923; https://doi.org/10.3390/cells14120923 - 18 Jun 2025
Viewed by 1083
Abstract
Melanomas quickly acquire resistance to vemurafenib, an important therapeutic for BRAFV600 mutant melanomas. Although combating vemurafenib resistance (VemR) to counter mitochondrial metabolic shift using mitochondria-targeting therapies has promise, no studies have analyzed the relationship between vemurafenib tolerance levels and metabolic plasticity. To determine [...] Read more.
Melanomas quickly acquire resistance to vemurafenib, an important therapeutic for BRAFV600 mutant melanomas. Although combating vemurafenib resistance (VemR) to counter mitochondrial metabolic shift using mitochondria-targeting therapies has promise, no studies have analyzed the relationship between vemurafenib tolerance levels and metabolic plasticity. To determine how vemurafenib endurance levels drive metabolic plasticity, we developed isogenic BRAFV600E VemR melanoma models with variant vemurafenib tolerances and performed an integrative analysis of metabolomic and transcriptome alterations using metabolome, Mitoplate-S1, Seahorse, and RNA-seq assays. Regardless of drug tolerance differences, both VemR models display resistance to MEK inhibitor and sensitivity to Wnt/β-catenin inhibitor, ICG-001. β-catenin, MITF, and ABCB5 levels are upregulated in both VemR models, and ICG-001 treatment restored vemurafenib sensitivity with reductions in MITF, ABCB5, phospho-ERK1/2, and mitochondrial respiration. Whereas β-catenin signaling induced TCA cycle and OXPHOS in highly drug tolerant A2058VemR cells, it activated pentose phosphate pathway in M14VemR cells with low vemurafenib tolerance, both of which are inhibited by ICG-001. These data implicate an important role for Wnt/β-catenin signaling in VemR-induced metabolic plasticity. Our data demonstrate that drug tolerance thresholds play a direct role in driving metabolic shifts towards specific routes, thus providing a new basis for delineating VemR melanomas for metabolism-targeting therapies. Full article
(This article belongs to the Collection Pathometabolism: Understanding Disease through Metabolism)
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