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Cell Death & Disease Jun 2024The cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically...
The cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically analyzed the structural and stability effects of all oncogenic Tyr-to-Cys mutations (Y126C, Y163C, Y205C, Y220C, Y234C, and Y236C) in the p53 DNA-binding domain (DBD). They were all highly destabilizing, drastically lowering the melting temperature of the protein by 8-17 °C. In contrast, two non-cancerous mutations, Y103C and Y107C, had only a moderate effect on protein stability. Differential stabilization of the mutants upon treatment with the anticancer agent arsenic trioxide and stibogluconate revealed an interesting proximity effect. Crystallographic studies complemented by MD simulations showed that two of the mutations, Y234C and Y236C, create internal cavities of different size and shape, whereas the others induce unique surface lesions. The mutation-induced pockets in the Y126C and Y205C mutant were, however, relatively small compared with that of the already druggable Y220C mutant. Intriguingly, our structural studies suggest a pronounced plasticity of the mutation-induced pocket in the frequently occurring Y163C mutant, which may be exploited for the development of small-molecule stabilizers. We point out general principles for reactivating thermolabile cancer mutants and highlight special cases where mutant-specific drugs are needed for the pharmacological rescue of p53 function in tumors.
Topics: Tumor Suppressor Protein p53; Humans; Mutation; Neoplasms; Arsenic Trioxide; Molecular Dynamics Simulation; Protein Stability; Antineoplastic Agents
PubMed: 38862470
DOI: 10.1038/s41419-024-06739-x -
Drug Target Insights 2024It is unclear whether induced spike protein-specific antibodies due to infections with SARS-CoV-2 or to the prototypic Wuhan isolate-based vaccination can immune-react...
INTRODUCTION
It is unclear whether induced spike protein-specific antibodies due to infections with SARS-CoV-2 or to the prototypic Wuhan isolate-based vaccination can immune-react with the emerging variants of SARS-CoV-2.
AIM/OBJECTIVES
The main objective of the study was to measure the immunoreactivity of induced antibodies postvaccination with Covishield™ (ChAdOx1 nCoV-19 coronavirus vaccines) or infections with SARS-CoV-2 by using selected peptides of the spike protein of wild type and variants of SARS-CoV-2.
METHODOLOGY
Thirty patients who had recovered from SARS-CoV-2 infections and 30 individuals vaccinated with both doses of Covishield™ were recruited for the study. Venous blood samples (5 mL) were collected at a single time point from patients within 3-4 weeks of recovery from SARS-CoV-2 infections or receiving both doses of Covishield™ vaccines. The serum levels of total immunoglobulin were measured in both study groups. A total of 12 peptides of 10 to 24 amino acids length spanning to the receptor-binding domain (RBD) of wild type of SARS-CoV-2 and their variants were synthesized. The serum levels of immune-reactive antibodies were measured using these peptides.
RESULTS
The serum levels of total antibodies were found to be significantly (p<0.001) higher in the vaccinated individuals as compared to COVID-19 recovered patients. Our study reported that the mutations in the RBD at the residues K417, E484, and N501 have been associated with reduced immunoreactivity with anti-sera of vaccinated people and COVID-19 recovered patients.
CONCLUSION
The amino acid substitutions at the RBD of SARS-CoV-2 have been associated with a higher potential to escape the humoral immune response.
PubMed: 38860262
DOI: 10.33393/dti.2024.3059 -
Global Medical Genetics Jun 2024Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological...
Myelodysplastic syndrome (MDS) is a malignant clonal disorder of hematopoietic stem cells which is characterized by morphologic dysplasia. However, the pathological characteristics of megakaryocytes (MKs) in MDS patients with gene mutation are not well established. Bone marrow MK specimens from 104 patients with primary MDS were evaluated, and all patients were distributed into two groups according to gene mutation associated with functional MKs. The morphologic and cellular characteristics of MKs and platelets were recorded and compared. The more frequently mutated genes in MDS patients were (11.54%), (8.65%), (5.77%), and the most common point mutation was p.(R307H) and p.(Q43P). Patients with MK mutation showed a decrease in adenosine diphosphate-induced platelet aggregation, high proportion of CD34 CD61 MKs (10.00 vs. 4.00%, = 0.012), and short overall survival (33.15 vs. 40.50 months, = 0.013). Further, patients with a higher percent of CD34 CD61 MKs (≧20.00%) had lower platelet counts (36.00 × 10 /L vs. 88.50 × 10 /L, = 0.015) and more profound emperipolesis ( = 0.001). By analyzing RNA-sequencing of MKs, differentially expressed mRNA was involved in physiological processes including platelet function and platelet activation, especially for MDS patients with high percent of CD34 CD61 MKs. The high levels of expression of CD62P, CXCL10, and S100A9 mRNA, shown by RNA sequencing, were validated by PCR assay. High proportion of CD34 CD61 MKs was a poor prognostic factor in MDS patients with MK mutation. CD62P, CXCL10, and S100A9 may be the potential targets to evaluate the molecular link between gene defects and platelet function.
PubMed: 38860162
DOI: 10.1055/s-0044-1787752 -
BioRxiv : the Preprint Server For... Jun 2024Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic...
Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, , and approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of , a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.
PubMed: 38854123
DOI: 10.1101/2024.05.28.596307 -
Cell & Bioscience Jun 2024The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show...
BACKGROUND
The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer.
RESULTS
Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins.
CONCLUSIONS
The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase's role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.
PubMed: 38849850
DOI: 10.1186/s13578-024-01249-x -
Brain Communications 2024Amyotrophic lateral sclerosis is an age-dependent cell type-selective degenerative disease. Genetic studies indicate that amyotrophic lateral sclerosis is part of a...
Amyotrophic lateral sclerosis is an age-dependent cell type-selective degenerative disease. Genetic studies indicate that amyotrophic lateral sclerosis is part of a spectrum of disorders, ranging from spinal muscular atrophy to frontotemporal dementia that share common pathological mechanisms. Amyotrophic lateral sclerosis Type 8 is a familial disease caused by mis-sense mutations in . VAPB is localized to the cytoplasmic surface of the endoplasmic reticulum, where it serves as a docking point for cytoplasmic proteins and mediates inter-organelle interactions with the endoplasmic reticulum membrane. A gene knock-in model of amyotrophic lateral sclerosis Type 8 based on the mutation and gene deletion has been generated in rats. These animals display a range of age-dependent phenotypes distinct from those previously reported in mouse models of amyotrophic lateral sclerosis Type 8. A loss of motor neurones in and animals is indicated by a reduction in the number of large choline acetyl transferase-staining cells in the spinal cord. animals exhibit a relative increase in cytoplasmic TDP-43 levels compared with the nucleus, but no large protein aggregates. Concomitant with these spinal cord pathologies , and animals exhibit age-dependent changes in paw placement and exerted pressures when traversing a CatWalk apparatus, consistent with a somatosensory dysfunction. Extramotor dysfunction is reported in half the cases of motor neurone disease, and this is the first indication of an associated sensory dysfunction in a rodent model of amyotrophic lateral sclerosis. Different rodent models may offer complementary experimental platforms with which to understand the human disease.
PubMed: 38846532
DOI: 10.1093/braincomms/fcae184 -
F1000Research 2021Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea's population resides in the malaria-endemic...
BACKGROUND
Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea's population resides in the malaria-endemic western lowlands of the country. , the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea.
METHODS
Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the (PF3D7_0417200), (PF3D7_0810800) and (PF3D7_1343700) partial gene regions.
RESULTS
Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant (S108N, C59R, N51I) haplotype, the double-mutant (N51I, S108N) haplotype, the single-mutant (K540E) haplotype, and the mixed-mutant (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous V45A mutation in four isolates, and a synonymous R449R in one isolate.
CONCLUSIONS
The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.
PubMed: 38840941
DOI: 10.12688/f1000research.54195.3 -
International Journal of Medical... Jun 2024Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S....
Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S. aureus infections have become increasingly challenging. Vancomycin is considered to be one of the last-resort drugs for treating most methicillin-resistant S. aureus (MRSA), so it is of great significance to further reveal the mechanism of vancomycin resistance. VraFG is one of the few important ABC (ATP-binding cassette) transporters in S. aureus that can form TCS (two-component systems)/ABC transporter modules. ABC transporters can couple the energy released from ATP hydrolysis to translocate solutes across the cell membrane. In this study, we obtained a strain with decreased vancomycin susceptibility after serial passaging and selection. Subsequently, whole-genome sequencing was performed on this laboratory-derived strain MWA2 and a novel single point mutation was discovered in vraF gene, leading to decreased sensitivity to vancomycin and daptomycin. Furthermore, the mutation reduces autolysis of S. aureus and downregulates the expression of lytM, isaA, and atlA. Additionally, we observed that the mutant has a less net negative surface charge than wild-type strain. We also noted an increase in the expression of the dlt operon and mprF gene, which are associated with cell surface charge and serve to hinder the binding of cationic peptides by promoting electrostatic repulsion. Moreover, this mutation has been shown to enhance hemolytic activity, expand subcutaneous abscesses, reflecting an increased virulence. This study confirms the impact of a point mutation of VraF on S. aureus antibiotic resistance and virulence, contributing to a broader understanding of ABC transporter function and providing new targets for treating S. aureus infections.
Topics: Virulence; Staphylococcal Infections; Anti-Bacterial Agents; Vancomycin; Animals; ATP-Binding Cassette Transporters; Bacterial Proteins; Staphylococcus aureus; Microbial Sensitivity Tests; Vancomycin Resistance; Whole Genome Sequencing; Daptomycin; Mice; Autolysis; Humans; Point Mutation; Mutation; Female
PubMed: 38838390
DOI: 10.1016/j.ijmm.2024.151624 -
OncoTargets and Therapy 2024The third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), osimertinib, is an effective first-line therapy for patients with common...
Characterization of Patients with Mutation-Positive NSCLC Following Emergence of the Osimertinib Resistance Mutations, L718Q or G724S: A Multicenter Retrospective Observational Study in France.
PURPOSE
The third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), osimertinib, is an effective first-line therapy for patients with common mutation-positive non-small cell lung cancer (NSCLC). However, almost all patients become resistant to treatment. In some patients, emergence of tertiary mutations is implicated as a resistance mechanism. This study describes patients with NSCLC who acquired the rare mutations, L718Q or G724S, following EGFR TKI treatment.
PATIENTS AND METHODS
This was a retrospective, observational study undertaken in France from Feb-Nov 2021, in patients with mutation-positive NSCLC with an acquired L718Q or G724S mutation. Primary objectives were description of tumor characteristics, progression, and progression under treatment.
RESULTS
Nine eligible patients were identified. Acquired resistance to initial EGFR TKI treatment was associated with T790M emergence in six patients, who then received osimertinib monotherapy. Overall, eight patients received osimertinib monotherapy treatment at some point (average treatment duration: 18.3 months). Following the emergence of L718Q or G724S, patients received chemotherapy (n = 4; two of whom subsequently received afatinib), nivolumab (n = 2), afatinib (n = 2), or immunochemotherapy (n = 1). In the four patients who received afatinib after identification of L718Q or G724S, 2 achieved a partial response, one had stable disease and one had progressive disease. Treatment duration was 1.6-31.7 months. In patients with controlled disease (n = 3), progression-free survival was 6.1-31.7 months. Two of these patients had previously received osimertinib.
CONCLUSION
Currently, there is no consensus regarding the treatment of mutation-positive NSCLC following emergence of the osimertinib resistance mutations, L718Q or G724S. Afatinib appears to be a promising treatment option in this setting.
PubMed: 38836187
DOI: 10.2147/OTT.S448909 -
MBio Jun 2024Group III hybrid histidine kinases are fungal-specific proteins and part of the multistep phosphorelay, representing the initial part of the high osmolarity glycerol...
UNLABELLED
Group III hybrid histidine kinases are fungal-specific proteins and part of the multistep phosphorelay, representing the initial part of the high osmolarity glycerol (HOG) pathway. TcsC, the corresponding kinase in was expected to be a cytosolic protein but is targeted to the nucleus. Activation of TcsC by the antifungal fludioxonil has lethal consequences for the fungus. The agent triggers a fast and TcsC-dependent activation of SakA and later on a redistribution of TcsC to the cytoplasm. High osmolarity also activates TcsC, which then exits the nucleus or concentrates in spot-like, intra-nuclear structures. The sequence corresponding to the N-terminal 208 amino acids of TcsC lacks detectable domains. Its loss renders TcsC cytosolic and non-responsive to hyperosmotic stress, but it has no impact on the antifungal activity of fludioxonil. A point mutation in one of the three putative nuclear localization sequences, which are present in the N-terminus, prevents the nuclear localization of TcsC, but not its ability to respond to hyperosmotic stress. Hence, this striking intracellular localization is no prerequisite for the role of TcsC in the adaptive response to hyperosmotic stress, instead, TcsC proteins that are present in the nuclei seem to modulate the cell wall composition of hyphae, which takes place in the absence of stress. The results of the present study underline that the spatiotemporal dynamics of the individual components of the multistep phosphorelay is a crucial feature of this unique signaling hub.
IMPORTANCE
Signaling pathways enable pathogens, such as , to respond to a changing environment. The TcsC protein is the major sensor of the high osmolarity glycerol (HOG) pathway of and it is also the target of certain antifungals. Insights in its function are therefore relevant for the pathogenicity and new therapeutic treatment options. TcsC was expected to be cytoplasmic, but we detected it in the nucleus and showed that it translocates to the cytoplasm upon activation. We have identified the motif that guides TcsC to the nucleus. An exchange of a single amino acid in this motif prevents a nuclear localization, but this nuclear targeting is no prerequisite for the TcsC-mediated stress response. Loss of the N-terminal 208 amino acids prevents the nuclear localization and renders TcsC unable to respond to hyperosmotic stress demonstrating that this part of the protein is of crucial importance.
PubMed: 38832777
DOI: 10.1128/mbio.01184-24