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Pitoulias, M.

Publications and source records attributed to Pitoulias, M..

3 recordsLinked to original sources

Targeting glucocorticoid signalling in dendritic cells for glioblastoma treatment

Glioblastoma (GBM) remains a devastating disease with few meaningful therapeutic advances over the past three decades. Dendritic cell (DC) vaccination is a promising immunotherapeutic strategy for GBM, but its efficacy is limited by the clinical use of dexamethasone to control cerebral oedema and associated symptoms. Here we show that steroid signalling is a central regulator of DC dysfunction in GBM. Through targeted metabolomics of primary GBM samples, we identified a steroid-rich tumour microenvironment in which dexamethasone is present at high levels. Across bulk and single-cell transcriptomic and epigenomic datasets, NR3C1 emerged as the dominant steroid receptor in GBM immune cells and was negatively associated with activated DC states. In patient-derived DCs, dexamethasone altered NR3C1 chromatin occupancy, induced broad transcriptional and chromatin remodelling, and suppressed co-stimulatory antigen-presentation and cytokine programmes. DC-specific deletion of Nr3c1 restricted syngeneic glioblastoma growth, enhanced DC activation, promoted cytotoxic CD8+ T cell responses and remodelled myeloid states in vivo. In GBM patient-derived DCs, pharmacological or non-viral CRISPR-mediated disruption of NR3C1 restored inflammatory, antigen presentation and T cell-stimulatory programmes, enhanced antigen-specific CD8+ T cell priming, and improved tumour lysate-loaded DC vaccination. Together, these findings identify glucocorticoid signalling as a key barrier to DC immunotherapy in GBM and establish NR3C1-targeted, steroid-resistant DCs as a potential therapeutic strategy.

immunology↗

Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation

SUMMARYCells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. Accumulating data suggest that this poorly understood control depends, at least in part, on Central Carbon Metabolism (CCM). In Bacillus subtilis, the glycolytic pyruvate kinase (PykA) is intricately linked to replication. This 585 amino-acid-long enzyme comprises a catalytic (Cat) domain that binds to phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP, and a C-terminal domain of unknown function. Interestingly, the C-terminal domain termed PEPut interacts with Cat and is homologous a domain that, in other metabolic enzymes, are phosphorylated at a conserved TSH motif at the expense of PEP and ATP to drive sugar import and catalytic or regulatory activities. To gain insights into the role of PykA in replication, DNA synthesis was analyzed in various Cat and PEPut mutants grown in a medium where the metabolic activity of PykA is dispensable for growth. Measurements of replication parameters (ori/ter ratio, C period and fork speed) and of the pyruvate kinase activity showed that PykA mutants exhibit replication defects resulting from side chain modifications in the PykA protein rather than from a reduction of its metabolic activity. Interestingly, Cat and PEPut have distinct commitments in replication: while Cat impacts positively and negatively replication fork speed, PEPut stimulates initiation through a process depending on Cat-PEPut interaction and growth conditions. Residues binding to PEP and ADP in Cat, stabilizing the Cat-PEPut interaction and belonging to the TSH motif of PEPut were found important for the commitment of PykA in replication. In vitro, PykA affects the activities of replication enzymes (the polymerase DnaE, helicase DnaC and primase DnaG) essential for initiation and elongation and genetically linked to pykA. Our results thus connect replication initiation and elongation to CCM metabolites (PEP, ATP and ADP), critical Cat and PEPut residues and to multiple links between PykA and the replication enzymes DnaE, DnaC and DnaG. We propose that PykA is endowed with a moonlighting activity that senses the concentration of signaling metabolites and interacts with replication enzymes to convey information on the cellular metabolic state to the replication machinery and adjust replication initiation and elongation to metabolism. This defines a new type of replication regulator proposed to be part of the metabolic control that gates replication in the cell cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

DNA replication initiation in Bacillus subtilis; Structural and functional characterisation of the essential DnaA-DnaD interaction

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we show that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail by NMR. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

biochemistry↗