Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements

The SH3 domain of Caskin1 binds to lysophosphatidic acid suggesting a direct role for the lipid in intracellular signaling

Src homology 3 or SH3 domains constitute one of the most common protein domains in signal transduction, generally characterized by their binding to proline-rich sequences on interacting signaling proteins. Caskin1, a scaffold protein regulating cortical actin filaments, enriched in neural synapses in mammals, has an atypical SH3 domain. Key aromatic residues necessary for ligand binding that are present in canonical SH3 domains are missing from Caskin1 SH3. In concordance, proline-rich interacting partner could not be identified yet. Based on previous reports that several SH3 domains are able to bind phospholipids, we sought for lipid interacting partners of the SH3 domain of human Caskin1. We investigated the signaling-born lysophospholipid mediators, such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) as potential binding partners for this SH3 domain. These lipid mediators as first messengers activate G protein-coupled receptors. They also exert several G protein-coupled receptor-independent functions but their intracellular target proteins are mostly unknown. Here we provide evidence that the SH3 domain of human Caskin1 selectively binds to LPA in vitro. The binding strength and stoichiometry depend on the association-state of the lipid, with nanomolar affinity to LPA-containing membraneous surfaces. The amino acids involved in the interaction are located in a β-strand structure and are distinct from those corresponding to the canonical proline-rich ligand-binding groove in the SH3 domain of Src kinase. Our results suggest that the SH3 domain of human Caskin1 is a lipid-binding domain rather than a proline-rich motif interacting domain

Characterization of Native and Human Serum Albumin-Bound Lysophosphatidic Acid Species and Their Effect on the Viability of Mesenchymal Stem Cells In Vitro

Scaffolds can provide a healthy environment for cell attachment, differentiation, proliferation, and migration in vitro and in vivo. Lysophosphatidic acid (LPA) is a naturally occurring bioactive phospholipid that is present in the serum mainly bound to albumin. The present study aims to investigate the biocompatibility of LPA. It also aims to determine the effect of different LPA species on the proliferation and migration of human bone marrow-derived mesenchymal stem cells (hBM-dMSCs) for LPA and human serum albumin (HSA) containing bone scaffold development. The HSA-LPA complex formation was assessed using Fourier-transform infrared (FTIR) spectroscopy. The effect of 18:1, 18:2, or 16:0 LPA alone, or in combination with 4% HSA, on cell viability and proliferation was determined by XTT. The cell migration was examined in a wound healing assay. The changes in the FTIR spectra of LPA-HSA compositions, compared with HSA alone, indicate the complex formation between the components. Our study showed that 18:1, 18:2, and 16:0 LPA species had no cytotoxic effects up to 10 µM concentration. The different LPA species increased the proliferation of hBM-dMSCs in a dose-dependent manner when administered in the presence of HSA, without an effect on the migration of this cell type. These findings make the in vivo application of LPA-HSA complex promising for bone regeneration

Stress-Induced, p53-Mediated Tumor Growth Inhibition of Melanoma by Modulated Electrohyperthermia in Mouse Models without Major Immunogenic Effects

Modulated electrohyperthermia (mEHT), an innovative complementary technique of radio-, chemo-, and targeted oncotherapy modalities, can induce tumor apoptosis and contribute to a secondary immune-mediated cancer death. Here, we tested the efficiency of high-fever range (~42 °C) mEHT on B16F10 melanoma both in cell culture and allograft models. In vivo, mEHT treatment resulted in significant tumor size reduction when repeated three times, and induced major stress response as indicated by upregulated cytoplasmic and cell membrane hsp70 levels. Despite the increased PUMA and apoptosis-inducing factor 1, and moderate rise in activated-caspase-3, apoptosis was not significant. However, phospho-H2AX indicated DNA double-strand breaks, which upregulated p53 protein and its downstream cyclin-dependent kinase inhibitors p21waf1 and p27kip. Combined in vitro treatment with mEHT and the p53 activator nutlin-3a additively reduced cell viability compared to monotherapies. Though mEHT promoted the release of damage-associated molecular pattern (DAMP) damage signaling molecules hsp70, HMGB1 and ATP to potentiate the tumor immunogenicity of melanoma allografts, it reduced MHC-I and melan-A levels in tumor cells. This might explain why the number of cytotoxic T cells was moderately reduced, while the amount of natural killer (NK) cells was mainly unchanged and only macrophages increased significantly. Our results suggest that mEHT-treatment-related tumor growth control was primarily mediated by cell-stress-induced p53, which upregulated cyclin-dependent kinase inhibitors. The downregulated tumor antigen-presenting machinery may explain the reduced cytotoxic T-cell response despite increased DAMP signaling. Decreased tumor antigen and MHC-I levels suggest that natural killer (NK) cells and macrophages were the major contributors to tumor eradication

The SH3 domain of Caskin1 binds to lysophosphatidic acid suggesting a direct role for the lipid in intracellular signaling

Src homology 3 or SH3 domains constitute one of the most common protein domains in signal transduction, generally characterized by their binding to proline-rich sequences on interacting signaling proteins. Caskin1, a scaffold protein regulating cortical actin filaments, enriched in neural synapses in mammals, has an atypical SH3 domain. Key aromatic residues necessary for ligand binding that are present in canonical SH3 domains are missing from Caskin1 SH3. In concordance, proline-rich interacting partner could not be identified yet. Based on previous reports that several SH3 domains are able to bind phospholipids, we sought for lipid interacting partners of the SH3 domain of human Caskin1. We investigated the signaling-born lysophospholipid mediators, such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) as potential binding partners for this SH3 domain. These lipid mediators as first messengers activate G protein-coupled receptors. They also exert several G protein-coupled receptor-independent functions but their intracellular target proteins are mostly unknown. Here we provide evidence that the SH3 domain of human Caskin1 selectively binds to LPA in vitro. The binding strength and stoichiometry depend on the association-state of the lipid, with nanomolar affinity to LPA-containing membraneous surfaces. The amino acids involved in the interaction are located in a β-strand structure and are distinct from those corresponding to the canonical proline-rich ligand-binding groove in the SH3 domain of Src kinase. Our results suggest that the SH3 domain of human Caskin1 is a lipid-binding domain rather than a proline-rich motif interacting domain. © 2017 Elsevier Inc

Angiotensin II-Induced Cardiac Effects Are Modulated by Endocannabinoid-Mediated CB1 Receptor Activation

Angiotensin II (Ang II) has various cardiac effects and causes vasoconstriction. Ang II activates the type-1 angiotensin receptor—Gq/11 signaling pathway resulting in the release of 2-arachidonoylglycerol (2-AG). We aimed to investigate whether cardiac Ang II effects are modulated by 2-AG-release and to identify the role of type-1 cannabinoid receptors (CB1R) in these effects. Expression of CB1R in rat cardiac tissue was confirmed by immunohistochemistry. To characterize short-term Ang II effects, increasing concentrations of Ang II (10−9–10−7 M); whereas to assess tachyphylaxis, repeated infusions of Ang II (10−7 M) were administered to isolated Langendorff-perfused rat hearts. Ang II infusions caused a decrease in coronary flow and ventricular inotropy, which was more pronounced during the first administration. CB agonist 2-AG and WIN55,212-2 administration to the perfusate enhanced coronary flow. The flow-reducing effect of Ang II was moderated in the presence of CB1R blocker O2050 and diacylglycerol-lipase inhibitor Orlistat. Our findings indicate that Ang II-induced cardiac effects are modulated by simultaneous CB1R-activation, most likely due to 2-AG-release during Ang II signalling. In this combined effect, the response to 2-AG via cardiac CB1R may counteract the positive inotropic effect of Ang II, which may decrease metabolic demand and augment Ang II-induced coronary vasoconstriction