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Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+‐releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two‐pore channels 1 (TPC1) and 2 (TPC2). NAADP‐induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony‐forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly–Phe β‐naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol‐1,4,5‐trisphosphate (InsP3)‐sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP‐induced Ca2+ release also required ER‐embedded InsP3 receptors. Finally, NAADP‐induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor‐induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine‐5′‐trisphosphate‐induced Ca2+ signaling. These findings demonstrated that NAADP‐induced TPC1‐mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs. [ABSTRACT FROM AUTHOR]

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold–sulfur bonds (Au–S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond. [ABSTRACT FROM AUTHOR]

The neurotransmitter glutamate increases cerebral blood flow (CBF) by activating postsynaptic neurons and presynaptic glial cells within the neurovascular (NVU) unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+]i) in the target cells, which activates the Ca2+/Calmodulin-dependent NO synthase (NOS) to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+]i and NO production. The present study assessed whether and how glutamate drives Ca2+-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose dependent oscillatory increase in [Ca2+]i, which was maximally activated at 200 µM and inhibited by MCPG, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by inositol-1,4,5-trisphosphate-sensitive receptors and NAADP-gated two-pore channel 1 (TPC1). Constitutive SOCE mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ wave. Of note, glutamate induced Ca2+-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain. [ABSTRACT FROM AUTHOR]

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca 2+ -releasing messenger that increases the intracellular Ca 2+ concentration by mobilizing the lysosomal Ca 2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca 2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca 2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca 2+ store and TPC1 in circulating ECFCs by harnessing Ca 2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca 2+ sequestration by acidic organelles, caused endogenous Ca 2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP 3 )-sensitive endoplasmic reticulum (ER) Ca 2+ pool. Likewise, the amount of ER releasable Ca 2+ was reduced by disrupting lysosomal Ca 2+ content. Liposomal delivery of NAADP induced a transient Ca 2+ signal that was abolished by disrupting the lysosomal Ca 2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca 2+ release also required ER-embedded InsP 3 receptors. Finally, NAADP-induced lysosomal Ca 2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca 2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca 2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca 2+ release can selectively be recruited to induce the Ca 2+ response to specific cues in circulating ECFCs., (© 2020 Wiley Periodicals LLC.)

Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca 2+ release. NAADP-induced EL Ca 2+ signals may be amplified by the endoplasmic reticulum (ER) through the Ca 2+ -induced Ca 2+ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca 2+ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca 2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca 2+ refilling of acidic organelles, caused massive Ca 2+ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP₃)-sensitive ER Ca 2+ store. Liposomal delivery of NAADP induced a transient Ca 2+ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca 2+ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP₃ receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca 2+ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.