Unique acyl chains mark phosphoinositides for recycling.

Two recent complementary studies show that, after phospholipase C cleavage, the characteristic acyl chain composition of phosphoinositide-derived diacylglycerol funnels them back into the PI cycle.

Metabolic Labeling-Based Chemoproteomics Establishes Choline Metabolites as Protein Function Modulators.

Choline is an essential nutrient for mammalian cells. Our understanding of the cellular functions of choline and its metabolites, independent of their roles as choline lipid metabolism intermediates, remains limited. In addition to fundamental cellular physiology, this knowledge has implications for cancer biology because elevated choline metabolite levels are a hallmark of cancer. Here, we establish a mammalian choline metabolite-interacting proteome by utilizing a photocrosslinkable choline probe. To design this probe, we performed metabolic labeling experiments with structurally diverse choline analogues that resulted in the serendipitous discovery of a choline lipid headgroup remodeling mechanism involving sequential dealkylation and methylation steps. We demonstrate that phosphocholine inhibits the binding of one of the proteins identified, the attractive anticancer target p32, to its endogenous ligands and to the promising p32-targeting anticancer agent, Lyp-1. Our results reveal that choline metabolites play vital roles in cellular physiology by serving as modulators of protein function.

All in One: Stimuli-Responsive, Efficient Mitotracking, and Single Source White Light Emission.

"All in one" type luminogens, possessing combined properties related to optical, materials, and biological implications, are of urgent demand today, mainly because of the combined application potential of such probes. To the best of our knowledge, until now, an "all in one" type white light emitter together with stimuli-responsive behavior and highly efficient mitochondrial-tracking ability has not been reported yet. In this contribution, for the first time, we have investigated a pair of luminogens exhibiting white light emission (CIE coordinates: 0.35, 0.35 (DPAEOA) and 0.29, 0.33 (DPAPMI)) with temperature-induced mechanochromic features of a centrosymmetrically packed probe (space group P-1). Most importantly, despite being neutral, our designed probe DPAEOA can specifically illuminate mitochondria with the highest Pearson coefficient value (0.93), which is rare, as almost all the commercially developed mitotrackers are cationic fluorophores. Thus, this study will pave a new avenue for the design of next generation "all in one" type organic luminogens exhibiting potential applications in notable optical, materials, and biological fields.

Sensing and modulation of amyloid fibrils by photo-switchable organic dots.

Abnormal aggregation of amyloidogenic proteins (like Aß 42, amylin, α-synuclein, insulin) and the deposition of these aggregates is believed to be associated with several diseases known as amyloidosis. The pathway of aggregation involves three distinct phases: the oligomeric, elongation and plateau phases. Among them, the oligomeric phase of Aß 42 and α-synuclein involves the generation of transient oligomeric species suspected to cause several neurological disorders, including Alzheimer's and Parkinson's diseases. Over the past few years, scientists have devoted much more effort to devising new fluorescent molecular probes to estimate the mechanisms of formation, and have gained vital information about possible therapeutic routes for amyloidosis. However, such fluorescent probes face serious limitations because of self-quenching at high concentrations of the probe; therefore, they are inappropriate for quantitative analysis and bio-imaging experiments. Hence, smart biocompatible fluorescent probes are indispensable, as they not only overcome the drawbacks of conventional fluorescent probes, but also have the potential ability to fight amyloidosis through modulation of the pathways involved. In this work, for the first time we introduce a series of promising photo-switchable aggregation-induced emission (AIE) dots (DPAPMI, CPMI) and aggregation caused quenching (ACQ) dots (DMAPMI) which can detect amyloid fibrils in terms of switching and enhancing their fluorescence emission. Interestingly, the organic dots enhance the aggregation rate of insulin by speeding up the microscopic processes, specifically secondary nucleation (with rate constant k2) and the elongation process (with rate constant k+). Moreover, the comparison of kinetics studies with ThT suggests that our organic dots can sense pre-fibrillar aggregates of insulin during the aggregation process, which may be beneficial for the early detection of amyloid fibrils. In summary, our study indicates that these organic dots can be used for the imaging and early stage detection of amyloid fibril formation and the modulation of amyloid formation pathways.

PLiMAP: Proximity-Based Labeling of Membrane-Associated Proteins.

Peripheral membrane proteins participate in numerous biological pathways. Thus, methods to analyze their membrane-binding characteristics have become important. In this report, we detail protocols for the synthesis and utilization of a photoactivable fluorescent lipid as a reporter to monitor membrane binding of proteins. The assay, referred to as proximity-based labeling of membrane-associated proteins (PLiMAP), is based on UV activation of a fluorescent lipid reporter, which in turn crosslinks with proteins bound to membranes and renders them fluorescent. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of BODIPY-diazirine phosphatidylethanolamine (BDPE) Basic Protocol 2: Preparation of BDPE-containing liposomes Basic Protocol 3: Performing PLiMAP with a candidate protein Basic Protocol 4: Quantitation of liposome-binding properties of the candidate protein from analyzing in-gel fluorescence Support Protocol: Purification of GST-2×P4M domain of SidM protein.

A facile, sensitive and quantitative membrane-binding assay for proteins.

Soluble proteins that bind membranes function in numerous cellular pathways yet facile, sensitive and quantitative methods that complement and improve sensitivity of widely used liposomes-based assays remain unavailable. Here, we describe the utility of a photoactivable fluorescent lipid as a generic reporter of protein-membrane interactions. When incorporated into liposomes and exposed to ultraviolet (UV), proteins bound to liposomes become crosslinked with the fluorescent lipid and can be readily detected and quantitated by in-gel fluorescence analysis. This modification obviates the requirement for high-speed centrifugation spins common to most liposome-binding assays. We refer to this assay as Proximity-based Labeling of Membrane-Associated Proteins (PLiMAP).

Copper(II) complex of methionine conjugated bis-pyrazole based ligand promotes dual pathway for DNA cleavage.

Three Cu(II) complexes of bis-pyrazole based ligands have been synthesized and structurally characterized by X-ray crystallography. One of the ligand (L2) contains a methionine ester conjugated to a bis-pyrazole carboxylate through an amide linkage. The binding constant for complexes 1-3 with CT DNA are of the order of 10(4) M(-1). The crystal structure suggests that the axial Cu-O bonds (ca. 2.31(4) Å) are relatively labile and hence during the redox cycle with ascorbic acid and oxygen one or both the axial Cu-O bonds might open to promote copper oxygen reaction and generate ROS. The chemical nuclease activity of complexes 1-3 in dark, show complete relaxation of supercoiled DNA at 100 µM concentration in presence of ascorbic acid (H2A). The mechanistic investigation suggests that the complexes 1 and 2 show involvement of peroxo species whereas 3 shows involvement of both singlet oxygen and peroxo species in DNA cleavage. The singlet oxygen formation in dark is otherwise unfavourable but the presence of methionine as pendant arm in L2 might activate the generation of singlet oxygen from the metal generated peroxo species. The results of DNA cleavage studies suggest that methionine based copper(II) complexes can promote dual pathway for DNA cleavage. Probing the cytotoxic activity of these complexes on MCF-7, human breast cancer cell line shows that 3 is the most effective one with an IC50 of 70(2) µM.

Histidine based fluorescence sensor detects Hg2+ in solution, paper strips, and in cells.

A chemosensor having a bipodal thiocarbamate scaffold attached to histidine moieties senses Hg(2+) with a remarkable selectivity. The binding results in a 50 nm blue shift in the fluorescence spectra and a 19-fold enhancement of the fluorescence quantum yield of the ligand. In addition to the detection of Hg(2+) visually under UV light in solution, the chemosensor was used for fabrication of paper strips that detected Hg(2+) in aqueous samples. The sensor was also used for imaging Hg(2+) in adult zebrafish and in human epithelial carcinoma HeLa S3 cells.

Singlet oxygen mediated DNA degradation by copper nanoparticles: potential towards cytotoxic effect on cancer cells.

The DNA degradation potential and anti-cancer activities of copper nanoparticles of 4-5 nm size are reported. A dose dependent degradation of isolated DNA molecules by copper nanoparticles through generation of singlet oxygen was observed. Singlet oxygen scavengers such as sodium azide and Tris [hydroxyl methyl] amino methane were able to prevent the DNA degradation action of copper nanoparticles confirming the involvement of activated oxygen species in the degradation process. Additionally, it was observed that the copper nanoparticles are able to exert cytotoxic effect towards U937 and Hela cells of human histiocytic lymphoma and human cervical cancer origins, respectively by inducing apoptosis. The growth characteristics of U937 and Hela cells were studied applying various concentrations of the copper nanoparticles.