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Most organophosphates (OPs) are hydrophobic, and after exposure, can sequester into lipophilic regions within the body, such as adipose tissue, resulting in long term chronic effects. Consequently, there is an urgent need for therapeutic agents that can decontaminate OPs in these hydrophobic regions. Accordingly, an enzyme-polymer surfactant nanocomplex is designed and tested comprising chemically supercharged phosphotriesterase (Agrobacterium radiobacter; arPTE) electrostatically conjugated to amphiphilic polymer surfactant chains ([cat.arPTE][S-]). Experimentally-derived structural data are combined with molecular dynamics (MD) simulations to provide atomic level detail on conformational ensembles of the nanocomplex using dielectric constants relevant to aqueous and lipidic microenvironments. These show the formation of a compact admicelle pseudophase surfactant corona under aqueous conditions, which reconfigures to yield an extended conformation at a low dielectric constant, providing insight into the mechanism underpinning cell membrane binding. Significantly, it demonstrated that [cat.arPTE][S-] spontaneously binds to human mesenchymal stem cell membranes (hMSCs), resulting in on-cell OP hydrolysis. Moreover, the nanoconstruct can endocytose and partition into the intracellular fatty vacuoles of adipocytes and hydrolyze sequestered OP.

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Microglial cells are recognized as very dynamic brain cells, screening the environment and sensitive to signals from all other cell types in health and disease. Apolipoprotein D (ApoD), a lipid-binding protein of the Lipocalin family, is required for nervous system optimal function and proper development and maintenance of key neural structures. ApoD has a cell and state-dependent expression in the healthy nervous system, and increases its expression upon aging, damage or neurodegeneration. An extensive overlap exists between processes where ApoD is involved and those where microglia have an active role. However, no study has analyzed the role of ApoD in microglial responses. In this work, we test the hypothesis that ApoD, as an extracellular signal, participates in the intercellular crosstalk sensed by microglia and impacts their responses upon physiological aging or damaging conditions. We find that a significant proportion of ApoD-dependent aging transcriptome are microglia-specific genes, and show that lack of ApoD in vivo dysregulates microglial density in mouse hippocampus in an age-dependent manner. Murine BV2 and primary microglia do not express ApoD, but it can be internalized and targeted to lysosomes, where unlike other cell types it is transiently present. Cytokine secretion profiles and myelin phagocytosis reveal that ApoD has both long-term pre-conditioning effects on microglia as well as acute effects on these microglial immune functions, without significant modification of cell survival. ApoD-triggered cytokine signatures are stimuli (paraquat vs. Aß oligomers) and sex-dependent. Acute exposure to ApoD induces microglia to switch from their resting state to a secretory and less phagocytic phenotype, while long-term absence of ApoD leads to attenuated cytokine induction and increased myelin uptake, supporting a role for ApoD as priming or immune training factor. This knowledge should help to advance our understanding of the complex responses of microglia during aging and neurodegeneration, where signals received along our lifespan are combined with damage-triggered acute signals, conditioning both beneficial roles and limitations of microglial functions.

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Small 30-nm vesicles containing the integral membrane protein Atg9 provide the initial membrane source for autophagy in yeast. Atg23 is an Atg9 binding protein that is required for Atg9 vesicle trafficking but whose exact function is unknown. In our recent paper, we explored the function of Atg23 using an approach combining cellular biology and biochemistry on purified protein. We determined that Atg23 is an elongated dimer spanning 320 Å in length. We also demonstrated that Atg23 is a membrane-binding and -tethering protein. Furthermore, we identified a series of amino acids residing in a putative coiled-coil region that when mutated prevent Atg23 dimer formation resulting in a stable Atg23 monomer. Last, we demonstrated that when monomeric Atg23 is expressed in yeast lacking Atg23, this leads to a loss of Atg23 puncta, a reduction in Atg9 puncta, a reduction in nonselective autophagy and a complete block in the cytoplasm-to-vacuole targeting (Cvt) pathway.

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Cerebral stroke is one of the leading causes of death in adults worldwide. However, the molecular mechanisms of stroke-induced neuron injury are not fully understood. Here, we obtained phosphoproteomic and proteomic profiles of the acute ischemic hippocampus by LC-MS/MS analysis. Quantitative phosphoproteomic analyses revealed that the dysregulated phosphoproteins were involved in synaptic components and neurotransmission. We further demonstrated that phosphorylation of Synaptotagmin-1 (Syt1) at the Thr112 site in cultured hippocampal neurons aggravated oxygen-glucose deprivation-induced neuronal injury. Immature neurons with low expression of Syt1 exhibit slight neuronal injury in a cerebral ischemia model. Administration of the Tat-Syt1T112A peptide protects neurons against cerebral ischemia-induced injury in vitro and in vivo. Surprisingly, potassium voltage-gated channel subfamily KQT member 2 (Kcnq2) interacted with Syt1 and Annexin A6 (Anxa6) and alleviated Syt1-mediated neuronal injury upon oxygen-glucose deprivation treatment. These results reveal a mechanism underlying neuronal injury and may provide new targets for neuroprotection after acute cerebral ischemia onset.

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ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

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Integrin receptors regulate normal cellular processes such as signaling, cell migration, adhesion to the extracellular matrix, and leukocyte function. Talin recruitment to the membrane is necessary for its binding to and activation of integrin. Vertebrates have two highly conserved talin homologs that differ in their expression patterns. The F1-F3 FERM subdomains of cytoskeletal proteins resemble a cloverleaf, but in talin1, its F1 subdomain and additional F0 subdomain align more linearly with its F2 and F3 subdomains. Here, we present the talin2 crystal structure, revealing that its F0-F1 di-subdomain displays another unprecedented constellation, whereby the F0-F1-F2 adopts a new cloverleaf-like arrangement. Using multiangle light scattering (MALS), fluorescence lifetime imaging (FLIM), and FRET analyses, we found that substituting the corresponding residues in talin2 that abolish lipid binding in talin1 disrupt the binding of talin to the membrane, focal adhesion formation, and cell spreading. Our results provide the molecular details of the functions of specific talin isoforms in cell adhesion.

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Vibrio cholerae cytolysin (VCC) is a membrane-damaging protein toxin with potent cytolytic/cytotoxic activity against wide range of eukaryotic cells. VCC is a ß-barrel pore-forming toxin (ß-PFT), and it inflicts damage to the target cell membranes by forming transmembrane heptameric ß-barrel pores. To exert pore-forming activity, VCC must bind to the cell membranes in an efficient manner. Efficient interaction with the cell membranes is an essential pre-requisite to trigger subsequent structural/conformational and organizational changes in the toxin molecules leading toward formation of the transmembrane oligomeric ß-barrel pores. Based on the large numbers of studies investigating the mode of action of VCC, it is now evident that VCC is capable of using multiple distinct mechanisms to recognize and bind to the membrane components and cell surface molecules. In this review article, we present an overview of our current understanding regarding the membrane interaction mechanisms of VCC, and their functional implications for the pore-forming activity of the toxin. © 2018 IUBMB Life, 70(4):260-266, 2018.

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Phox-homology (PX) domains target proteins to the organelles of the secretary and endocytic systems by binding to phosphatidylinositol phospholipids (PIPs). Among all the structures of PX domains that have been solved, only three have been solved in a complex with the main physiological ligand: PtdIns3P. In this work, molecular dynamic simulations have been used to explore the structure and dynamics of the p40(phox) -PX domain and the SNX17-PX domain and their interaction with membrane-bound PtdIns3P. In the simulations, both PX domains associated spontaneously with the membrane-bound PtdIns3P and formed stable complexes. The interaction between the p40(phox) -PX domain and PtdIns3P in the membrane was found to be similar to the crystal structure of the p40(phox) -PX-PtdIns3P complex that is available. The interaction between the SNX17-PX domain and PtdIns3P was similar to that observed in the p40(phox) -PX-PtdIns3P complex; however, some residues adopted different orientations. The simulations also showed that nonspecific interactions between the ß1-ß2 loop and the membrane play an important role in the interaction of membrane bound PtdIns3P and different PX domains. The behaviour of unbound PtdIns3P within a 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) membrane environment was also examined and compared to the available experimental data and simulation studies of related molecules.

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