Improved Tolerance to Freeze and Salt Stress in Mitochondria of Drosophilia melanogaster Cells Conferred by LEA Protein

Mechanisms that govern anhydrobiosis involve the accumulation of highly hydrophilic macromolecules, such as late embryogenesis abundant (LEA) proteins. Group 1 LEA proteins comprised of 181 (AfLEA1.1) and 197 (AfLEA1.3) amino acids were cloned from embryos of Artemia franciscana and expressed in Drosophila melanogaster cells (Kc167). Confocal microscopy revealed accumulations of green fluorescence protein (GFP) and AfLEA1.3 constructs in the mitochondria (AfLEA1.3-GFP) and AfLEA1.1-GFP constructs in the cytoplasm. In the presence of mixed substrates, oxygen consumption was similar for permeabilized Kc167 control and Kc167-AfLEA1.3 cells. Acute titrations of NaCl (up to 500 mM) led to successive drops in oxygen flux of permeabilized cells and were ameliorated by 18% in Kc167-AfLEA1.3 cells compared to Kc167 controls. Mitochondria were isolated from both cell types and resuspended in a sucrose-based buffer solution. The purified mitochondria from Kc167 control cells exhibited larger reductions in respiratory capacities after one freeze-thaw cycle (-80°C) compared to mitochondria isolated from Kc167-AfLEA1.3 cells. My data demonstrates that AfLEA1.3 exerts a protective influence on mitochondrial function during freezing and osmotically challenging events

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation ‘chromatin switch’ on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation

Structure and flexibility of the yeast NuA4 histone acetyltransferase complex

The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation

Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

<div><p>Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery for membrane protein targets.</p></div

Screening strategy effectively eliminates false positives.

<p>(a) Screening assays used to evaluate the effect of Spectrum Collection molecules on AβO binding. The arrows indicate the reduction in compounds resulting at each step. (b) Compiled data from the primary AlphaScreen assay are shown in a single graph normalized to POPC and SMPL in-plate controls. (c) Schematics of counterscreening assays designed to identify false positive compounds acting on off-target elements of the primary screening assay (dashed red lines). Assays use AlphaScreen donor and acceptor beads linked together by either biotinylated hexahistidine (top) or Nanodiscs containing biotinylated synaptic proteins (bottom). (d) Data from the biotinylated hexahistidine counterscreen. Black symbols denote compounds classified as likely false positives. Blue symbols denote compounds that were retested in dose-response format (Examples shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125263#pone.0125263.g007" target="_blank">Fig 7</a>), and the compounds showing significant signal reduction at 1 μM are shown as open red circles. (e) Secondary, orthogonal assays to verify compound efficacy in preventing AβO binding include a dot immunoblot test for AβO binding to rat cortical synaptosomes (Top; shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125263#pone.0125263.g008" target="_blank">Fig 8</a>) and an immunocytochemical analysis of AβO binding to cultures of rat hippocampal neurons (Bottom; shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125263#pone.0125263.g009" target="_blank">Fig 9</a> for ATA). Red squares in panels b and d identify the data points associated with ATA.</p

SMPL Nanodiscs provide the basis for a high-throughput assay for AβO binding antagonists.

<p>A schematic of the AlphaScreen assay adapted to measure AβO binding to synaptic Nanodiscs (a). Biotinylated AβOs and His-tagged MSP molecules link Nanodiscs to AlphaScreen donor and acceptor beads. The proof-of-concept assay produces high dynamic range (b). NU2 oligomer-specific antibodies were used as a drug stand-in to test the assay’s response to an applied treatment (c).</p

Synaptic Nanodiscs contain an AβO binding protein that interacts selectively with high molecular weight oligomers.

<p>A Nanodisc-based assay for AβO binding indicates the transfer of the AβO binding site into Nanodiscs (a) (n = 3; mean +/- SD; * p<0.05). Nanodiscs containing a range of detergent-solubilized precursor membranes or lipid mixtures were applied to the AβO binding assay (b; SPM—synaptic plasma membrane, Trypsinized SPM—SPM treated with trypsin prior to Nanodisc incorporation, Synaptic Lipids—lipids extracted from SPM, POPC—100% POPC lipid, PC/PS—1:1 POPC:POPS; n = 3; mean +/- SD; * p<0.05). AβOs were separated into populations smaller (diagonal fill) and larger (horizontal fill) than 50 kDa and assayed for binding to synaptosomes (c) and Nanodiscs (d). (n = 2; mean +/- SD; * p<0.05)</p

Nanodiscs preserve synaptic protein composition and structure.

<p>(a) Schematic of SMPL Nanodisc formation using synaptic plasma membranes. (b) Nanodiscs containing biotinylated synaptic membranes (solid curve) or POPC (dashed curve) were separated by size exclusion chromatography. Fractions eluting from the synaptic Nanodisc run were collected and analyzed by dot blot to locate biotinylated synaptic proteins (red curve; Mean +/-SD; n = 2). The population of biotinylated membrane proteins inserted into Nanodiscs was analyzed by SDS-PAGE, probing for biotin (c) or using antibodies against specific proteins related to AβO binding (d). ³H glutamate binding to SMPL Nanodiscs was assessed in the absence and presence of a 100-fold excess of cold glutamate (Mean +/- SD; n = 3; * p<0.05) (e). Enzymatic activity was assessed in synaptic plasma membranes (Syn PM) and SMPL Nanodiscs (SMPL) by probing for tyrosine phosphorylation in the absence and presence of ATP (f). Insulin receptor activity was probed using an antibody recognizing IR_βpTyr^1162/1163 (g).</p

Impact of selected compounds on synaptosome binding.

<p>Selected compounds were repurchased and tested for an impact on AβO binding to rat cortical synaptosomes in a dot immunoblot assay. n = 3; Mean+/-SEM.</p

AβO binding is receptor-mediated but PrP^C-independent in Nanodiscs and mature neurons.

<p>Immobilized SMPL Nanodiscs were titrated with AβOs and bound AβOs were detected with NU2 oligomer-specific antibody coupled to an HRP-based colorimetric assay. Nonspecific binding was measured in the presence of excess ATA, which blocks AβO/receptor binding, and used to calculate the specific binding component (blue). n = 3; mean +/- SD. (a). To analyze Nanodisc proteins co-immunoprecipitating with AβOs, Nanodiscs containing biotinylated synaptic plasma membranes were affinity precipitated and visualized by SDS-PAGE immunoblot with biotin detection (b). To test the prediction that PrP^C mediates AβO binding, immobilized Nanodiscs were split into four equivalent reactions and pre-treated with 0, 0.05, 0.1, or 0.2 units of PIPLC to remove PrP^C before exposing to AβOs and probing with NU2 as in (a). PrP^C removal was verified by Western blotting after the colorimetric assay was complete. NU2 bound to each immobilized Nanodisc/AβO complex is detected in the blot by anti-mouse secondary antibodies used to probe for the mouse antibody against PrP^C (c). The effect of PrP^C removal on AβO binding was tested using mature hippocampal cultures treated with PIPLC (d). PrP^C was detected using an antibody (red) and fluorescence-conjugated AβOs were visualized directly (green).</p