Biophysical Chemistry

Glycation, a non-enzymatic reaction occurring between sugars and biological macromolecules, plays a critical role in ageing and disease pathogenesis. Methylglyoxal (MG) is a highly reactive -oxoaldehyde that leads to the formation of endogenous advanced glycation end products (AGEs). These AGEs are associated with diabetes and many other diseases, including neurodegeneration and cancer. This is often through interactions with the receptor for advanced glycation end products (RAGE). Inhibition of glycation/AGEs formation using natural products to target cancer is an area of recent interest. In vitro AGEs formation was observed by browning of samples, increased fluorescence, and carbonyl stress. MG induced changes in the structure of BSA were analysed using electrophoresis, spectroscopy, TEM, AFM, DLS, and CD spectroscopy. Our results show that AGEs form random structures, oligomeric aggregates, and {beta}-sheets. Thioflavin T and Congo red staining further validated these findings. Galangin and Caffeic acid demonstrated significant antiglycation activity, suppressing AGEs formation in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/737425v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@113b391org.highwire.dtl.DTLVardef@7208a1org.highwire.dtl.DTLVardef@94c2e1org.highwire.dtl.DTLVardef@867b85_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMethylglyoxal-induced Advanced Glycation End Products were prepared in vitro C_LIO_LIMethylglyoxal -induced structural modifications in BSA C_LIO_LIAGEs were characterised using various parameters C_LIO_LIBoth fluorescent and non-fluorescent AGEs were formed. C_LIO_LIPhytochemical treatment induced inhibition of AGEs formation C_LI

The aggregation of -synuclein (Syn) is a molecular hallmark of Parkinson's disease (PD) and other synucleinopathies. Understanding the molecular mechanisms that determine the aggregation of this protein may thus facilitate the development of disease-modifying therapies. While Syn is most commonly expressed as a 140-residue protein (Syn-140), recent evidence suggests an involvement of alternatively spliced Syn isoforms in disease onset and progression. Here, we report and characterise the interaction between Syn-140 and the aggregation-prone Syn-112 variant, one of the most abundant Syn splice isoforms. We found that amounts as low as 1% of Syn-112 accelerate the nucleation and aggregation of Syn-140. To further investigate this phenomenon, we employed MALDI-MS and NMR spectroscopy, confirming that Syn-140 and Syn-112 monomers interact strongly with one another. Furthermore, to assess the association of Syn-112 with disease pathology, we performed immunohistochemical staining combined with confocal microscopy on PD brain samples. Thereby, we found an increase in the number as well as the area of Syn-112 immunoreactive aggregates compared to healthy controls. These results illustrate how low-abundance Syn splice isoforms can modulate the aggregation landscape of Syn-140 and in turn contribute to the molecular heterogeneity of synucleinopathies.

Production of recombinant proteins frequently yields inclusion bodies that must undergo refolding to yield active protein. Here, we optimized the refolding conditions for the recombinant leucyl aminopeptidase (rPepL) from Lactocaseibacillus casei expressed in inclusion bodies from E. coli. Several chemical additives were assessed for how well they facilitated an increase in refolding efficiency. The best, 0.5 M L-arginine, yielded 50.8% refolding. The addition of stabilizers, such as sucrose and glycerol, with L-arginine further increased yields to 85%. Urea at lower concentrations (0.25-0.5 M) also facilitated an increase in the refolding yield when co-added with L-arginine, whereas guanidinium chloride inhibited it. Sugars and polyols exhibited dose-dependent effects, with ranges for optima also defined. Fluorescence spectroscopy verified enhancements in the refolding under the optimized conditions. Molecular dynamics simulation under mixed solvent conditions provided atomic insights about stabilizing interactions that are likely to facilitate increased refolding. The results show that a series of aggregation suppressors and protein stabilizers can, in a collaborative way, increase the refolding efficiency for the recombinant proteins from the inclusion bodies. The protocol with the optimization using the additives L-arginine, sucrose, and glycerol is an efficient method for the production of active rPepL. This article outlines the best refolding method to recover recombinant leucyl aminopeptidase from inclusion bodies of E. coli using L-arginine combined with sucrose and glycerol. The combined experimental observations and computational simulations elucidate the molecular process of additive-induced stabilization, which elucidates how aggregation inhibition and hydrogen-bonded stabilization act synergistically. The results presented herein answer both mechanistic understanding and experimental guidance for improving protein refolding.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

The protozoan parasite Trypanosoma brucei is implicated in deadly African sleeping sickness. Experimental studies show that T. brucei codes for three aquaporin homologs (TbAQP1 to TbAQP3). TbAQP2 has been established as the high affinity drug transporter of drugs pentamidine and melarsoprol. Mutation in TbAQP2 or its loss result in pentamidine-melarsoprol cross-resistance. TbAQP2 is also shown to transport water, glycerol and other solutes to respond to osmoregulation in the infected hosts or glycerol metabolism. Experimentally determined structures of TbAQP2 shows that it adopts the same aquaporin-like hourglass helical fold. However, the so called aromatic/arginine selectivity filter (Ar/R SF) in TbAQP2 has neither arginine nor aromatic residue and all four residues are hydrophobic. Mutation and functional studies have demonstrated the role of Ar/R SF residues in the transport and selectivity of solutes in aquaporin homologs. The intriguing question is how the completely hydrophobic Ar/R SF region enables the transport of water and glycerol molecules. In this study, we used computational approach to elucidate the molecular mechanism of water and glycerol transport. Our equilibrium molecular dynamics simulations showed that the number of water molecules transported by TbAQP2 is almost one order of magnitude higher than that of prototype water channel AQP1. Moreover, the residence time within TbAQP2 channel is much less compared to that found in AQP1. The relatively wider constriction, interactions of water molecules with the selectivity filter residues and the contact duration, all contribute to a large number of water molecules transported through TbAQP2 channel. Our umbrella sampling studies show that when glycerol is transported through TbAQP2, it participates in interactions with channel residues that can be considered as complimentary to that observed in prototype glycerol transporter GlpF. Our studies reveal the molecular mechanism of water and glycerol transport in TbAQP2 and establish that TbAQP2 is an efficient water transporter. Statement of SignificanceTrypanosoma brucei causes African sleeping sickness and a homolog of aquaporin, TbAQP2, is involved in the transport of drugs that are used to treat this disease. Developing anti-parasitic drugs requires the knowledge of molecular mechanism of the proteins function. TbAQP2 has been shown to transport water and glycerol. Permeating solutes have to pass through a narrow constriction region formed by all hydrophobic residues. In the present study, equilibrium molecular dynamics simulations showed that TbAQP2 transports water molecules faster in large quantity in comparison with mammalian AQP1. Higher water transport is due to relatively wider constriction and minimum water interactions with selectivity filter hydrophobic residues. Permeating glycerol molecule is involved in complementary interactions with the channel residues. Our studies reveal how water and glycerol are transported through hydrophobic selectivity filter in TbAQP2.

Magnetic treatment in tomato seeds (Solanum lycopersicum L.) has been studied as a biotechnological technique to induce a reduction in germination times and enhance plant development. However, the modified cellular mechanisms involved in the reduction of germination times or the improvement of development parameters are not yet clearly established. To explore a possible altered cellular mechanism, the effect of homogeneous static magnetic fields on the structure of the cyclic nucleotide-gated channel 6 (CNGC6), the modification in the organization of POPC lipids in the plasma membrane, and changes in calcium ion mobility were evaluated. For this purpose, coarse-grained molecular dynamics simulations were performed using the Martini 3 model in GROMACS, applying five different magnetic flux densities (0.000, 0.001, 0.010, 0.100, 1.000, and 10.000) T over 1 000 ns. The results showed an anisotropic effect in the longitudinal direction of the protein, which generated heterogeneous behavior among the chains of the homotetramer; this altered the conformation of the CNGC6 channel and modified the pore bottleneck. In contrast, no significant changes were observed in the conformational order of the POPC phospholipid chains. As a preliminary, single-replicate exploratory study, these results suggest that homogeneous static magnetic fields may induce specific structural modifications in the CNGC6 ion channel of Solanum lycopersicum L. without compromising the integrity of the lipid bilayer or the dynamics of ion transport within the analyzed timescale; these preliminary findings provide a molecular-level structural basis for future experimental and computational investigations of magnetic field effects on plant cyclic nucleotide-gated channels.

Sickle cell disease (SCD) is caused by a single amino acid substitution in the {beta}S globin chain at 6th position (6E[->]V). This results in polymerization of deoxy state of sickle hemoglobin (HbS), followed by its precipitation and subsequent sickling of erythrocytes. These deformed cells can block small capillaries (vaso-occlusion), causing cardiovascular complications, ultimately leading to ischemia-reperfusion injury, severe oxygen deficiency, and progressive systemic damage. Occasionally, patients with SCD have been observed to produce exorbitantly high levels of fetal hemoglobin (HbF), which has been linked with the inhibition of HbS polymerization. One of the effects of hydroxyurea, the most commonly used therapeutic for SCD, is to elevate HbF levels. However, the mechanism of inhibitory role of HbF on HbS polymerization is largely unknown. This study attempts to gain insights into the mechanisms involved in this process by means of native mass spectrometry, ion mobility mass spectrometry, and hydrogen deuterium exchange-based mass spectrometry (H/DX-MS). The conformational flexibility of asymmetric hemoglobin, HbFS (2{gamma}{beta}S), for the observed regions in the tetrameric molecule appears to be more in the deoxy state as compared to the oxy state, eventually leading to reduced polymerization of sickle hemoglobin in patients with SCD that express elevated HbF levels.

Mechanistic elucidation and inhibition of the pathogenic aberrant mitochondrial localization of the RNA/DNA-binding protein, TDP-43, can help in the therapeutics of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A mitochondrial localization sequence of TDP-43, M1, is largely solvent inaccessible, therefore, how it interacts with the mitochondrial import machinery to facilitate TDP-43s transit to mitochondria is unclear. Towards this, we examined the unfolding TDP-43s N-terminal domain (NTD) that hosts M1, using equilibrium all-atom molecular dynamics (MD) simulations, and observed an early loss of the hydrogen-bonded interactions between {beta}4-{beta}5 bridge and the interactions involving residues Phe-35 and Gly-40 of M1, indicating structural lability of M1 to become solvent-accessible that may enhance its interaction with the mitochondrial receptor(s) for import. Furthermore, via virtual screening of 2,115 FDA-approved and 515,545 non-FDA-approved small molecules from ZINC15 database towards binding to M1 and inhibiting TDP-43s mitochondrial import, we identified a molecule, ZINC73240059, that was previously characterized as an inhibitor of MAP kinase-activating protein kinase 2 (MAPKAPK2). ZINC73240059 remains stably bound to M1 of NTD during MD simulations manifesting negative Gibbs free energy ({Delta}G) with significant contribution from Pro-36 of M1. Overall, ZINC73240059 can be a molecule of interest towards thwarting TDP-43s pathogenic mitochondrial localization in ALS.

The Tar-DNA Binding Protein-43 C-terminal region, TDP43LC, has been previously shown to form amyloid-like fibrils with distinct folds in ALS and FTD. In both diseases, proteinaceous inclusions contain TDP43 C-terminal protein fragments as well as phosphorylated TDP43. Here, we use solution NMR to show that soluble phosphomimetic TDP43LC, P-TDP43LC, is structurally similar to wild-type TDP43LC. Disperse P-TDP43LC, like wild-type protein, contains a central helical region flanked by long disordered regions. Despite this similarity, our turbidity measurements, imaging, and kinetic assays show that P-TDP43LC has different aggregation behavior than wild-type protein. Using solid state NMR measurements we find that that phosphomimetic mutations alter the wild-type fibril conformation. Electrostatic repulsion from negatively charged sidechains, despite having little effect on the soluble proteins structure, perturbs amyloid-like fibril formation and selects for a different conformation in vitro. These results shed light on the structural role of TDP43LC phosphorylation in fibril formation in disease. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/725298v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1c63aforg.highwire.dtl.DTLVardef@1d48ed6org.highwire.dtl.DTLVardef@1ed8fd3org.highwire.dtl.DTLVardef@17d67a8_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisPhosphomimetic mutations at ALS and FTD neurodegeneration-associated sites in an amyloid forming protein perturbs the aggregated structure compared to wild-type protein.

Eukaryotic cap-dependent translation initiation is regulated by binding of the predominantly folded eukaryotic initiation factor 4E (eIF4E) to the intrinsically disordered eIF4E binding proteins (4E-BPs). Here, we report full-length atomistic conformational ensembles generated by IDPConformerGenerator and optimized by X-EISDv2 workflow for both apo 4E-BP2, the neuronal 4E-BP, and 4E-BP2 in complex with eIF4E, using data from single-molecule fluorescence and nuclear magnetic resonance (NMR), together with select coordinates from a 4E-BP1:eIF4E crystal structure. Structural sampling within dynamic complexes is often under-appreciated, with NMR and crystal structure data for 4E-BP:eIF4E suggesting different degrees of structural heterogeneity. Our ensemble models validated by solution spectroscopy data enable comparison of free 4E-BP2 and its complex with eIF4E. This shows a delocalization of contacts around canonical regions, which supports previous findings of unidirectional conditional occupancy of the binding sites. Two new contact regions emerged: one between the disordered N-termini of eIF4E and 4E-BP2, which may play an allosteric role in tuning the binding affinity, and the other between the C-terminus of 4E-BP2 and an extended region of eIF4E, which is consistent with the extended, dynamic binding interface that we reported previously. These results support a model of translation regulation in which the dynamic 4E-BP2:eIF4E complex facilitates accessibility of regulatory sites of 4E-BP2 when bound.

Glioblastoma is a very aggressive brain tumor with few therapeutics options. Type I and II Interferons (IFNs) co-formulation HeberFERON has been used in cancer treatment, with promising results in high grade brain tumors. High throughput techniques in easy-to-handle models have been important to interrogate biomolecules changes, describe mechanisms and find pharmacodynamic biomarkers. This study aims to elucidate the effect of HeberFERON over the cell proteome in comparison to its individual IFNs components. Proteomic changes with HeberFERON in the glioblastoma-derived cell line U-87MG, in comparison with individual IFN-2b and IFN-{gamma}, were studied using a nanoLC instrument EasyLC coupled to Velos Pro mass spectrometer; Maxquant and Perseus were also used. Several enrichment tools, networking analysis and canSAR for drug targets were employed. Translation, RNA processing, mitotic cell cycle, cytoskeleton and chromosome organization, apoptosis, autophagy, DNA repair are enriched to limit cellular growing together with changes in immune response components, supporting HeberFERON as a multitarget treatment. This co-formulation is distinguished at modulating RNA splicing with SMN complex, cytoskeleton organization and microtubule-based movement, nuclear envelope breakdown, DNA conformational changes, and oxidative phosphorylation, with a better drawing of effects over a variety of systems inside the tumoral cell. Together with previous microarray experiment, informative genes and proteins as pharmacodynamic biomarkers for antiproliferative effects showed up (ex. STAT1/2, CENPE, ATRIP, MAP1B, LIMA1, VCP, several ribosomal, spliceosome and proteasomal complexes proteins). This study complements transcriptomic and phosphoproteomic previous experiments in this model and underscore HeberFERON as a glioblastoma therapeutic.

Amyloid {beta} peptides (A{beta}) trigger Alzheimers disease (AD) but how has remained elusive. A{beta} stimulates the {beta}2 adrenergic receptor ({beta}2AR), which forms a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. LTCCs have been implicated in the etiology of dementia and AD. We show that A{beta} acutely potentiates CaV1.2 via the {beta}2AR, which triggers postsynaptic recruitment of Ca2+ permeable (CP) AMPARs in hippocampal cultures and impairs LTP in hippocampal slices within minutes. The long-term consequence is a loss of postsynaptic structure of glutamatergic synapses and neurotoxicity. Disrupting this signaling cascade with highly specific tools prevented all of these effects, unifying a number of currently divergent findings on A{beta} synaptotoxicity including dysregulation of AMPARs and synaptic plasticity. TEASERAmyloid {beta} peptide is the primary pathological agent in Alzheimers disease. It affects the nanoscale structure and function of glutamatergic synapses. The molecular mechanisms are largely unknown except for identification of several binding proteins including the {beta}2 adrenergic receptor. We show that this binding potently (EC50<100 nM) augments Ca2+ influx through the L-type Ca channel CaV1.2. This effect leads to improper recruitment of Ca2+-permeable glutamate receptors to postsynaptic sites (EC50<100 nM), synaptic dysfunction and ultimately neuronal death. This work identifies an essential mechanism in amyloid {beta} neurotoxicity and explains many of the observed postsynaptic alterations. HighlightsImmediate effects of A{beta}-induced stimulation of {beta}2AR on Cav1.2: O_LIA{beta} induces phosphorylation of Cav1.2 on S1928 by PKA C_LIO_LIA{beta} augments Cav1.2 activity via {beta}2AR-induced S1928 phosphorylation within seconds C_LI A{beta}-induced {beta}2AR - Cav1.2 signaling has the following synaptotoxic effects. O_LIA{beta} induces postsynaptic accumulation of Ca-permeable AMPARs via {beta}2AR - Cav1.2 signaling within 20 min C_LIO_LIA{beta} impairs long-term potentiation (LTP) via {beta}2AR - Cav1.2 signaling C_LIO_LIA{beta} impairs postsynaptic structure and neuronal viability over 24 h C_LIO_LIPotency of A{beta} in all the above effects is very high (100 nM A{beta} is saturating!) C_LIO_LIAll effects are prevented in S1928A KI mice and acute displaces {beta}2AR from Cav1.2 with tat-Pep1923 C_LI

A priori assessment of target proteins' druggability remains an unsolved problem in the field of drug development. The empirical approaches widely used to solve this problem demonstrate low efficiency. In this work, we investigated the factor of hydration of a representative set of 65 evolutionarily and structurally unrelated human enzymes in a water environment. This factor depends only on the structure of the proteins, and not on the physical and chemical properties of any potential ligands. The results show that, unlike the widely used approaches based on calculations of the accessible surface area (ASA), the content of low-entropy water molecules (LEW) in the active sites of human enzymes is systematically higher than that in other areas of their surface, including inactive cavities. Optimal criteria and a step-by-step procedure for identifying protein ligand binding sites are proposed. The proposed approach, based on the calculation of the LEW content in the first hydration layer of potentially interesting target proteins, makes it possible to evaluate their medicinal suitability even before the development of any ligands. The article also presents the results of a comparative analysis of experimental Raman spectroscopy data and the results of molecular dynamics simulations of water hydrogen bonds using three widely used water models (TIP3P, OPC3, and TIP5P) and standard algorithms for calculating hydrogen bond networks.

Alzheimers Disease and Related Dementias (ADRDs) are linked to reduced bone integrity and increased fracture risk, but the mechanisms that underlie this risk remain poorly defined. Current research suggests that environmental factors, such as diet, sleep, and light exposure can modulate the brain-bone axis, increasing susceptibility to bone loss and fractures. Circadian disruption (CD) associated with ADRDs may exacerbate the effects of disease and aging in the bone. In particular, regulation of bone marrow progenitors may be acutely susceptible to disruption along this axis. Here, we explore the interplay among genetic and environmental factors that influence bone structure, marrow progenitor cell activity, and monocyte-derived macrophages. The APP/PS1 transgenic mouse model (AP) is used as an in vivo model of amyloid-beta deposition. High-resolution micro-computed tomography (CT) identified sex- and genotype-specific responses in trabecular morphometry. Follow-up analysis with Raman spectroscopy (RS) found accumulation of non-enzymatic modifications of the organic matrix and notched three-point bending identified concomitant loss of bone toughness due to both CD and AP. Single-cell RNA sequencing (scRNA-seq) confirmed the presence of oxidative stress signals in the cellular populations of the bone marrow. We further mapped significantly differentially expressed genes (DEGs) from monocytes in the bone marrow to circadian-regulated proteins in monocyte-derived macrophages, revealing dysregulation of circadian timing in macrophages in vitro. These findings offer new insights into how environmental disruptions can exacerbate the progression of neurodegenerative disease and bone degradation. LAY SUMMARYPatients with Alzheimers disease have an increased bone fracture risk, but the biological link between brain and bone disease is not well understood. Everyday factors such as altered light exposure (shift work, screens late at night, etc.) can worsen outcomes in the brain and skeleton. Using a mouse model of Alzheimers disease, we found that both genetic risk and circadian disruption contribute to weaker bone and altered bone quality. We also identified inflammation and stress responses in bone marrow cells, suggesting that bone marrow may play a key role in linking brain disease to bone fragility.

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

Sleep abnormalities and dysfunction of gamma band (30-80 Hz) activity generated by parvalbumin (PV) interneurons are early characteristics of Alzheimers disease (AD) which correlate with the severity of amyloid-{beta} deposition (A{beta}) and cognitive impairment. However, the timing of these alterations in vivo with respect to disease progression is unclear. Here, in longitudinal recordings from APP/PS1/PV-cre (AD mice) from 3-6 months, we found reduced sleep slow-wave power (0.5-4 Hz) in hippocampus and medial prefrontal cortex in AD mice as young as 3 months old, compared to non-AD (PV-cre) mice, well before overt pathology. This finding was primarily due to reductions in the NREM delta range (1.5-4 Hz), a hallmark of restorative functions of sleep. In contrast, beta (15-30 Hz) power linked to insomnia was significantly higher across all sleep-wake states. Loss of deep NREM sleep was not compensated by an increase in NREM sleep time, instead NREM sleep during the dark (active) phase was slightly but significantly lower in AD mice. 40-Hz auditory steady-state responses and associated evoked calcium responses of hippocampal PV neurons recorded using fiber photometry were also impaired by 3 months old. However, Y-maze performance in 3- and 6-month-old AD mice was not significantly different from non-AD mice. These results reveal reduced deep sleep and PV-associated 40-Hz activity as very early changes amenable to early intervention occurring prior to cognitive deficits. Furthermore, they establish APP/PS1 mice as a good model to causally test the relationship between sleep, PV neuronal activity and amyloid-mediated pathology.

BackgroundRising antimicrobial resistance rates, require new therapeutic approaches such as antimicrobial peptides (AMPs), which are part of the innate immune defense, as alternatives to antibiotics. In this study, we aim to unravel the antibacterial activity of human histone H1.2 peptide against Pseudomonas aeruginosa and its potential immune modulatory role. MethodsWe used a hemofiltrate peptide database for antimicrobial peptide prediction to identify novel human AMPs. Thirteen sequences of histone H1 were identified as putative AMPs, synthesized, and tested against bacterial ESKAPE pathogens in a radial diffusion assay. SYTOX green assay, electrophoretic mobility shift assay, and differential proteomics assays were conducted to determine the mode of action of H1.2 peptide fragment. A crystal violet assay was performed to evaluate the inhibition of biofilm formation. The cytotoxicity of the peptide was tested in LDH and Alamar assays. Finally, to visualize the contributions of H1.2 in NETs formation, scanning electron microscopy was performed. ResultsThe H1.2 peptide inhibited the growth of P. aeruginosa in a dose and pH-dependent manner without cytotoxicity towards mammalian THP-1 cells. It acts on intracellular targets to inhibit the growth of P. aeruginosa. STRING analysis from the differential proteomics assay showed that H1.2 targets the downregulation of proteins involved in the biogenesis of outer membrane proteins, including the folding and trafficking of outer membrane proteins across the cytoplasmic membrane. Scanning electron microscopy images showed that H1.2 forms NET-like structures capable of trapping and immobilizing P. aeruginosa. ConclusionThe characterized antimicrobial activity of H1.2 points to a role for human histone H1 fragments in innate immunity and may represent a promising approach for the development of novel antibacterial therapies. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/724237v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1778ddborg.highwire.dtl.DTLVardef@26430org.highwire.dtl.DTLVardef@ffbfa2org.highwire.dtl.DTLVardef@7e38ae_HPS_FORMAT_FIGEXP M_FIG C_FIG Sec transport and BAM complex system including chaperone proteins and quality control proteases are inhibited by H1.2 in Pseudomonas aeruginosa.Outer membrane proteins (OMPs) are synthesized in the cytoplasm and transported across the inner membrane via the Sec translocase, assisted by SecA/SecB or ribosomes. In the periplasm, they are escorted by chaperones such as SurA to the BAM complex for insertion into the outer membrane. Here, we show that H1.2, an antimicrobial peptide, targets membrane biogenesis in P. aeruginosa through downregulating Sec translocase (SecA/SecB and SecYEG), SurA, and BAM complex. Therefore, leading to improper transfer, folding and insertion of OMPs into the outer membrane. Normally, misfolded proteins are degraded by the protease MucD to prevent toxic aggregation in the bacteria. However, with H1.2 inhibiting MucD the proteotoxic stress is exacerbated, ultimately compromising bacterial homeostasis and viability. Figure created using BioRender.com.

GFAP is a type III intermediate filament primarily found within astrocytes and is known to maintain proper cell structure and mechanical strength. Mutations in GFAP are implicated in the pathology of Alexander disease, a neurodegenerative disease characterized by cytoplasmic inclusions of protein, known as Rosenthal fibers. GFAP has a typical type III intermediate filament domain structure, consisting of a highly conserved alpha-helical rod domain bracketed by an intrinsically disordered N-terminal head and C-terminal tail domains. While the general domain organization of monomeric GFAP and the assembly process for higher order quaternary structures are known, we lack an atomic resolution mechanistic understanding of GFAP assembly into mature filaments. Understanding the structure of GFAP filaments and how mutations disrupt this structure will provide vital information into how mutations produce Alexander disease pathology. As a first step towards a mechanistic description, we characterized GFAP wild type tetrameric and filamentous assemblies using solid state NMR and compared the results to those obtained from an assembly-deficient GFAP mutant. For wild-type GFAP, we observe surprisingly uniform rigid alpha helical structure and can spectroscopically resolve highly mobile intrinsically disordered regions in the filament assemblies. Wild type tetramers show increased mobility, likely arising from the head and tail domains. Mutation of the highly conserved cysteine at position 294 to serine results in an inability to form full-length filament assemblies. We show that the rigid regions of the C294S mutant assemblies largely remain structurally consistent with wild type tetrameric assemblies but differ from wild-type filament assemblies. There is an increase in highly mobile regions for the C294S mutant relative to the wild-type. Our results provide a foundation for developing solid state NMR approaches to characterize intermediate filament assembly mechanisms and the interfering effect of disease mutations.

Significant cellular processes, including protein sorting, signal transduction, and pathogen entry, amongst others, are associated with membrane microdomains, also known as lipid rafts. Lipid rafts, due to their unique biophysical properties compared to their surrounding environment, which stem from their distinct lipid and protein profiles, have garnered interest in methods and techniques that tune their coexisting liquid-ordered/liquid-disordered state, aiming to disrupt or destabilize them. Since cholesterol stabilizes the membrane domain, cholesterol-depleting compounds like cyclodextrin can be used to destabilize and disrupt the membrane rafts. Overall, given the membrane rafts importance in biological processes, it is crucial to understand the biophysical factors that influence its stability. In this study, we present a new method for disrupting and dissolving lipid rafts in a model system of phase-separated supported lipid bilayer (SLB) patches composed of DOPC, DPPC, and cholesterol. Using fluorescence microscopy to monitor the liquid ordered (Lo) and liquid disordered (Ld) phases of the SLB patches, we observed that adding DOPC liposomes causes a transformation of the co-existing Ld and Lo phases into a single-phase bilayer. On the other hand, adding liposomes that match the lipid content of the phase-separated SLB patch increase the areas of the existing Ld and Lo phases. This work also offers a new method for redistributing raft-localized molecules, confirmed by tracking the redistribution of cholera toxin bound to GM1 after domain dissolution with DOPC liposomes. The work describes an alternative method for dynamically altering membrane composition and dissolving domains via liposome addition, rather than lipid depletion or exchange.

Prion diseases are neurodegenerative disorders associated with the structural conversion of the cellular prion protein (PrPc) into its misfolded infectious isoform (PrPSc). Despite substantial efforts, no disease-modifying therapy or cure is currently available. Here, we present an integrated computational-experimental pipeline for the rational design of cyclic peptides targeting PrPc to inhibit its pathogenic conversion. Starting from crystal structures of antibody-bound mouse PrPc, we develop a rational design strategy combined with iterative molecular dynamics simulations and sequence optimization to generate peptides with enhanced binding and structural impact. Three candidates were selected for experimental validation. Our results show that PH1 (49YGPDPSDSYT58, antibody numbering) that binds stably to the &alpha2-&alpha3 interface most effectively reduced PrPSc levels in GT1-7 cells, essentially by inducing allosteric rearrangements that reinforce the intramolecular helical bundle. PL1 (89GQSNTKPYT97) and PL2 (89RQSNTWPYT97) binding the &beta1-&alpha1/&alpha3 junction exerted more modest effects due to the potential competition of the flexible tail to bind at this site. These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.