Identification of allosteric fingerprints of alpha-synuclein aggregates in matrix metalloprotease-1 and substrate-specific virtual screening with single molecule insights.

Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previous reports showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. This paper reports quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is unavailable, binding poses were predicted by molecular docking of MMP1 with aSyn using ClusPro. MMP1 dynamics were simulated using predicted binding poses and compared with the experimental interdomain dynamics to identify an appropriate pose. The selected aSyn-MMP1 binding pose near aSyn residue K45 was simulated and analyzed to define conformational changes at the catalytic site. Allosteric residues in aSyn-bound MMP1 exhibiting strong correlations with the catalytic motif residues were compared with allosteric residues in free MMP1, and aSyn-specific residues were identified. The allosteric residues in aSyn-bound MMP1 are K281, T283, G292, G327, L328, E329, R337, F343, G345, N346, Y348, G353, Q354, D363, Y365, S366, S367, F368, P371, R372, V374, K375, A379, F391, A394, R399, M414, F419, V426, and C466. Shannon entropy was defined to quantify MMP1 dynamics. Virtual screening was performed against a site on selected aSyn-MMP1 binding poses, which showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for potential substrate-specific control of MMP1 activity in the future. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Lysine-arginine advanced glycation end-product cross-links and the effect on collagen structure: A molecular dynamics study.

The accumulation of advanced glycation end-products is a fundamental process that is central to age-related decline in musculoskeletal tissues and locomotor system function and other collagen-rich tissues. However, although computational studies of advanced glycation end-product cross-links could be immensely valuable, this area remains largely unexplored given the limited availability of structural parameters for the derivation of force fields for Molecular Dynamics simulations. In this article, we present the bonded force constants, atomic partial charges and geometry of the arginine-lysine cross-links DOGDIC, GODIC, and MODIC. We have performed in vacuo Molecular Dynamics simulations to validate their implementation against quantum mechanical frequency calculations. A DOGDIC advanced glycation end-product cross-link was then inserted into a model collagen fibril to explore structural changes of collagen and dynamics in interstitial water. Unlike our previous studies of glucosepane, our findings suggest that intra-collagen DOGDIC cross-links furthers intra-collagen peptide hydrogen-bonding and does not promote the diffusion of water through the collagen triple helices.

Measurement properties of Patient-Reported Outcome Measures used to assess the sleep quality in adults with high prevalence chronic pain conditions: a systematic review.

OBJECTIVES: To provide a comprehensive overview of the measurement properties of patient-reported outcome measures (PROMs) used to assess sleep quality in adult patients with prevalent pain-related conditions. METHODS: Without language restrictions PubMed, Scopus, and Embase were searched from their inception to January 2020. Independent reviewers screened and selected studies, extracted data, assessed the methodological quality using the Consensus-Based Standards for the Selection of Health Measurement Instruments (COSMIN) Risk of Bias checklist, and performed an evidence synthesis for each measurement property. The results were classified as sufficient, insufficient, inconsistent, or indeterminate, and quality of evidence was evaluated using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. RESULTS: Nineteen studies were included investigating twelve PROMs and six pain-related conditions. Reliability, internal consistency, structural and construct validity were the most prevalent measurement properties investigated across the studies. Three questionnaires were investigated in more than one study (Jenkins Sleep Scale [JSS] and Pittsburgh Sleep Quality Scale [PSQI] and the Medical Outcomes Study-Sleep Scale [MOS-SS]). High-quality evidence was reported for sufficient content validity in the JSS and indeterminate responsiveness in the MOS-SS. Moderate to high evidence was reported for sufficient structural validity in the MOS-SS and PSQI and sufficient and insufficient construct validity in the JSS And MOS-SS. Low to high evidence was reported for sufficient internal consistency in the MOS-SS and PSQI, while low to moderate evidence for sufficient reliability in the JSS, MOS-SS, and PSQI. CONCLUSION: Studies reporting the most measurement properties included the JSS, MOS-SS, and PSQI in multiple prevalent pain-related conditions. Given this, not all measurement properties have been reported for these as well as other questionnaires, and much investigation is needed to ensure the quality of these questionnaires within high prevalence chronic pain conditions. PROSPERO: CRD42019136623.

Allosteric Communications between Domains Modulate the Activity of Matrix Metalloprotease-1.

An understanding of the structure-dynamics relationship is essential for understanding how a protein works. Prior research has shown that the activity of a protein correlates with intradomain dynamics occurring at picosecond to millisecond timescales. However, the correlation between interdomain dynamics and the function of a protein is poorly understood. Here, we show that communications between the catalytic and hemopexin domains of matrix metalloprotease-1 (MMP1) on type 1 collagen fibrils correlate with its activity. Using single-molecule Förster resonance energy transfer, we identified functionally relevant open conformations in which the two MMP1 domains are well separated, which were significantly absent for catalytically inactive point mutant (E219Q) of MMP1 and could be modulated by an inhibitor or an enhancer of activity. The observed relevance of open conformations resolves the debate about the roles of open and closed MMP1 structures in function. We fitted the histograms of single-molecule Förster resonance energy transfer values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We used a two-state Poisson process to describe the dynamics and calculate the kinetic rates from the fit parameters. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an interdomain separation facilitates opening up the catalytic pocket so that the collagen chains come closer to the MMP1 active site. Coordination of functional conformations at different parts of MMP1 occurs via allosteric communications that can take place via interactions mediated by collagen even if the linker between the domains is absent. Modeling dynamics as a Poisson process enables connecting the picosecond timescales of molecular dynamics simulations with the millisecond timescales of single-molecule measurements. Water-soluble MMP1 interacting with water-insoluble collagen fibrils poses challenges for biochemical studies that the single-molecule tracking can overcome for other insoluble substrates. Interdomain communications are likely important for multidomain proteins.

Common transcriptional signatures of neuropathic pain.

ABSTARCT: The dorsal root ganglia (DRG) are key structures in nociception and chronic pain disorders. Several gene expression studies of DRG in preclinical pain models have been performed, but it is unclear if consistent gene changes are identifiable. We, therefore, compared several recent RNA-Seq data sets on the whole DRG in rodent models of nerve injury. Contrary to previous findings, we show hundreds of common differentially expressed genes and high positive correlation between studies, despite model and species differences. We also find, in contrast to previous studies, that 60% of the common rodent gene response after injury is likely to occur in nociceptors of the DRG. Substantial expression changes are observed at a 1-week time-point, with smaller changes in the same genes at a later 3- to 4-week time-point. However, a subset of genes shows a similar magnitude of changes at both early and late time-points, suggesting their potential involvement in the maintenance of chronic pain. These genes are centred around suppression of endogenous opioid signalling. Reversal of this suppression could allow endogenous and exogenous opioids to exert their analgesic functions and may be an important strategy for treating chronic pain disorders. Currently used drugs, such as amitriptyline and duloxetine, do not seem to appropriately modulate many of the critical pain genes and indeed may transcriptionally suppress endogenous opioid signalling further.

The aggregation of striped nanoparticles in mixed phospholipid bilayers.

The unique and adjustable properties of nanoparticles present enormous opportunities for their use as targeted drug delivery vectors. For example, nanoparticles functionalized with key surface ligands have been shown to pass through phospholipid bilayers without causing localised disruption. However, the further effects nanoparticles have on multi-component phospholipid bilayers remain unclear. We use coarse-grained computational models to investigate the structural properties of mixed phospholipid bilayers in the presence of ligand-functionalized nanoparticles. Model bilayers are composed of DPPC, DUPC, DFPC and cholesterol, and the nanoparticles are striped with a hydrophobic-ligand band and charged-ligand spherical caps. Our results show that nanoparticles aggregate near unsaturated phospholipid regions, phospholipid bilayer phase-separation is promoted in the presence of nanoparticles, and the heterogeneous components of a phospholipid bilayer play a significant role in the lateral organization of nanoparticles. This study highlights the need for considering the complexity of realistic phospholipid bilayers when optimising ligand functionalized nanoparticles for efficient drug delivery vectors.

Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen.

Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule's size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules.

Lack of IFNγ signaling attenuates spread of influenza A virus in vivo and leads to reduced pathogenesis.

IFNγ is a key regulator of inflammatory responses but its role in influenza A virus (IAV) pathogenesis is unclear. Our studies show that infection of mice lacking the IFNγ receptor (IFNγR-/-) at a dose which caused severe disease in wild type 129 Sv/Ev (WT) mice resulted in milder clinical symptoms and significantly lower lung virus titers by 6 days post-infection (dpi). Viral spread was reduced in IFNγR-/- lungs at 2 and 4 dpi. Levels of inflammatory cytokines and chemokines were lower in IFNγR-/- mice at 2 dpi and there was less infiltration of monocyte/macrophage lineage cells than in WT mice. There was no difference in CD4+ and CD8+ T cells and alveolar macrophages in the bronchoalveolar lavage fluid (BALF) at 2 and 4 dpi but by 4 dpi IFNγR-/- mice had significantly higher percentages of neutrophils. Our data strongly suggest that IAV can use the inflammatory response to promote viral spread.

Glucosepane is associated with changes to structural and physical properties of collagen fibrils.

Collagen glycation, and in particular the formation of advanced glycation end-product (AGE) crosslinks, plays a central role in the ageing process and in many of the long-term complications of diabetes. Glucosepane, the most abundant and relevant AGE crosslink, has been suggested to increase the stiffness of tissue and reduce its solubility, although no evidence is available concerning the mechanisms. We have used a combination of computational and experimental techniques to study a collagen-rich tissue with a relatively simple organisation to further our understanding of the impact of glucosepane on the structural and physical properties of collagen fibrils. Our work shows that glucosepane levels increase dramatically in aged tendon tissue and are associated with the reduced density of collagen packing and increased porosity to water molecules. Our studies provide the basis to understand many of the tissue dysfunctions associated with ageing and diabetes across a range of different tissues types.

MARDy: Mycology Antifungal Resistance Database.

Summary: The increase of antifungal drug resistance is a major global human health concern and threatens agriculture and food security; in order to tackle these concerns, it is important to understand the mechanisms that cause antifungal resistance. The curated Mycology Antifungal Resistance Database (MARDy) is a web-service of antifungal drug resistance mechanisms, including amino acid substitutions, tandem repeat sequences and genome ploidy. MARDy is implemented on a Linux, Apache, MySQL and PHP web development platform and includes a local installation of BLASTn of the database of curated genes. Availability and implementation: MARDy can be accessed at http://www.mardy.net and is free to use. The complete database can be retrieved, ordered by organism, gene and drug. Missing or new mycological antifungal resistance data can be relayed to the development team through a contribute entry form. Updates and news will be publicized via a dedicated Twitter feed: @MARDYfungi.

Modelling water diffusion in plasticizers: development and optimization of a force field for 2,4-dinitroethylbenzene and 2,4,6-trinitroethylbenzene.

A classical all-atom force field has been developed for 2,4,6-trinitroethylbenzene and 2,4-dinitroethylbenzene and applied in molecular dynamics simulations of the two pure and two mixed plasticizer systems. Bonding parameters and partial charges were derived through electronic and geometry optimization of the single molecules. The other required parameters were derived from values already available in the literature for generic nitro aromatic compounds, which were adjusted to reproduce to a high level of accuracy the densities of 2,4-dinitroethylbenzene, 2,4,6-trinitroethylbenzene and the energetic plasticizers K10 and R8002. This force field has been applied to both K10 and R8002, which when used as plasticizers form an energetic binder with nitrocellulose. Nitrocellulose decomposes in storage, under varying conditions, but in particular where it may become increasingly dry. Following the derivation of the force field, we have therefore applied it to calculate water diffusion coefficients for each of the different materials at 298 K and 338 K, thereby providing a starting point for understanding water behaviour in a nitrocellulose binder.

Simulations of CYP51A from Aspergillus fumigatus in a model bilayer provide insights into triazole drug resistance.

Azole antifungal drugs target CYP51A in Aspergillus fumigatus by binding with the active site of the protein, blocking ergosterol biosynthesis. Resistance to azole antifungal drugs is now common, with a leucine to histidine amino acid substitution at position 98 the most frequent, predominantly conferring resistance to itraconazole, although cross-resistance has been reported in conjunction with other mutations. In this study, we create a homology model of CYP51A using a recently published crystal structure of the paralog protein CYP51B. The derived structures, wild type, and L98H mutant are positioned within a lipid membrane bilayer and subjected to molecular dynamics simulations in order improve the accuracy of both models. The structural analysis from our simulations suggests a decrease in active site surface from the formation of hydrogen bonds between the histidine substitution and neighboring polar side chains, potentially preventing the binding of azole drugs. This study yields a biologically relevant structure and set of dynamics of the A. fumigatus Lanosterol 14 alpha-demethylase enzyme and provides further insight into azole antifungal drug resistance.

ForceGen: atomic covalent bond value derivation for Gromacs.

A large number of crystallographic protein structures include ligands, small molecules and post-translational modifications. Atomic bond force values for computational atomistic models of post-translational or non-standard amino acids, metal binding active sites, small molecules and drug molecules are not readily available in most simulation software packages. We present ForceGen, a Java tool that extracts the bond stretch and bond angle force values and equilibrium values from the Hessian of a Gaussian vibrational frequency analysis. The parameters are compatible with force fields derived using the second order tensor of the Hessian. The output is formatted with the Gromacs topology in mind. This study further demonstrates the use of ForceGen over the quantum mechanically derived structures of a small organic solvent, a naturally occurring protein crosslink derived from two amino acids following post-translational modification and the amino acid ligands of a zinc ion. We then derive Laplacian bond orders to understand how the resulting force values relate back to the quantum mechanical model. The parameterisation of the organic solvent, toluene, was verified using Molecular Mechanics simulations. The structural data from the simulation compared well with the quantum mechanical structure and the system density compared well with experimental values.

Combining nano-physical and computational investigations to understand the nature of "aging" in dermal collagen.

The extracellular matrix of the dermis is a complex, dynamic system with the various dermal components undergoing individual physiologic changes as we age. Age-related changes in the physical properties of collagen were investigated in particular by measuring the effect of aging, most likely due to the accumulation of advanced glycation end product (AGE) cross-links, on the nanomechanical properties of the collagen fibril using atomic force microscope nano-indentation. An age-related decrease in the Young's modulus of the transverse fibril was observed (from 8.11 to 4.19 GPa in young to old volunteers, respectively, P<0.001). It is proposed that this is due to a change in the fibril density caused by age-related differences in water retention within the fibrils. The new collagen-water interaction mechanism was verified by electronic structure calculations, showing it to be energetically feasible.

Mapping intermolecular interactions and active site conformations: from human MMP-1 crystal structure to molecular dynamics free energy calculations.

The zinc-dependent Matrix Metalloproteinases (MMPs) found within the extracellular matrix (ECM) of vertebrates are linked to pathological processes such as arthritis, skin ulceration and cancer. Although a general backbone proteolytic mechanism is understood, crystallographic data continue to suggest an active site that is too narrow to encompass the respective substrate. We present a fully parameterised Molecular Dynamics (MD) study of the structural properties of an MMP-1-collagen crystallographic structure (Protein Data Bank - 4AUO), followed by an exploration of the free energy surface of a collagen polypeptide chain entering the active site, using a combined meta-dynamics and umbrella sampling (MDUS) approach. We conclude that the interactions between MMP-1 and the collagen substrate are in good agreement with a number of experimental studies. As such, our unrestrained MD simulations and our MDUS results, which indicate an energetic barrier for a local uncoiling and insertion event, can inform future investigations of the collagen-peptide non-bonded association steps with the active site prior to proteolytic mechanisms. The elucidation of such free energy barriers provides a better understanding of the role of the enzyme in the ECM and is important in the design of future MMP inhibitors.

Computational study of glucosepane-water and hydrogen bond formation: an electron topology and orbital analysis.

The collagen protein provides tensile strength to the extracellular matrix in addition to localising cells, proteins and protein cofactors. Collagen is susceptible to a build up of glycation modifications as a result of an exceptionally long half-life. Glucosepane is a collagen cross-linking advanced glycation end product; the structural and mechanical effects of glucosepane are still the subjects of much debate. With the prospect of an ageing population, the management and treatment of age-related diseases is becoming a pressing concern. One area of interest is the isolation of hydrated glucosepane, which has yet to be reported at an atomistic level. This study presents a series of glucosepane-water complexes within an implicit aqueous environment. Electronic structure calculations were performed using density functional theory and a high level basis set. Hydrogen bonds between glucosepane and explicit water were identified by monitoring changes to covalent bonds, calculating levels of electron donation from Natural Bonding Orbital analysis and the detection of bond critical points. Hydrogen bond strength was calculated using second-order perturbation calculations. The combined results suggest that glucosepane is very hydrophilic, with the imidazole feature being energetically more attractive to water than either hydroxyl group, although all hydrogen bonds, regardless of bond strength, were electrostatic in nature. Our results are in growing support of an earlier hypothesis that cross-links may result in an increase in interstitial water retention, which would permit the collagen fibril to swell, thereby potentially affecting the tensile and compression properties and biological function of connective tissues.

Intra-molecular lysine-arginine derived advanced glycation end-product cross-linking in Type I collagen: A molecular dynamics simulation study.

Covalently cross-linked advanced glycation end products (AGE) are among the major post-translational modifications to proteins as a result of non-enzymatic glycation. The formation of AGEs has been shown to have adverse effects on the properties of the collagenous tissue; they are even linked to a number of age related disorders. Little is known about the sites at which these AGEs form or why certain sites within the collagen are energetically more favourable than others. In this study we have used a proven fully atomistic molecular dynamics approach to identify six sites where the formation of the intra-molecular 3-deoxyglucosone-derived imidazolium cross-link (DOGDIC) is energetically favourable. We have also conducted a comparison of these positions with those of the more abundant glucosepane cross-link, to determine any site preference. We show that when we consider both lysine and arginine AGEs, they exhibit a prevalence to form within the gap region of the collagen fibril.

Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype.

The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection.

Preferential sites for intramolecular glucosepane cross-link formation in type I collagen: A thermodynamic study.

The extracellular matrix (ECM) undergoes progressive age-related stiffening and loss of proteolytic digestibility due to an increase in concentration of advanced glycation end products (AGEs). The most abundant AGE, glucosepane, accumulates in collagen with concentrations over 100 times greater than all other AGEs. Detrimental collagen stiffening properties are believed to play a significant role in several age-related diseases such as osteoporosis and cardiovascular disease. Currently little is known of the potential location of covalently cross-linked glucosepane formation within collagen molecules; neither are there reports on how the respective cross-link sites affect the physical and biochemical properties of collagen. Using fully atomistic molecular dynamics simulations (MD) we have identified six sites where the formation of a covalent intra-molecular glucosepane cross-link within a single collagen molecule in a fibrillar environment is energetically favourable. Identification of these favourable sites enables us to align collagen cross-linking with experimentally observed changes to the ECM. For example, formation of glucosepane was found to be energetically favourable within close proximity of the Matrix Metalloproteinase-1 (MMP1) binding site, which could potentially disrupt collagen degradation.

De novo design of transmembrane helix-helix interactions and measurement of stability in a biological membrane.

Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu "scaffold" was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix-helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔGapp) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (<1.4 kcal mol(-1)) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes).