Search

BACKGROUND:Novel cryo-techniques are continuously being developed that may better improve cryogenic survival in plants, with the aim of reducing exposure times to otherwise toxic cryoprotective agents whilst maximising regeneration rates. OBJECTIVE:This study used cryo-mesh and vacuum infiltration vitrification with two vitrification solutions (PVS2 and PVS3) to develop an optimised cryopreservation protocol for Arabidopsis thaliana . MATERIALS AND METHODS:Shoot tips from 10-day old seedlings of wild type A. thaliana were cryopreserved using either vacuum infiltration vitrification or the cryo-mesh technique. Shoot tips were treated for up to 60 min in increments of 10 min with PVS2 and PVS3, and for an additional 180 and 300 min incubation for cryo-mesh prior to exposure to liquid nitrogen. RESULTS:Both methods resulted in very high regeneration rates, but which decreased after longer exposure to the vitrification solutions. The highest regeneration rate for vacuum-infiltration vitrification was attained after only 30 min incubation in PVS2 (92.5%) and 50 min incubation in PVS3 (93.55%). In the case of cryo-mesh the highest regeneration was observed after 180 min incubation in either PVS2 (100%) or PVS3 (92.2%). CONCLUSION: Vacuum- infiltration vitrification is more effective than cryo-mesh by reducing exposure times to cryoprotective solutions whilst achieving very high regeneration rates of shoot tips of A. thaliana .

Human islet amyloid polypeptide (hIAPP) is a naturally occurring, intrinsically disordered protein (IDP) whose abnormal aggregation into toxic soluble oligomers and insoluble amyloid fibrils is a pathological feature in type-2 diabetes. Rat IAPP (rIAPP) differs from hIAPP by only six amino acids yet has a reduced tendency to aggregate or form fibrils. The structures of the monomeric forms of IAPP are difficult to characterize due to their intrinsically disordered nature. Molecular dynamics simulations can provide a detailed characterization of the monomeric forms of rIAPP and hIAPP in near-physiological conditions. In this work, the conformational landscapes of rIAPP and hIAPP as a function of secondary structure content were predicted using well-tempered bias exchange metadynamics simulations. Several combinations of commonly used biomolecular force fields and water models were tested. The predicted conformational preferences of both rIAPP and hIAPP are typical of IDPs, exhibiting dominant random coil structures but showing a low propensity for transient α-helical conformations. Predicted nuclear magnetic resonance Cα chemical shifts reveal different preferences with each force field towards certain conformations, with AMBERff99SBnmr2/TIP4Pd showing the best agreement with the experiment. Comparisons of secondary structure content demonstrate residue-specific differences between hIAPP and rIAPP that may reflect their different aggregation propensities.

Intrinsically disordered peptides, such as amyloid β42 (Aβ42), lack a well-defined structure in solution. Aβ42 can undergo abnormal aggregation and amyloidogenesis in the brain, forming fibrillar plaques, a hallmark of Alzheimer's disease. The insoluble fibrillar forms of Aβ42 exhibit well-defined, cross β-sheet structures at the molecular level and are less toxic than the soluble, intermediate disordered oligomeric forms. However, the mechanism of initial interaction of monomers and subsequent oligomerization is not well understood. The structural disorder of Aβ42 adds to the challenges of determining the structural properties of its monomers, making it difficult to understand the underlying molecular mechanism of pathogenic aggregation. Certain regions of Aβ42 are known to exhibit helical propensity in different physiological conditions. NMR spectroscopy has shown that the Aβ42 monomer at lower pH can adopt an α-helical conformation and as the pH is increased, the peptide switches to β-sheet conformation and aggregation occurs. CD spectroscopy studies of aggregation have shown the presence of an initial spike in the amount of α-helical content at the start of aggregation. Such an increase in α-helical content suggests a mechanism wherein the peptide can expose critical non-polar residues for interaction, leading to hydrophobic aggregation with other interacting peptides. We have used molecular dynamics simulations to characterize in detail the conformational landscape of monomeric Aβ42 in solution to identify molecular properties that may mediate the early stages of oligomerization. We hypothesized that conformations with α-helical structure have a higher probability of initiating aggregation because they increase the hydrophobicity of the peptide. Although random coil conformations were found to be the most dominant, as expected, α-helical conformations are thermodynamically accessible, more so than β-sheet conformations. Importantly, for the first time α-helical conformations are observed to increase the exposure of aromatic and hydrophobic residues to the aqueous solvent, favoring their hydrophobically driven interaction with other monomers to initiate aggregation. These findings constitute a first step toward characterizing the mechanism of formation of disordered, low-order oligomers of Aβ42.

Pathogenic fungal diseases in crops are mediated by the release of effector proteins that facilitate infection. Characterising the structure of these fungal effectors is vital to understanding their virulence mechanisms and interactions with their hosts, which is crucial in the breeding of plant cultivars for disease resistance. Several effectors have been identified and validated experimentally; however, their lack of sequence conservation often impedes the identification and prediction of their structure using sequence similarity approaches. Structural similarity has, nonetheless, been observed within fungal effector protein families, creating interest in validating the use of computational methods to predict their tertiary structure from their sequence. We used Rosetta ab initio modelling to predict the structures of members of the ToxA-like and MAX effector families for which experimental structures are known to validate this method. An optimised approach was then used to predict the structures of phenotypically validated effectors lacking known structures. Rosetta was found to successfully predict the structure of fungal effectors in the ToxA-like and MAX families, as well as phenotypically validated but structurally unconfirmed effector sequences. Interestingly, potential new effector structural families were identified on the basis of comparisons with structural homologues and the identification of associated protein domains.

The discovery of new fungal effector proteins is necessary to enable the screening of cultivars for disease resistance. Sequence-based bioinformatics methods have been used for this purpose, but only a limited number of functional effector proteins have been successfully predicted and subsequently validated experimentally. A significant obstacle is that many fungal effector proteins discovered so far lack sequence similarity or conserved sequence motifs. The availability of experimentally determined three-dimensional (3D) structures of a number of effector proteins has recently highlighted structural similarities amongst groups of sequence-dissimilar fungal effectors, enabling the search for similar structural folds amongst effector sequence candidates. We have applied template-based modelling to predict the 3D structures of candidate effector sequences obtained from bioinformatics predictions and the PHI-BASE database. Structural matches were found not only with ToxA- and MAX-like effector candidates but also with non-fungal effector-like proteins—including plant defensins and animal venoms—suggesting the broad conservation of ancestral structural folds amongst cytotoxic peptides from a diverse range of distant species. Accurate modelling of fungal effectors were achieved using RaptorX. The utility of predicted structures of effector proteins lies in the prediction of their interactions with plant receptors through molecular docking, which will improve the understanding of effector–plant interactions.

The barrier imposed by the outer layer of the skin, the stratum corneum, creates an almost impermeable environment for exogenous substances. Few lipophilic drugs with low molecular mass can passively diffuse through this layer, highlighting the need to develop methods to enable the delivery of more drugs via the transdermal route. The prodrug approach involves modifying the structure of a drug molecule to enhance its permeability across the skin, but it is often difficult to predict how exactly changes in chemical structure affect permeation. This study uses molecular dynamics simulations to predict permeability values and adequately characterise the molecular mechanism of permeation of the prodrugs Me-5ALA and its parent compound 5ALA across a molecular model of the lipid bilayers of the human stratum corneum. The influence of increased hydrophobicity in Me-5ALA on its permeation revealed a reduction in hydrogen bonding capability that enables it to interact more favourably with the hydrophobic region of the bilayer and diffuse at a faster rate with less resistance, thus making it a better permeant compared to its more hydrophilic parent compound. This molecular simulation approach offers a promising route for the rational design of drug molecules that can permeate effectively across the stratum corneum.

Cryopreservation allows the long-term storage of plant germplasm, but can cause damage to plant tissues, which must be repaired for survival to occur. This repair process is fuelled by the metabolic function of mitochondria; however, little is known about how metabolic function is affected by the cryopreservation process in plants. We compared metabolic rates of shoot tips of two Australian native species, Androcalva perlaria and Anigozanthos viridis. Overall, cryopreservation resulted in a significant reduction in the metabolic rates of shoot tips from both species, even in tissues that regenerated after cryopreservation. Metabolic rate did not increase within 48 h after of thawing, even in shoot tips which later regenerated. When examined in isolation, both pre-treatment on desiccation medium and exposure to cryoprotective agents significantly decreased metabolic rates in regenerating shoot tips of A. viridis, however both caused a significant increase in shoot tips of A. perlaria, suggesting diversity of response to cryopreservation stresses across species. Measurements of shoot tip metabolic rate during cryopreservation will inform investigations into cellular energy production and provide critical information on the state of shoot health after exposure to different cryoprotective treatments, which could play a useful role in guiding protocol optimisation for threatened species to maximise post-cryopreservation regeneration.

Graphical abstract, The human ATP-binding cassette B5 (ABCB5) transporter, a member of the ABC transporter superfamily, is linked to chemoresistance in tumour cells by drug effluxion. However, little is known about its structure and drug-binding sites. In this study, we generated an atomistic model of the full-length human ABCB5 transporter with the highest quality using the X-ray crystal structure of mouse ABCB1 (Pgp1), a close homologue of ABCB5 and a well-studied member of the ABC family. Molecular dynamics simulations were used to validate the atomistic model of ABCB5 and characterise its structural properties in model cell membranes. Molecular docking simulations of known ABCB5 substrates such as taxanes, anthracyclines, camptothecin and etoposide were then used to identify at least three putative binding sites for chemotherapeutic drugs transported by ABCB5. The location of these three binding sites is predicted to overlap with the corresponding binding sites in Pgp1. These findings will serve as the basis for future in vitro studies to validate the nature of the identified substrate-binding sites in the full-length ABCB5 transporter.

Cell membranes protect and compartmentalise cells and their organelles. The semi-permeable nature of these membranes controls the exchange of solutes across their structure. Characterising the interaction of small molecules with biological membranes is critical to understanding of physiological processes, drug action and permeation, and many biotechnological applications. This review provides an overview of how molecular simulations are used to study the interaction of small molecules with biological membranes, with a particular focus on the interactions of water, organic compounds, drugs and short peptides with models of plasma cell membrane and stratum corneum lipid bilayers. This review will not delve on other types of membranes which might have different composition and arrangement, such as thylakoid or mitochondrial membranes. The application of unbiased molecular dynamics simulations and enhanced sampling methods such as umbrella sampling, metadynamics and replica exchange are described using key examples. This review demonstrates how state-of-the-art molecular simulations have been used successfully to describe the mechanism of binding and permeation of small molecules with biological membranes, as well as associated changes to the structure and dynamics of these membranes. The review concludes with an outlook on future directions in this field.

The potentially damaging action of dimethyl sulfoxide (DMSO) on phospholipid bilayers remains a matter of controversy. We have conducted a series of long-scale molecular dynamics simulations of 1,2-dioleoyl

Background: Type 2 diabetes related human islet amyloid polypeptide (hIAPP) plays a dual role in Alzheimer’s disease (AD). hIAPP has neuroprotective effects in AD mouse models whereas, high hIAPP concentrations can promote co-aggregation with amyloid-β (Aβ) to promote neurodegeneration. In fact, both low and high plasma hIAPP concentration has been associated with AD. Therefore, non-aggregating hIAPP analogues have garnered interest as a treatment for AD. The aromatic amino acids F23 and I26 in hIAPP have been identified as the key residues involved in self-aggregation and Aβ cross-seeding. Objective: Three novel IAPP analogues with single and double alanine mutations (A1 = F23, A2 = I26, and A3 = F23 + I26) were assessed for their ability to aggregate, modulate Aβ oligomer formation, and alter neurotoxicity. Methods: A range of biophysical methods including Thioflavin-T, gel electrophoresis, photo-crosslinking, circular dichroism combined with cell viability assays were utilized to assess protein aggregation and toxicity. Results: All IAPP analogues showed significantly less self-aggregation than hIAPP. Co-aggregated Aβ42-A2 and A3 also showed reduced aggregation compared to Aβ42-hIAPP mixtures. Self- and co-oligomerized A1, A2, and A3 exhibited random coil conformations with reduced beta sheet content compared to hIAPP and Aβ42-hIAPP aggregates. A1 was toxic at high concentrations compared to A2 and A3. However, co-aggregated Aβ42-A1, A2, or A3 showed reduced neurotoxicity compared to Aβ42, hIAPP, and Aβ42-hIAPP aggregates. Conclusion: These findings confirm that hIAPP analogues with non-aromatic residues at positions 23 and 26 have reduced self-aggregation and the ability to neutralize Aβ42 toxicity. This warrants further characterization of their protective effects in pre-clinical AD models.

The local intermolecular structure and related dynamics in the liquid cis-trans N-methylformamide mixture at ambient temperature and density conditions have been systematically studied by employing Born-Oppenheimer molecular dynamics simulation techniques. Particular attention has been paid to the local structure around the cis- and trans- conformers and the formation and dynamics of hydrogen bonds with their closest neighbors. The calculated atom-atom radial distribution functions are in very good agreement with available experimental data and reveal the existence of different types of hydrogen bonding intermolecular interactions. The average number of hydrogen bonds formed by the cis-conformers is higher in comparison with the one corresponding to the trans-conformers. Moreover, the lifetimes of the hydrogen bonds formed in the liquid are longer when the cis-conformers participate in the bond formation, either as donors or acceptors. These findings clearly indicate that the local structural network around the cis-conformers in the liquid is more cohesive. The latter finding is also reflected in the slower reorientational dynamics of the cis-conformers and the low- and high-frequency region of the spectral densities of the atomic velocity time correlation functions. Finally, the calculated average dipole moments of the trans- and cis- conformers are significantly higher than their corresponding gas-phase values, signifying the importance of polarization effects in this particular polar liquid solvent.

The mechanism by which cryosolvents such as alcohols modify and penetrate cell membranes as a function of their concentration and hydration state remains poorly understood. We conducted molecular dynamics simulations of 1,2-dioleoyl

Cryopreservation has several advantages over other ex situ conservation methods, and indeed is the only viable storage method for the long term conservation of most plant species. However, despite many advances in this field, it is increasingly clear that some species are ill-equipped to overcome the intense stress imposed by the cryopreservation process, making protocol development incredibly difficult using traditional trial and error methods. Cryobiotechnology approaches have been recently recognised as a strategic way forward, utilising intimate understanding of biological systems to inform development of more effective cryopreservation protocols. Mitochondrial function is a model candidate for a cryobiotechnological approach, as it underpins not only energy provision, but also several other key determinants of germplasm outcome, including stress response, reduction-oxidation status, and programmed cell death. Extensive research in animal cell and tissue cryopreservation has established a clear link between mitochondrial health and cryopreservation survival, but also indicates that mitochondria are routinely subject to damage from multiple aspects of the cryopreservation process. Evidence is already emerging that mitochondrial dysfunction may also occur in plant cryopreservation, and this research can be greatly expanded by using considered applications of innovative technologies. A range of mitochondria-targeted prophylactic and therapeutic interventions already exist with potential to improve cryopreservation outcomes through mitochondrial function.

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

Sugar-membrane interactions are believed to be responsible for cell preservation during desiccation and freezing, but the molecular mechanism by which they achieve this is still not well understood. The associated decrease of the main phase transition temperature of phospholipid bilayers is explained by two opposing views on the matter: the direct sugar-phospholipid interaction at the bilayer interface (water replacement hypothesis) and an entropy-driven phase transition with sugar molecules concentrating away from the lipid interface (hydration forces explanation). Both mechanisms are supported by experiments but molecular dynamics (MD) simulations have overwhelmingly shown the occurrence of direct sugar-phospholipid interactions. We have performed MD simulations of 1,2-dioleoyl

Protein electrochemistry studies at a polarized interface between two immiscible electrolyte solutions (ITIES) indicate that the detection mechanism of a protein at the interface involves a combination of protein-anion complexation and interfacial adsorption processes. A detailed characterization of the protein-facilitated mechanism of ion transfer at the ITIES will allow the development of new label-free biomolecular detection tools. Molecular dynamics simulations were performed to describe the mechanism of transfer of the hydrophobic anion tetraphenylborate (TPB-) from a 1,2-dichloroethane (organic) phase to an aqueous phase mediated by lysozyme as a model protein under the action of an external electric field. The anion migrated to the protein at the interface and formed multiple contacts. The side chains of positively charged Lys and Arg residues formed electrostatic interactions with the anion. Nonpolar residues like Trp, Met, and Val formed hydrophobic contacts with the anion as it moved along the protein surface. During this process, lysozyme adopted multiple, partially unfolded conformations at the interface, all involving various anion-protein complexes with small free-energy barriers between them. The general mechanism of protein-facilitated ion transfer at a polarized liquid-liquid interface thus likely involves the movement of a hydrophobic anion along the protein surface through a combination of electrostatic and hydrophobic interactions.

‘Effectors’ are a broad class of cytotoxic or virulence-promoting molecules that are released from plant-pathogen cells to cause disease in their host. Fungal effectors are a core research area for improving host disease resistance; however, because they generally lack common distinguishing features or obvious sequence similarity, discovery of effectors remains a major challenge. This study presents a novel tool and pipeline for effector prediction - Predector - which interfaces with multiple software tools and methods, aggregates disparate features that are relevant to fungal effector proteins, and ranks effector candidate proteins using a pairwise learning to rank approach. Predector outperformed alternative effector prediction methods that were applied to a curated set of confirmed effectors derived from multiple species. We present Predector as a useful tool for the prediction and ranking of effector candidates, which aggregates and reports additional supporting information relevant to effector and secretome prediction in a simple, efficient, and reproducible manner. Predector is available from https://github.com/ccdmb/predector and associated data from https://github.com/ccdmb/predector-data.

Graphical abstract, High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) molecule that triggers the progression of several pro-inflammatory diseases such as diabetes, Alzheimer’s disease and cancer, by inducing signals upon interaction with the receptors such as the receptor for advanced glycation end-products (RAGE) and toll-like receptors (TLRs). The acidic C-terminal tail of HMGB1 is an intrinsically disordered region of the protein which is known to determine the interaction of HMGB1 to DNA and histones. This study characterizes its structural properties using a combination of circular dichroism (CD) and molecular dynamics (MD) simulations. The full-length and tail-less forms of HMGB1 were compared to rationalise the role of the acidic tail in maintaining the stability of the entire structure of HMGB1 in atomistic detail. Consistent with experimental data, the acidic tail was predicted to adopt an extended conformation that allows it to make a range of hydrogen-bonding and electrostatic interactions with the box-like domains that stabilize the overall structure of HMGB1. Absence of the acidic tail was predicted to increase structural fluctuations of all amino acids, leading to changes in secondary structure from α-helical to more hydrophilic turns along with increased exposure of multiple amino acids to the surrounding solvent. These structural changes reveal the intrinsic conformational dynamics of HMGB1 that are likely to affect the accessibility of its receptors.

Summary The electrochemistry of proteins at the interface between two immiscible electrolyte solutions (ITIES) is discussed, with detection capabilities based on protein-facilitated anion transfer, and which has enabled protein detection via adsorptive stripping voltammetry. Targeting an enzymatic biomarker has achieved detection at picomolar concentrations, whilst studies into structural aspects of proteins at the ITIES have revealed formation of oligomers (cytochrome c) and unfolded protein (lysozyme) together with alterations to the protein secondary structures.

The beta amyloid protein (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis and its interaction with cell membranes in known to promote mutually disruptive structural perturbations that contribute to amyloid deposition and neurodegeneration in the brain. In addition to protein aggregation at the membrane interface and disruption of membrane integrity, growing reports demonstrate an important role for the membrane in modulating Aβ production and uptake into cells. The aim of this review is to highlight and summarize recent literature that have contributed insight into the implications of altered membrane composition on amyloid precursor protein (APP) proteolysis, production of Aβ, its internalization in to cells via permeabilization and receptor mediated uptake. Here, we also review the various membrane model systems and experimental tools used for probing Aβ-membrane interactions to investigate the key mechanistic aspects underlying the accumulation and toxicity of Aβ in AD.

The neural-specific transcription factor Engrailed 1 - is overexpressed in basal-like breast tumours. Synthetic interference peptides - comprising a cell-penetrating peptide/nuclear localisation sequence and the Engrailed 1-specific sequence from the N-terminus have been engineered to produce a strong apoptotic response in tumour cells overexpressing EN1, with no toxicity to normal or non Engrailed 1-expressing cells. Here scaled molecular dynamics simulations were used to study the conformational dynamics of these interference peptides in aqueous solution to characterise their structure and dynamics. Transitions from disordered to α-helical conformation, stabilised by hydrogen bonds and proline-aromatic interactions, were observed throughout the simulations. The backbone of the wild-type peptide folds to a similar conformation as that found in ternary complexes of anterior Hox proteins with conserved hexapeptide motifs important for recognition of pre-B-cell leukemia Homeobox 1, indicating that the motif may possess an intrinsic preference for helical structure. The predicted NMR chemical shifts of these peptides are consistent with the Hox hexapeptides in solution and Engrailed 2 NMR data. These findings highlight the importance of aromatic residues in determining the structure of Engrailed 1 interference peptides, shedding light on the rational design strategy of molecules that could be adopted to inhibit other transcription factors overexpressed in other cancer types, potentially including other transcription factor families that require highly conserved and cooperative protein-protein partnerships for biological activity.