Evolutionary and structural constraints influencing apolipoprotein A-I amyloid behavior.

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.

Post-Translational Modifications at the Coarse-Grained Level with the SIRAH Force Field.

Post-translational modifications (PTMs) on proteins significantly enlarge the physicochemical diversity present in biological macromolecules, altering function, localization, and interactions. Despite their critical role in regulating cellular processes, theoretical methods are not yet fully capable of coping with this diversity. These limitations are particularly more marked for coarse-grained (CG) models, in which comprehensive and self-consistent parametrizations are less frequent. Here we present a set of topologies and interaction parameters for the most common PTMs, fully compatible with the SIRAH force field. The PTMs introduced here reach the same level of structural description of the existing SIRAH force field, expanding the chemical spectrum with promising applications in dynamical protein-protein interactions in large and complex cellular environments.

Mechanistic and biological characterisation of novel N5-substituted paullones targeting the biosynthesis of trypanothione in Leishmania.

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.

Searching for improved mimetic peptides inhibitors preventing conformational transition of amyloid-ß42 monomer.

A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.

Comparative analysis reveals amino acids critical for anticancer activity of peptide CIGB-552.

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Early stages in Aß1-42 spontaneous aggregation: An unbiased dataset from coarse-grained molecular dynamics simulations.

The small soluble aggregates of Aß1-42 are broadly documented as potential targets for the development of new compounds with the capacity to inhibit the early stages of Alzheimer´s disease. Nevertheless, Aß1-42 peptides show an intrinsically disordered character with a high propensity for aggregation, which complicates the identification of conserved structural patterns. Because of this, experimental techniques find substantial difficulties in the characterization of such soluble oligomers. Theoretical techniques, such as molecular dynamics (MD) simulations, provide a possible workaround for this problem. However, the computational cost associated with comprehensively sampling the vast conformational space accessible to these peptides might become prohibitive. In this sense, coarse-grained (CG) simulations can effectively overcome that hurdle at a fraction of the computational cost. In this dataset, we furnish an extensive collection of Aß1-42 peptides in dimeric conformation generated with the SIRAH force field for CG MD simulation. It comprises 25 independent trajectories in .xtc (gromacs) format of Aß1-42 couples of peptides that evolve towards dimeric states along eleven µs-long unbiased simulations. Thanks to the backmapping capabilities of our force field, pseudo atomistic coordinates can be straightforwardly recovered from MD trajectories reported here and analyzed with popular molecular editing programs. This set of simulations performed at room conditions and physiological salt concentrations may furnish a complete collection of inter-peptide interfaces that can be used in high-throughput docking or as new starting states for peptide oligomerization seeding of Aß1-42 dimerization.

The stressed life of a lipid in the Zika virus membrane.

Protein-lipid interactions modulate a plethora of physiopathologic processes and have been the subject of countless studies. However, these kinds of interactions in the context of viral envelopes have remained relatively unexplored, partially because the intrinsically small dimensions of the molecular systems escape to the current resolution of experimental techniques. However, coarse-grained and multiscale simulations may fill that niche, providing nearly atomistic resolution at an affordable computational price. Here we use multiscale simulations to characterize the lipid-protein interactions in the envelope of the Zika Virus, a prominent member of the Flavivirus genus. Comparisons between the viral envelope and simpler molecular systems indicate that the viral membrane is under extreme pressures and asymmetric forces. Furthermore, the dense net of protein-protein contacts established by the envelope proteins creates poorly solvated regions that destabilize the external leaflet leading to a decoupled dynamics between both membrane layers. These findings lead to the idea that the Flaviviral membrane may store a significant amount of elastic energy, playing an active role in the membrane fusion process.

Assessing SIRAH's Capability to Simulate Intrinsically Disordered Proteins and Peptides.

The challenges posed by intrinsically disordered proteins (IDPs) to atomistic and coarse-grained (CG) simulations are boosting efforts to develop and reparametrize current force fields. An assessment of the dynamical behavior of IDPs' and unstructured peptides with the CG SIRAH force field suggests that the current version achieves a fair description of IDPs' conformational flexibility. Moreover, we found a remarkable capability to capture the effect of point mutations in loosely structured peptides.

Dissecting the role of glutamine in seeding peptide aggregation.

Poly glutamine and glutamine-rich peptides play a central role in a plethora of pathological aggregation events. However, biophysical characterization of soluble oligomers -the most toxic species involved in these processes- remains elusive due to their structural heterogeneity and dynamical nature. Here, we exploit the high spatio-temporal resolution of coarse-grained simulations as a computational microscope to characterize the aggregation propensity and morphology of a series of polyglutamine and glutamine-rich peptides. Comparative analysis of ab-initio aggregation pinpointed a double role for glutamines. In the first phase, glutamines mediate seeding by pairing monomeric peptides, which serve as primers for higher-order nucleation. According to the glutamine content, these low molecular-weight oligomers may then proceed to create larger aggregates. Once within the aggregates, buried glutamines continue to play a role in their maturation by optimizing solvent-protected hydrogen bonds networks.

A homogeneous dataset of polyglutamine and glutamine rich aggregating peptides simulations.

This dataset contains a collection of molecular dynamics (MD) simulations of polyglutamine (polyQ) and glutamine-rich (Q-rich) peptides in the multi-microsecond timescale. Primary data from coarse-grained simulations performed using the SIRAH force field has been processed to provide fully atomistic coordinates. The dataset encloses MD trajectories of polyQs of 4 (Q4), 11 (Q11), and 36 (Q36) amino acids long. In the case of Q11, simulations in presence of Q5 and QEQQQ peptides, which modulate aggregation, are also included. The dataset also comprises MD trajectories of the gliadin related p31-43 peptide, and Insulin's C-peptide at pH=7 and pH=3.2, which constitute examples of Q-rich and Q-poor aggregating peptides. The dataset grants molecular insights on the role of glutamines in spontaneous and unbiased ab-initio aggregation of a series of peptides using a homogeneous set of simulations [1]. The trajectory files are provided in Protein Data Bank (PDB) format containing the Cartesian coordinates of all heavy atoms in the aggregating peptides. Further analyses of the trajectories can be performed directly using any molecular visualization/analysis software suites.

Role of membrane curvature on the activation/deactivation of Carnitine Palmitoyltransferase 1A: A coarse grain molecular dynamic study.

Carnitine Palmitoyltransferase 1A (CPT 1A) is an enzyme anchored to the outer mitochondrial membrane (OMM), where it regulates the passage of fatty acids into the mitochondria and intervenes in the process of ß-oxidation of long-chain fatty acids. Although CPT 1A is inhibited by malonyl-CoA, its activity is also modulated by the curvature of OMM. This modulation depends on the behavior of the N-terminal domain (NTD), which can be adsorbed onto the OMM (nonactive CPT 1A) or interacting with the C-terminal domain (active CPT 1A). Aimed to provide mechanistic insights on the regulatory mechanism of CPT 1A, we studied the influence of the bilayer curvature on the NTD behavior through a series of coarse-grained (CG) molecular dynamics simulations using curved and planar membranes. Comparative analysis suggests that the main determinant for the activation/deactivation of the enzyme is the tilt angle orientation of the transmembrane (TM) domains. Planar membranes induce a wide variation on the tilt angle orientation of TM helices, while curved geometries promote small angles with the membrane normal. Our results identify the first TM domain as an important component of the membrane sensing mechanism.

Structural conformation and self-assembly process of p31-43 gliadin peptide in aqueous solution. Implications for celiac disease.

Celiac disease (CeD) is a highly prevalent chronic immune-mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α-gliadin peptide, p31-43, induces proinflammatory responses, observed by in vitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31-43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31-43 secondary structure by different biophysical and in silico techniques. By dynamic light scattering and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy analysis showed p31-43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self-organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with ß-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased ß-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.

Fat SIRAH: Coarse-Grained Phospholipids To Explore Membrane-Protein Dynamics.

The capability to handle highly heterogeneous molecular assemblies in a consistent manner is among the greatest challenges faced when deriving simulation parameters. This is particularly the case for coarse-grained (CG) simulations in which chemical functional groups are lumped into effective interaction centers for which transferability between different chemical environments is not guaranteed. Here, we introduce the parametrization of a set of CG phospholipids compatible with the latest version of the SIRAH force field for proteins. The newly introduced lipid species include different acylic chain lengths and partial unsaturation, as well as polar and acidic head groups that show a very good reproduction of structural membrane determinants, such as areas per lipid, thickness, order parameter, etc., and their dependence with temperature. Simulation of membrane proteins showed unprecedented accuracy in the unbiased description of the thickness-dependent membrane-protein orientation in systems where this information is experimentally available (namely, the SarcoEndoplasmic Reticulum Calcium-SERCA-pump and its regulator Phospholamban). The interactions that lead to this faithful reproduction can be traced down to the single amino acid-lipid interaction level and show full agreement with biochemical data present in the literature. Finally, the present parametrization is implemented in the GROMACS and AMBER simulation packages facilitating its use by a wide portion of the biocomputing community.

Complex I and II Subunit Gene Duplications Provide Increased Fitness to Worms.

Helminths use an alternative mitochondrial electron transport chain (ETC) under hypoxic conditions, such as those found in the gastrointestinal tract. In this alternative ETC, fumarate is the final electron acceptor and rhodoquinone (RQ) serves as an electron carrier. RQ receives electrons from reduced nicotinamide adenine dinucleotide through complex I and donates electrons to fumarate through complex II. In this latter reaction, complex II functions in the opposite direction to the conventional ETC (i.e., as fumarate reductase instead of succinate dehydrogenase). Studies in Ascaris suum indicate that this is possible due to changes in complex II, involving alternative succinate dehydrogenase (SDH) subunits SDHA and SDHD, derived from duplicated genes. We analyzed helminth genomes and found that distinct lineages have different gene duplications of complex II subunits (SDHA, SDHB, SDHC, and SDHD). Similarly, we found lineage-specific duplications in genes encoding complex I subunits that interact with quinones (NDUF2 and NDUF7). The phylogenetic analysis of ETC subunits revealed a complex history with independent evolutionary events involving gene duplications and losses. Our results indicated that there is not a common evolutionary event related to ETC subunit genes linked to RQ. The free-living nematode Caenorhabditis elegans uses RQ and has two genes encoding SDHA (sdha-1 and sdha-2) and two genes encoding NDUF2 (nduf2-1 and nduf2-2). sdha-1 and nduf2-1 are essential genes and have a similar expression pattern during C. elegans lifecycle. Using knockout strains, we found that sdha-2 and nduf2-2 are not essential, even in hypoxia. Yet, sdha-2 and nduf2-2 expression is increased in the early embryo and in dauer larvae, stages where there is low oxygen tension. Strikingly, sdha-1 and sdha-2 as well as nduf2-1 and nduf2-2 showed inverted expression profiles during the C. elegans life cycle. Finally, we found that sdha-2 and nduf2-2 knockout mutant strain progeny is affected. Our results indicate that different complex I and II subunit gene duplications provide increased fitness to worms.

The SIRAH 2.0 Force Field: Altius, Fortius, Citius.

A new version of the coarse-grained (CG) SIRAH force field for proteins has been developed. Modifications to bonded and non-bonded interactions on the existing molecular topologies significantly ameliorate the structural description and flexibility of a non-redundant set of proteins. The SIRAH 2.0 force field has also been ported to the popular simulation package AMBER, which along with the former implementation in GROMACS expands significantly the potential range of users and performance of this CG force field on CPU/GPU codes. As a non-trivial example of its application, we undertook the structural and dynamical analysis of the most abundant and conserved calcium-binding protein, calmodulin (CaM). CaM is composed of two calcium-binding motifs called EF-hands, which in the presence of calcium specifically recognize a cognate peptide by embracing it. CG simulations of CaM bound to four calcium ions in the presence or absence of a binding peptide (holo and apo forms, respectively) resulted in good and stable ion coordination. The simulation of the holo form starting from an experimental structure sampled near-native conformations, retrieving quasi-atomistic precision. Removing the binding peptide enabled the EF-hands to perform large reciprocal movements, comparable to those observed in NMR structures. On the other hand, the isolated peptide starting from the helical conformation experienced spontaneous unfolding, in agreement with previous experimental data. However, repositioning the peptide in the neighborhood of one EF-hand not only prevented the peptide from unfolding but also drove CaM to a fully bound conformation, with both EF-hands embracing the cognate peptide, resembling the experimental holo structure. Therefore, SIRAH 2.0 shows the capacity to handle a number of structurally and dynamically challenging situations, including metal ion coordination, unbiased conformational sampling, and specific protein-peptide recognition.

p31-43 Gliadin Peptide Forms Oligomers and Induces NLRP3 Inflammasome/Caspase 1- Dependent Mucosal Damage in Small Intestine.

Celiac disease (CD) is a chronic enteropathy elicited by a Th1 response to gluten peptides in the small intestine of genetically susceptible individuals. However, it remains unclear what drives the induction of inflammatory responses of this kind against harmless antigens in food. In a recent work, we have shown that the p31-43 peptide (p31-43) from α-gliadin can induce an innate immune response in the intestine and that this may initiate pathological adaptive immunity. The receptors and mechanisms responsible for the induction of innate immunity by p31-43 are unknown and here we present evidence that this may reflect conformational changes in the peptide that allow it to activate the NLRP3 inflammasome. Administration of p31-43, but not scrambled or inverted peptides, to normal mice induced enteropathy in the proximal small intestine, associated with increased production of type I interferon and mature IL-1ß. P31-43 showed a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro and induced activation of the ASC speck complex. In parallel, the enteropathy induced by p31-43 in vivo did not occur in the absence of NLRP3 or caspase 1 and was inhibited by administration of the caspase 1 inhibitor Ac-YVAD-cmk. Collectively, these findings show that p31-43 gliadin has an intrinsic propensity to form oligomers which trigger the NLRP3 inflammasome and that this pathway is required for intestinal inflammation and pathology when p31-43 is administered orally to mice. This innate activation of the inflammasome may have important implications in the initial stages of CD pathogenesis.

Modeling DMPC lipid membranes with SIRAH force-field.

Coarse-grained simulation schemes are increasingly gaining popularity in the scientific community because of the significant speed up granted, allowing a considerable expansion of the accessible time and size scales accessible to molecular simulations. However, the number of compatible force fields capable of representing ensembles containing different molecular species (i.e., Protein, DNA, etc) is still limited. Here, we present a set of parameters and simplified representation for lipids compatible with the SIRAH force field for coarse-grained simulations ( http://www.sirahff.com ). We show that the present model not only achieves a correct reproduction of structural parameters as area per lipid and thickness, but also dynamic descriptors such as diffusion coefficient, order parameters, and proper temperature driven variations. Adding phospholipid membranes to the existing aqueous solution, protein and DNA representations of the SIRAH force field permit considering the most common problems tackled by the biomolecular simulation community.