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Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically impacts virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes.In situcryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

Ebola viruses (EBOVs) are filamentous particles, whose shape and stability are determined by the VP40 matrix. Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

Over the past thirty years, researchers have highlighted the role played by a class of proteins or polypeptides that forms pathogenic amyloid aggregates in vivo, including i) the amyloid Abeta peptide, which is known to form senile plaques in Alzheimer's disease; ii) alpha-synuclein, responsible for Lewy body formation in Parkinson's disease and iii) IAPP, which is the protein component of type 2 diabetes-associated islet amyloids. These proteins, known as intrinsically disordered proteins (IDPs), are present as highly dynamic conformational ensembles. IDPs can partially (mis) fold into (dys) functional conformations and accumulate as amyloid aggregates upon interaction with other cytosolic partners such as proteins or lipid membranes. In addition, an increasing number of reports link the toxicity of amyloid proteins to their harmful effects on membrane integrity. Still, the molecular mechanism underlying the amyloidogenic proteins transfer from the aqueous environment to the hydrocarbon core of the membrane is poorly understood. This review starts with a historical overview of the toxicity models of amyloidogenic proteins to contextualize the more recent lipid-chaperone hypothesis. Then, we report the early molecular-level events in the aggregation and ion-channel pore formation of Abeta, IAPP, and alpha-synuclein interacting with model membranes, emphasizing the complexity of these processes due to their different spatial-temporal resolutions. Next, we underline the need for a combined experimental and computational approach, focusing on the strengths and weaknesses of the most commonly used techniques. Finally, the last two chapters highlight the crucial role of lipid-protein complexes as molecular switches among ion-channel-like formation, detergent-like, and fibril formation mechanisms and their implication in fighting amyloidogenic diseases., Comment: 45 pages, 3 figures

International audience; Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer’s disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly β-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aβ(1–40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

An increasing number of human diseases has been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-β, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer's, and Parkinson's, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-β, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal that this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-β, and α-synuclein) and nonamyloidogenic (rat IAPP, β-synuclein) proteins. Based on these results, we propose a "lipid-chaperone" hypothesis as a unifying framework for protein-membrane poration.

The Bcl-2 protein family comprises both proand antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family mem-bers can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

The metabotropic glutamate receptor (mGluR) 2 plays a key role in the central nervous system. mGluR2 has been shown to be regulated by its surrounding lipid environment, especially by cholesterol, by an unknown mechanism. Here, using a combination of biochemical approaches, photo-cross-linking experiments, and molecular dynamics simulations we show the interaction of cholesterol with at least two, but potentially five more, preferential sites on the mGluR2 transmembrane domain. Our simulations demonstrate that surface matching, rather than electrostatic interactions with specific amino acids, is the main factor defining cholesterol localization. Moreover, the cholesterol localization observed here is similar to the sterol-binding pattern previously described in silico for other members of the mGluR family. Biochemical assays suggest little influence of cholesterol on trafficking or dimerization of mGluR2. Nevertheless, simulations revealed a significant reduction of residue-residue contacts together with an alteration in the internal mechanical stress at the cytoplasmic side of the helical bundle when cholesterol was present in the membrane. These alterations may be related to destabilization of the basal state of mGluR2. Due to the high sequence conservation of the transmembrane domains of mGluRs, the molecular interaction of cholesterol and mGluR2 described here is also likely to be relevant for other members of the mGLuR family.

Increasing number of human diseases have been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-β, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer’s, and Parkinson’s, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-β, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-β, and α-synuclein) and non-amyloidogenic (rat IAPP, β-synuclein) proteins. Based on these results, we propose a “lipid-chaperone” hypothesis as a unifying framework for protein-membrane poration.

Synaptic neurotransmission has recently been proposed to function via either a membrane-independent or a membrane-dependent mechanism, depending on the neurotransmitter type. In the membrane-dependent mechanism, amphipathic neurotransmitters first partition to the lipid headgroup region and then diffuse along the membrane plane to their membrane-buried receptors. However, to date, this mechanism has not been demonstrated for any neurotransmitter-receptor complex. Here, we combined isothermal calorimetry measurements with a diverse set of molecular dynamics simulation methods to investigate the partitioning of an amphipathic neurotransmitter (dopamine) and the mechanism of its entry into the ligand-binding site. Our results show that the binding of dopamine to its receptor is consistent with the membrane-dependent binding and entry mechanism. Both experimental and simulation results showed that dopamine favors binding to lipid membranes especially in the headgroup region. Moreover, our simulations revealed a ligand-entry pathway from the membrane to the binding site. This pathway passes through a lateral gate between transmembrane alpha-helices 5 and 6 on the membrane-facing side of the protein. All in all, our results demonstrate that dopamine binds to its receptor by a membrane-dependent mechanism, and this is complemented by the more traditional binding mechanism directly through the aqueous phase. The results suggest that the membrane-dependent mechanism is common in other synaptic receptors, too. publishedVersion

The abnormal deposition of Aβ amyloid deposits in the brain is a hallmark of Alzheimer's disease (AD). Based on this evidence, many current therapeutic approaches focus on the development of small molecules halting Aβ aggregation. However, due to the temporary and elusive structures of amyloid assemblies, the rational design of aggregation inhibitors remains a challenging task. Here we combine ThT assays and MD simulations to study Aβ aggregation in the presence of the natural compounds tyrosol (TY), 3-hydroxytyrosol (HDT), and 3-methoxytyrosol (homovanillyl alcohol - HVA). We show that albeit HDT is a potent inhibitor of amyloid growth, TY and HVA catalyze fibril formation. An inspection of MD simulations trajectories revealed that the different effects of these three molecules on Aβ1-40 aggregation are ascribable to their capacity to arrange H-bonds network between the ligand (position C-3) and the peptide (Glu22). We believe that our results may contribute to the design of more effective and safe small molecules able to contrast pathogenic amyloid aggregation.

Amyloidogenic proteins are involved in many diseases, including Alzheimer's, Parkinson's, and type II diabetes. These proteins are thought to be toxic for cells because of their abnormal interaction with the cell membrane. Simpler model membranes (LUVs) have been used to study the early steps of membrane-protein interactions and their subsequent evolution. Phospholipid LUVs formed in water solution establish a chemical equilibrium between self-assembled LUVs and a small amount of phospholipids in water solution (CMC). Here, using both experimental and molecular dynamics simulations approach we demonstrate that the insertion of IAPP, an amyloidogenic peptide involved in diabetes, in membranes is driven by free lipids in solution in dynamic equilibrium with the self-assembled lipids of the bilayer. It is suggested that this could be a general mechanism lying at the root of membrane insertion processes of self-assembling peptides.

FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces., Legrand et al. identify two lysine residues on molecular surface of Fibroblast Growth Factor 2 (FGF2) essential for its interaction with α1 subunit of the Na,K-ATPase. They further conclude that this interaction precedes interaction of the FGF2 with PI(4,5)P2 and facilitates its unconventional secretion across the membrane, which is impaired by Ouabain, an Na,K-ATPase inhibitor.

SummaryFibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is exported from cells by an unconventional secretory pathway. This process is based on direct translocation of FGF2 across the plasma membrane. FGF2 membrane translocation depends on PI(4,5)P2-induced formation of membrane-inserted FGF2 oligomers followed by extracellular trapping of FGF2 at the outer leaflet mediated by cell surface heparan sulfate proteoglycans. Beyond the well-characterized core mechanism of FGF2 membrane translocation, the Na,K-ATPase has been proposed to play a so far unknown role in unconventional secretion of FGF2. Here, we define a direct physical interaction of FGF2 with a subdomain of the cytoplasmic part of the α1 subunit of the Na,K-ATPase. Employing NMR spectroscopy and molecular dynamics simulations, we identified two lysine residues on the molecular surface of FGF2 that are shown to be essential for its interaction with α1. In intact cells, the corresponding lysine-to-glutamate variants of FGF2 were characterized by inefficient secretion and reduced recruitment to the inner plasma membrane leaflet as shown by single molecule TIRF microscopy. Our findings suggest that α1 acts upstream of PI(4,5)P2 facilitating efficient membrane translocation of FGF2 to the cell surface of tumor cells.

The most accredited hypothesis links the toxicity of amyloid proteins to their harmful effects on membrane integrity through the formation of prefibrillar-transient oligomers able to disrupt cell membranes. However, damage mechanisms necessarily assume a first step in which the amyloidogenic protein transfers from the aqueous phase to the membrane hydrophobic core. This determinant step is still poorly understood. However, according to our lipid-chaperon hypothesis, free lipids in solution play a crucial role in facilitating this footfall. Free phospholipid concentration in the aqueous phase acts as a switch between ion channel-like pore and fibril formation, so that high free lipid concentration in solution promotes pore and repress fibril formation. Conversely, low free lipids in the solution favor fibril and repress pore formation. This behavior is due to the formation of stable lipid-protein complexes. Here, we hypothesize that the helix propensity is a fundamental requirement to fulfill the lipid-chaperon model. The alpha-helix region seems to be responsible for the binding with amphiphilic molecules fostering the proposed mechanism. Indeed, our results show the dependency of protein-lipid binding from the helical structure presence. When the helix content is substantially lower than the wild type, the contact probability decreases. Instead, if the helix is broadening, the contact probability increases. Our findings open a new perspective for in silico screening of secondary structure-targeting drugs of amyloidogenic proteins.

Single-particle tracking (SPT) is an experimental technique that allows one to follow the dynamics of individual molecules in biological membranes with unprecedented precision. Given the importance of lipid and membrane protein diffusion in the formation of nanoscale functional complexes, it is critical to understand what exactly is measured in SPT experiments. To clarify this issue, we employed nanoscale computer simulations designed to match SPT experiments that exploit streptavidin-functionalized Au nanoparticles (AuNPs). The results show that lipid labeling interferes critically with the diffusion process; thus, the diffusion measured in SPT is a far more complex process than what has been assumed. It turns out that the influence of AuNP-based labels on the dynamics of probe lipids includes not only the AuNP-induced viscous drag that is the more significant the larger the NP but, more importantly, also the effects related to the interactions of the streptavidin linker with membrane lipids. Due to these effects, the probe lipid moves in a concerted manner as a complex with the linker protein and numerous unlabeled lipids, which can slow down the motion of the probe by almost an order of magnitude. Furthermore, our simulations show that nonlinker streptavidin tetramers on the AuNP surface are able to interact with the membrane lipids, which could potentially lead to multivalent labeling of the NPs by the probe lipids. Our results further demonstrate that in the submicrosecond time domain the motion of the probe lipid is uncorrelated with the motion of the AuNP, showing that there is a 1 mu s limit for the temporal resolution of the SPT technique. However, this limit for the temporal resolution depends on the nanoparticle size and increases rapidly with growing AuNPs. Overall, the results provide a molecular-scale framework to accurately interpret SPT data and to design protocols that minimize label-induced artifacts.

We report a structural investigation on IAPP, Aβ(1–40) and their equi-molar mixture aggregation pathway at nano-molar concentration using the Surface Enhanced Raman Spectroscopy (SERS) effect induced by silver metal colloids prepared by laser processes in solution and molecular dynamics simulations. Our data show the ability of silver NPs coupled with SERS to detect secondary structures of IAPP, Aβ(1–40) and their 1 : 1 molar ratio mixture in the oligomeric state. The preparation of silver colloids shows superior performance with respect to chemically prepared nano-particles. SERS spectroscopy shows both selectivity and sensitivity in detecting the secondary structures of hIAPP and Aβ(1–40) and to recognize both proteins in their mixture. On the other hand, molecular dynamics simulations confirm SERS structural data and the given atomistic details about the structural organization of IAPP and Aβ(1–40) oligomers. Our study shows an inhomogeneity in the chemical composition of IAPP/Aβ(1–40) oligomer aggregates.

The self-assembling of the amyloid β (Aβ) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer’s disease (AD). Based on the “amyloid hypothesis”, many efforts have been devoted to designing molecules able to halt disease progression by inhibiting Aβ self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. Despite TEM analysis demonstrating that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment 35MVGGVV40 of Aβ40. Consequently to silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments 17LVFF20 and 27NKGAII32 of Aβ40 which shows a high tendency to form bend, turn, and β-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment 17LVFF20 but lead to the formation of different small-sized, ThT-positive Aβ aggregates. Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aβ indicated that silybin B is the most effective of the four compounds in counteracting Aβ proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to silybin B as a promising lead compound for further development in anti-AD therapeutics.

International audience; Monolayer-protected gold nanoparticles are emerging as promising candidates for drug delivery due to their ability to permeate through plasma membranes. Understanding the molecular mechanisms of such complex systems is crucial to control cell permeation and to develop efficient biomedical delivery applications based on nanoscale gold nanoparticles. Here, neutron reflectometry (NR) and molecular dynamics (MD) simulations were used to shed light on the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes and the consequences thereof. Atomistic simulations predicted that there is a free energy barrier that has to be overcome to enforce AuNPs to partition into a DSPC bilayer. NR experiments confirmed the prediction, showing that AuNP encapsulation takes place only at temperatures higher than ∼330 K. Meanwhile, in a mixture of DSPC-DSPG (3:1), experiments showed that the adsorption of AuNPs to the membrane is weak; the nanoparticles were readily released during annealing. Coarse-grained MD simulations used to interpret this behaviour indicated that in this mixture DSPGs migrate around AuNPs, thereby weakening their interaction with the surface and leading to detachment of AuNPs at high temperatures. NR experiments also confirmed this by showing that in the presence of negative lipids (DSPG), desorption of AuNPs is associated with a reduced coverage of the floating bilayer, suggesting that some lipids, which comprise both DSPC and DSPG, are extracted and left the bilayer. Finally, the results indicated that the crowding of lipids over the nanoparticles shields the electrostatic interaction between cationic nanoparticles, thus fostering their aggregation on the membrane surface and driving the membrane composition to be locally asymmetric, causing membrane instability.

Our knowledge of the molecular events underlying type 2 diabetes mellitus-a protein conformational disease characterized by the aggregation of islet amyloid polypeptide (IAPP) in pancreatic β cells-is limited. However, amyloid-mediated membrane damage is known to play a key role in IAPP cytotoxicity, and therefore the effects of lipid composition on modulating IAPP-membrane interactions have been the focus of intense research. In particular, membrane cholesterol content varies with aging and consequently with adverse environmental factors such as diet and lifestyle, but its role in the development of the disease is controversial. In this study, we employ a combination of experimental techniques and in silico molecular simulations to shed light on the role of cholesterol in IAPP aggregation and the related membrane disruption. We show that if anionic POPC/POPS vesicles are used as model membranes, cholesterol has a negligible effect on the kinetics of IAPP fibril growth on the surface of the bilayer. In addition, cholesterol inhibits membrane damage by amyloid-induced poration on membranes, but enhances leakage through fiber growth on the membrane surface. Conversely, if 1:2 DOPC/DPPC raft-like model membranes are used, cholesterol accelerates fiber growth. Next, it enhances pore formation and suppresses fiber growth on the membrane surface, leading to leakage. Our results highlight a twofold effect of cholesterol on the amyloidogenicity of IAPP and help explain its debated role in type 2 diabetes mellitus.

Amyloid aggregation of islet amyloid polypeptide (IAPP) in pancreatic tissues is a typical feature of type 2 diabetes mellitus. Resveratrol, a natural product extensively studied for its wide range of biological effects, has been shown to inhibit IAPP aggregation. However, the mechanism by which resveratrol inhibits IAPP aggregation is still far from complete elucidation. Now, an increasing knowledge of the mechanism of amyloid toxicity shifts the target of research towards the development of compounds which can prevent amyloid-mediated membrane damage rather than merely inhibit fiber formation. In this study we used all atom molecular dynamics to investigate the interaction of resveratrol with full-length human IAPP in a negatively charged membrane environment. Our results show that the presence of resveratrol induces the formation of secondary structures (sheets and helices) by inserting in a hydrophobic pocket between the interaction surface of two IAPP molecules in aqueous solution. On the other hand, resveratrol significantly perturbs the interaction of IAPP with negatively charged membranes by anchoring specific hydrophobic regions (23FGA25 and 32VGS34) of the peptide and forming a stable 1:2 IAPP:resveratrol complex at the water/membrane interphase.

The glucose transporter 4 (GLUT4) is one of the most important glucose transporter proteins for the absorption of glucose from the plasma circulation after a meal or during exercise. It is present in skeletal muscle, adipose tissue cells, cardiac muscle, and it has also been found in brain cells. It plays a significant role in the development of various diseases such as type 2 diabetes, cancer, and cardiac diseases. Inside a cell, GLUT4 is transported towards the cell membrane upon an insulin stimulus, leading to a 10- to 40-fold increase in the glucose uptake. In spite of its importance, the molecular mechanism of glucose transport by GLUT4 is still not clear. There is no crystal structure available for the GLUT4 protein either. However, using existing structural information of the other solved glucose transporter structures, we have modeled and validated the GLUT4 structure in three conformations: the outward-open, outward-occluded, and inward-open conformation. The GLUT4 models with glucose bound were subsequently embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane and simulated over microsecond time scales using unbiased atomistic molecular dynamics simulations. In addition, we also carried out random acceleration molecular dynamics simulations to explore all possible pathways for glucose transport across the membrane. Our simulation studies revealed specific GLUT4 residues that captured the conformational changes in GLUT4 structure on glucose binding. Overall, the study provides atomistic level structural information on glucose transport and also provides one with ideas for the development of therapeutic agents blocking the function of GLUT4. This development work is important given that cancer cells express elevated levels of glucose transporter proteins and depend on increased glucose uptake for proliferation.

Neurotransmission by chemical synapses is often described as a simple process, which starts from neurotransmitters release from presynaptic vesicles, is followed by neurotransmitters diffusion through the synaptic cleft to the opposite neuron, and is finally completed by the binding of neurotransmitters to their receptors, thus initializing the activation of a postsynaptic neuron. This description, however, may be too simplified given that there are numerous macromolecules in the synaptic cleft, which interact with neurotransmitters water-soluble domains in a non-specific way. This suggests that the binding of certain neurotransmitters to their receptor's binding site does not take place directly from the water phase but is instead membrane mediated. This possibility is largely overlooked in the literature. Our recent study of 13 neurotransmitters showed that neurotransmitters can be categorized into two groups: membrane-binding (e.g., dopamine, serotonin, epinephrine, norepinephrine, melatonin, and adenosine) and membrane-nonbinding molecules [1]. This categorization corresponds to the receptor types of their neurotransmitters: receptors of membrane-binding neurotransmitters are predominately G-protein coupled receptors with the entry to the binding pocket located close to the lipid membrane surface, while receptors of membrane-nonbinding neurotransmitters are ligand-gated ion channels with a binding pocket located far from the lipid membrane surface. These observations allowed us to postulate the existence of a membrane-mediated mechanism for neurotransmission where neurotransmitters first attach to the membrane surface and then through lateral diffusion find and enter their receptors binding site. Following this idea, we performed studies of dopamine and adenosine entry pathways to the binding pockets in their receptors. Using accelerated molecular dynamics simulations, free energy calculations, and atomistic molecular dynamics simulations we confirmed that the most probable path of dopamine and adenosine entry into their receptor takes place via the membrane-water interface. [1] P. Postila, I. Vattulainen, T. Rog. 2016. Selective effect of synaptic membrane on neurotransmission. Sci. Rep. 6:19345.

The protein transport inside a cell is a complex phenomenon that goes through several difficult steps. The facilitated transport requires sophisticated machineries involving protein assemblies. In this work, we developed a diffusion-reaction model to simulate co-transport kinetics of proteins and lipids. We assume the following: (a) there is always a small lipid concentration of order of the Critical Micellar Concentration (CMC) in equilibrium with the membrane; (b) the binding of lipids to proteins modulates the hydrophobicity of the complexes and, therefore, their ability to interact and merge with the bilayer; and (c) some lipids leave the bilayer to replenish those bound to proteins. The model leads to a pair of integral equations for the time-evolution of the adsorbed proteins in the lipid bilayer. Relationships between transport kinetics, CMC, and lipid-protein binding constants were found. Under particular conditions, a perturbation analysis suggests the onset of kinks in the protein adsorption kinetics. To validate our model, we performed leakage measurements of vesicles composed by either high or low CMC lipids interacting with Islet Amyloid PolyPeptide (IAPP) and Aβ (1-40) used as sample proteins. Since the lipid-protein complex stoichiometry is not easily accessible, molecular dynamics simulations were performed using monomeric IAPP interacting with an increasing number of phospholipids. Main results are the following: (a) 1:1 lipid-protein complexes generally show a faster insertion rate proportional to the complex hydrophobicity and inversely related to lipid CMC; (b) on increasing the number of bound lipids, the protein insertion rate decreases; and (c) at slow lipids desorption rate, the lipid-assisted proteins transport might exhibit a discontinuous behavior and does non-linearly depend on protein concentration.