New insights on the mechanism of polyethylenimine transfection and their implications on gene therapy and DNA vaccines

Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called “proton sponge” effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccinesThis work was supported by the Spanish "Ministerio de Ciencia, Innovación y Universidades" (Project PID2019–109517RB-I00)S

Persistent Neuroadaptations in the Expression of Genes Involved in Cholesterol Homeostasis Induced by Chronic, Voluntary Alcohol Intake in Rats

Alcohol use disorder (AUD) is associated with persistent adaptations in the brain that are believed to participate in the long-lasting vulnerability to relapse after abstinence. Cholesterol, the major sterol compound found in the central nervous system (CNS), plays a major role in maintenance of neuronal morphology, synaptogenesis and synaptic communication and may be involved in alcohol-induced neuroadaptations. In this study, we investigated whether alcohol consumption in a two-bottle choice paradigm followed by 3 weeks of abstinence could alter the expression of genes encoding proteins involved in cholesterol homeostasis in brain regions involved in addiction and relapse, namely the prefrontal cortex (PFC), the nucleus accumbens (NAc), the mesencephalon and the amygdala. We found that voluntary alcohol intake followed by 3 weeks of forced abstinence produces changes in the transcription of several genes encoding proteins directly involved in cholesterol synthesis such as 3-hydroxyl-3-methylglutaryl-coenzyme A (HMGCoA) reductase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1) and farnesyl diphosphate synthase (FDPS) and in its regulation such as sterol regulatory element-binding factor-2 (SREBF2), in cholesterol transport such as ATP-binding cassette subfamily A member 1 (ABCA1) and in cholesterol degradation such as CYP46A1. Interestingly, these changes appeared to be region-specific and suggest that previous chronic exposure to alcohol might durably increase cholesterol metabolism in the PFC, the NAc and the mesencephalon and decrease cholesterol metabolism in the amygdala. Altogether, these results suggest that alcohol consumption leads to durable deregulations in cholesterol metabolism in key areas involved in loss of control over drug use and addiction. These long-term neuroadaptations may participate in the changes in brain structure and functioning that are responsible for the long-lasting risks of relapse to alcohol

A direct interaction of cholesterol with the dopamine transporter prevents its out-to-inward transition

<div><p>Monoamine transporters (MATs) carry out neurotransmitter reuptake from the synaptic cleft, a key step in neurotransmission, which is targeted in the treatment of neurological disorders. Cholesterol (CHOL), a major component of the synaptic plasma membrane, has been shown to exhibit a modulatory effect on MATs. Recent crystal structures of the dopamine transporter (DAT) revealed the presence of two conserved CHOL-like molecules, suggesting a functional protein-CHOL direct interaction. Here, we present extensive atomistic molecular dynamics (MD) simulations of DAT in an outward-facing conformation. In the absence of bound CHOL, DAT undergoes structural changes reflecting early events of dopamine transport: transition to an inward-facing conformation. In contrast, in the presence of bound CHOL, these conformational changes are inhibited, seemingly by an immobilization of the intracellular interface of transmembrane helix 1a and 5 by CHOL. We also provide evidence, from coarse grain MD simulations that the CHOL sites observed in the DAT crystal structures are preserved in all human monoamine transporters (dopamine, serotonin and norepinephrine), suggesting that our findings might extend to the entire family.</p></div

Η κατανομή της Can1 στα εισοσώματα και ο ρόλος της ως μηχανισμός ρύθμισης

Η πλασματική μεμβράνη του ζυμομύκητα έχει δειχθεί ότι αποτελείται από πολλές, αλληλεπικαλυπτόμενες περιοχές. Ενώ είναι γνωστό ότι κάποιες μεμβρανικές πρωτεΐνες κατανέμονται δυναμικά μεταξύ περιοχών, λίγα είναι γνωστά για τους παράγοντες που καθορίζουν αυτή τη συμπεριφορά ή τη φυσιολογική της σημασία. Η παρούσα εργασία εστιάζει σε μία τέτοια πρωτεΐνη, την Can1, τον μεταφορέα αργινίνης του Saccharomyces cerevisiae, ο οποίος συγκεντρώνεται στα εισοσώματα αλλά βρίσκεται και σε άλλες μεμβρανικές περιοχές. Με τη χρήση συνεστιακής λέιζερ μικροσκοπίας, ποσοτικοποιήθηκε ο βαθμός της συγκέντρωσης αυτής και δείχνεται πως εξαρτάται από τη λειτουργικότητα και τη στερεοδιάταξη του μεταφορέα, ενώ είναι ανεξάρτητη της κατάστασης ουβικιτινίωσης. Φαίνεται πώς τα σύνθετα σφιγγολιπίδια παίζουν σημαντικό ρόλο, καθώς αναστολή της βιοσυνθετικής τους πορείας οδηγεί σε εξάλειψη του φαινομένου. Συνδυάζοντας προηγούμενα αποτελέσματα στο πεδίο, προκύπτει ένα μηχανιστικό μοντέλο για την κατανομή της Can1 στην πλασματική μεμβράνη, καθώς και ένας πιθανός ρόλος για τα ίδια τα εισοσώματα.The plasma membrane of yeast has been shown to be highly compartmentalized, composed of numerous, overlapping domains. While it is known that some membrane proteins dynamically localize between compartments, little information is available regarding the factors affecting this dynamic behavior or its physiological significance. This thesis focuses on one such protein, Can1, the arginine permease of yeast, which preferentially clusters in EMCs but is also found in other compartments. Using confocal laser microscopy, the degree of this enrichment was quantified to show that it depends on the activity and conformation of the transporter, while being independent of its ubiquitination status. Complex sphingolipids appear to play an essential role, as inhibition of the relevant biosynthesis pathway results in the extinction of the phenomenon. By integrating previous observations in the field, a mechanistic model thus emerges for the localization behavior of Can1, as well as a possible function of EMC/eisosomes in general

HEADS OR TAILS: LIPID INHIBITORS OF THE GLYCINE TRANSPORTER, GLYT2

Membrane proteins are influenced by the dynamic lipid membrane environment, which can impart stability, mediate protein interactions, and provide highly selective contacts essential for function. Membrane proteins can also bind endogenous lipid ligands or are able to be allosterically modulated by lipids, many of which are thought to access their specific binding sites via the cell membrane. N-arachidonyl glycine (NAGly) is a bioactive lipid that is found in its highest concentrations within the spinal cord and may play an important role in endogenous regulation of glycinergic neurotransmission and pain perception through inhibition of the glycine transporter, GlyT2. In addition to NAGly, a number of lipid inhibitors of GlyT2 have been identified. These compounds are comprised of a long flexible unsaturated acyl tail conjugated to an amino acid or amino acid derivative head group. The aims of my study were two-fold; first to identify new, more potent, lipid inhibitors and develop a structure activity relationship for these compounds; and second, to elucidate the molecular mechanisms of inhibition. Wild type and mutant GlyT2 transporters were expressed in Xenopus laevis oocytes with glycine transport and the subsequent inhibition of transport measured using two-electrode voltage clamp electrophysiology, and radiolabelled uptake of glycine. A library of 55 N-acyl amino acids with varying head and tail groups were synthesised and tested at both GlyT2 and the closely related glycine transporter, GlyT1. Two distinct groups of compounds were tested: the first group maintaining a glycine head group and altering the lipid tail; and the second conjugating the [C18 ω9] oleoyl tail to amino acids with varying properties. I found the lipid constituent of the acyl-glycine analogues is essential for specific interactions and the mechanism of inhibition and is not merely a non-selective, sticky adjunct. There was an ideal chain length, with an order of potency C18 > C16 > C14, and stringently defined double bond conformation and position. Conservative differences between compounds are sufficient to impart or remove inhibitory activity which validates highly specific binding to a subtype specific, allosteric pocket. While changing the tail did not greatly alter potency, analogues where the head group was altered significantly influenced apparent affinity. Acyl amino acids containing an aromatic or positively charged side chain conferred the highest apparent affinity, with C16 ω3 L-Lys possessing the highest potency (10.7 nM). 12 compounds inhibited GlyT2 < 100nM, and one of these inhibitors, oleoyl D-Lys, is also metabolically stable and produces analgesia in a rat model of neuropathic pain. Mutagenesis of extracellular loop 4 (EL4), and transmembrane helices TM5 and TM8 suggest that the allosteric binding site is comprised of a cluster of aromatic residues which may strongly coordinate aromatic or positively charged head groups of the most potent analogues. Additionally, changing the properties of a membrane facing residue alters the otherwise slow washout of lipids. From these results, in addition to dynamic docking studies, it is proposed that acyl amino acids may first diffuse into the lipid bilayer and interact with regions of GlyT2 at the protein-membrane interface. Acyl amino acids then access their final binding site formed by aliphatic and aromatic residues from TM5, TM8, and EL4. It has previously been shown that EL4 undergoes important conformational changes in this family of transporters, where EL4 shifts into the outward facing vestibule to occlude the extracellular side and continue the transport cycle. Acyl amino acids may therefore inhibit GlyT2 by stabilising EL4 in a conformation that does not favour transport. The combination of structure-activity studies with molecular insights provides key information on the mechanism of inhibition which will drive further generation of GlyT2 inhibitors for the treatment of neuropathic pain

The interplay between a dietary preference for fat and sugar, gene expression in the dopaminergic system and executive cognition in humans

Obesity is a health issue of both individual and global importance. Evidence from rodent literature suggests that dietary preferences for fat and sugar might influence dopaminergic signaling in the brain and thus executive cognition. These diet-related changes could provide a mechanistic basis potentially explaining obesity-promoting behaviour. However, valid evidence for this link in humans is still scarce. This thesis aimed to add to this gap by studying dopamine-related gene expression profiles in peripheral cells and executive cognition in a human sample (n = 75). The results provide indications for an association between dietary preference and alterations in dopamingeric sigaling on a peripheral gene expression level even though the group differences were not statistically significant. A link to cognition could not be established with the methods applied. Yet, several targets for future research are suggested to further explore this interplay

Computational Modeling of Realistic Cell Membranes

Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead

Emerging Diversity in Lipid-Protein Interactions

Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions