Glucose restriction in Saccharomyces cerevisiae modulates the phosphorylation pattern of the 20S proteasome and increases its activity.

Caloric restriction is known to extend the lifespan and/or improve diverse physiological parameters in a vast array of organisms. In the yeast Saccharomyces cerevisiae, caloric restriction is performed by reducing the glucose concentration in the culture medium, a condition previously associated with increased chronological lifespan and 20S proteasome activity in cell extracts, which was not due to increased proteasome amounts in restricted cells. Herein, we sought to investigate the mechanisms through which glucose restriction improved proteasome activity and whether these activity changes were associated with modifications in the particle conformation. We show that glucose restriction increases the ability of 20S proteasomes, isolated from Saccharomyces cerevisiae cells, to degrade model substrates and whole proteins. In addition, threonine 55 and/or serine 56 of the α5-subunit, were/was consistently found to be phosphorylated in proteasomes isolated from glucose restricted cells, which may be involved in the increased proteolysis capacity of proteasomes from restricted cells. We were not able to observe changes in the gate opening nor in the spatial conformation in 20S proteasome particles isolated from glucose restricted cells, suggesting that the changes in activity were not accompanied by large conformational alterations in the 20S proteasome but involved allosteric activation of proteasome catalytic site.

Energetics and Kinetic Assembly Pathways of Hepatitis B Virus Capsids in the Presence of Antivirals.

Capsid assembly modulators (CAMs) are antiviral molecules that disturb the formation of icosahedral viral capsids, in particular, those of the Hepatitis B virus (HBV). We report an integrated, physics-driven study elucidating quantitatively the effects of two classes of CAMs on the HBV capsid assembly. Time-resolved small-angle X-ray scattering measurements revealed accelerated self-assembly processes that implied the increase of subunit binding energy from 9- up to 18-fold the thermal energy due to CAMs. Cryotransmission electron microscopy images showed that both classes induce various changes in capsid morphology: from a slight elongation, unrecognized in previous work, to a strong deformation with a capsid size more than twice as large. The observed capsid morphologies were closely reproduced in coarse-grained simulations by varying the Föppl-von-Kármán number, thus pointing out the role of CAMs in altering the capsid elastic energy. Our results illuminate the mechanisms of action of CAMs on HBV capsid assembly at high spatiotemporal resolution and may bring perspectives on virus-derived nanocapsules with tunable morphologies.

Engineering of ion permeable planar membranes and polymersomes based on ß-cyclodextrin-cored star copolymers.

For polymersome-based nanoreactor purposes, we herein present the synthesis and characterization of well-defined star amphiphilic copolymers composed of a beta-cyclodextrin (ßCD) core and seven poly(butylene oxide)-block-polyglycidol (PBO-PGL) arms per side (ßCD-(PBO-PGL)14). The self-assembly behavior of 14-armed ßCD-(PBO-PGL)14 and PGL-PBO-PGL (linear analogues without the ßCD segment) was investigated using scattering techniques for comparison. The morphologies, including vesicles and micelles, are governed by the hydrophobic-to-hydrophilic (weight) ratio, regardless of the polymer architecture (linear or star). Interestingly, despite notable differences in polymer conformation, the produced supramolecular structures were evidenced to be fairly similar on the structural point of view. We subsequently investigated the ion permeability of the membranes of the self-assemblies focusing on the impact of the presence of ßCD. The results demonstrated that the ßCD-containing vesicular membranes are less permeable to H+, compared with ßCD-free vesicular membranes. The presence of ßCD in planar membranes also influences the K+Cl- permeability to some extent. Thus, ßCD-containing membranes can be considered as potential candidates in designing nano-containers towards applications where precise changes in environmental pH are required.

Synthesis of Double Hydrophilic Block Copolymers Poly(2-isopropyl-2-oxazoline-b-ethylenimine) and their DNA Transfection Efficiency.

Gene delivery is now a part of the therapeutic arsenal for vaccination and treatments of inherited or acquired diseases. Polymers represent an opportunity to develop new synthetic vectors for gene transfer, with a prerequisite of improved delivery and reduced toxicity compared to existing polymers. Here, the synthesis in a two-step's procedure of linear poly(ethylenimine-b-2-isopropyl-2-oxazoline) block copolymers with the linear polyethylenimine (lPEI) block of various molar masses is reported; the molar mass of the poly(2-isopropyl-2-oxazoline) (PiPrOx) block has been set to 7 kg mol-1 . Plasmid DNA condensation is successfully achieved, and in vitro transfection efficiency of the copolymers is at least comparable to that obtained with the lPEI of same molar mass. lPEI-b-PiPrOx block copolymers are however less cytotoxic than their linear counterparts. PiPrOx can be a good alternative to PEG which is often used in drug delivery systems. The grafting of histidine moieties on the lPEI block of lPEI-b-PiPrOx does not provide any real improvement of the transfection efficiency. A weak DNA condensation is observed, due to increased steric hindrance along the lPEI backbone. The low cytotoxicity of lPEI-b-PiPrOx makes this family a good candidate for future gene delivery developments.

Glucose restriction in Saccharomyces cerevisiae modulates the phosphorylation pattern of the 20S proteasome and increases its activity

Caloric restriction is known to extend the lifespan and/or improve diverse physiological parameters in a vast array of organisms. In the yeast Saccharomyces cerevisiae, caloric restriction is performed by reducing the glucose concentration in the culture medium, a condition previously associated with increased chronological lifespan and 20S proteasome activity in cell extracts, which was not due to increased proteasome amounts in restricted cells. Herein, we sought to investigate the mechanisms through which glucose restriction improved proteasome activity and whether these activity changes were associated with modifications in the particle conformation. We show that glucose restriction increases the ability of 20S proteasomes, isolated from Saccharomyces cerevisiae cells, to degrade model substrates and whole proteins. In addition, threonine 55 and/or serine 56 of the α5-subunit, were/was consistently found to be phosphorylated in proteasomes isolated from glucose restricted cells, which may be involved in the increased proteolysis capacity of proteasomes from restricted cells. We were not able to observe changes in the gate opening nor in the spatial conformation in 20S proteasome particles isolated from glucose restricted cells, suggesting that the changes in activity were not accompanied by large conformational alterations in the 20S proteasome but involved allosteric activation of proteasome catalytic site.

Fluorescent Marangoni Flows under Quasi-Steady Conditions.

Marangoni flow is among the most intriguing effects in complex fluids and interfacial science. We report here on a fluorescent surfactant that enables to monitor Marangoni flows under quasi-steady conditions, without the need of invasive tracers. The Marangoni zone is clearly visible, and its dynamics can be quantitatively probed both at the air-water interface and within the bulk. In particular, we show that the Marangoni zone exhibits unexpected dependencies with the container size and water depth with the pyrene-tailed surfactant. Additionally, recirculation flows are evidenced by fluorescence near the bottom of the container. This fluorescent probe may find other useful applications in deciphering the complexity of the ubiquitous Marangoni effect.

Cryo-EM reconstructions of BMV-derived virus-like particles reveal assembly defects in the icosahedral lattice structure.

The increasing interest in virus-like particles (VLPs) has been reflected by the growing number of studies on their assembly and application. However, the formation of complete VLPs is a complex phenomenon, making it difficult to rationally design VLPs with desired features de novo. In this paper, we describe VLPs assembled in vitro from the recombinant capsid protein of brome mosaic virus (BMV). The analysis of VLPs was performed by Cryo-EM reconstructions and allowed us to visualize a few classes of VLPs, giving insight into the VLP self-assembly process. Apart from the mature icosahedral VLP practically identical with native virions, we describe putative VLP intermediates displaying non-icosahedral arrangements of capsomers, proposed to occur before the final disorder-order transition stage of icosahedral VLP assembly. Some of the described VLP classes show a lack of protein shell continuity, possibly resulting from too strong interaction with the cargo (in this case tRNA) with the capsid protein. We believe that our results are a useful prerequisite for the rational design of VLPs in the future and lead the way to the effective production of modified VLPs.

Relationships between RNA topology and nucleocapsid structure in a model icosahedral virus.

The process of genome packaging in most of viruses is poorly understood, notably the role of the genome itself in the nucleocapsid structure. For simple icosahedral single-stranded RNA viruses, the branched topology due to the RNA secondary structure is thought to lower the free energy required to complete a virion. We investigate the structure of nucleocapsids packaging RNA segments with various degrees of compactness by small-angle x-ray scattering and cryotransmission electron microscopy. The structural differences are mild even though compact RNA segments lead on average to better-ordered and more uniform particles across the sample. Numerical calculations confirm that the free energy is lowered for the RNA segments displaying the larger number of branch points. The effect is, however, opposite with synthetic polyelectrolytes, in which a star topology gives rise to more disorder in the capsids than a linear topology. If RNA compactness and size account in part for the proper assembly of the nucleocapsid and the genome selectivity, other factors most likely related to the host cell environment during viral assembly must come into play as well.

Nonsymmetrical Dynamics of the HBV Capsid Assembly and Disassembly Evidenced by Their Transient Species.

As with many protein multimers studied in biophysics, the assembly and disassembly dynamical pathways of hepatitis B virus (HBV) capsid proteins are not symmetrical. Using time-resolved small-angle X-ray scattering and singular value decomposition analysis, we have investigated these processes in vitro by a rapid change of salinity or chaotropicity. Along the assembly pathway, the classical nucleation-growth mechanism is followed by a slow relaxation phase during which capsid-like transient species self-organize in accordance with the theoretical prediction that the capture of the few last subunits is slow. By contrast, the disassembly proceeds through unexpected, fractal-branched clusters of subunits that eventually vanish over a much longer time scale. On the one hand, our findings confirm and extend previous views as to the hysteresis phenomena observed and theorized in capsid formation and dissociation. On the other hand, they uncover specifics that may directly relate to the functions of HBV subunits in the viral cycle.

Ultrashort Peptide Theranostic Nanoparticles by Microfluidic-Assisted Rapid Solvent Exchange.

Ultrashort peptides (USPs), composed of three to seven amino acids, can self-assemble into nanofibers in pure water. Here, using hydrodynamic focusing and a solvent exchange method on a microfluidic setup, we convert these nanofibers into globular nanoparticles with excellent dimensional control and polydispersity. Thanks to USP nanocarriers' structure, different drugs can be loaded. We used Curcumin as a model drug to evaluate the performance of USP nanocarriers as a novel drug delivery vehicle. These nanoparticles can efficiently cross the cell membrane and possess nonlinear optical properties. Therefore, we envisage USP nanoparticles as promising future theranostic nanocarriers.

How a Virus Circumvents Energy Barriers to Form Symmetric Shells.

Previous self-assembly experiments on a model icosahedral plant virus have shown that, under physiological conditions, capsid proteins initially bind to the genome through an en masse mechanism and form nucleoprotein complexes in a disordered state, which raises the question as to how virions are assembled into a highly ordered structure in the host cell. Using small-angle X-ray scattering, we find out that a disorder-order transition occurs under physiological conditions upon an increase in capsid protein concentrations. Our cryo-transmission electron microscopy reveals closed spherical shells containing in vitro transcribed viral RNA even at pH 7.5, in marked contrast with the previous observations. We use Monte Carlo simulations to explain this disorder-order transition and find that, as the shell grows, the structures of disordered intermediates in which the distribution of pentamers does not belong to the icosahedral subgroups become energetically so unfavorable that the caps can easily dissociate and reassemble, overcoming the energy barriers for the formation of perfect icosahedral shells. In addition, we monitor the growth of capsids under the condition that the nucleation and growth is the dominant pathway and show that the key for the disorder-order transition in both en masse and nucleation and growth pathways lies in the strength of elastic energy compared to the other forces in the system including protein-protein interactions and the chemical potential of free subunits. Our findings explain, at least in part, why perfect virions with icosahedral order form under different conditions including physiological ones.

Polyglycidol-Stabilized Nanoparticles as a Promising Alternative to Nanoparticle PEGylation: Polymer Synthesis and Protein Fouling Considerations.

We herein demonstrate the outstanding protein-repelling characteristic of star-like micelles and polymersomes manufactured from amphiphilic block copolymers made by poly(butylene oxide) (PBO) hydrophobic segments and polyglycidol (PGL) hydrophilic outer shells. Although positively charged proteins (herein modeled by lysozyme) may adsorb onto the surface of micelles and polymersomes where the assemblies are stabilized by short PGL chains (degree of polymerization smaller than 15), the protein adsorption vanishes when the degree of polymerization of the hydrophilic segment (PGL) is higher than ∼20, regardless the morphology. This has been probed by using three different model proteins which are remarkably different concerning molecular weight, size, and zeta potential (bovine serum albumin (BSA), lysozyme, and immunoglobulin G (IgG)). Indeed, the adsorption of the most abundant plasma protein (herein modeled as BSA) is circumvented even by using very short PGL shells due to the highly negative zeta potential of the produced assemblies which presumably promote protein-nanoparticle electrostatic repulsion. The negative zeta potential, on the other hand, enables lysozyme adsorption, and the phenomenon is governed by electrostatic forces as evidenced by isothermal titration calorimetry. Nevertheless, the protein coating can be circumvented by slightly increasing the degree of polymerization of the hydrophilic segment. Notably, the PGL length required to circumvent protein fouling is significantly smaller than the one required for PEO. This feature and the safety concerns regarding the synthetic procedures on the preparation of poly(ethylene oxide)-based amphiphilic copolymers might make polyglycidol a promising alternative toward the production of nonfouling spherical particles.

Improved histidinylated lPEI polyplexes for skeletal muscle cells transfection.

Linear Polyethylenimine (lPEI) is an efficient cationic polymer for transfecting cells, both in vitro and in vivo, but poses concerns regarding cytotoxicity. Histidinylated lPEI (His-lPEI) exhibits also high transfection efficiency but lower cytotoxicity than lPEI. For the first time, we tested polyfection efficiency of polyplexes comprising both lPEI and His-lPEI. A series of pDNA polyplexes was prepared with mixtures of lPEI and His-lPEI and the amount of each polymer within His-lPEI/lPEI polyplexes was determined by flow cytometry. We show that His-lPEI/lPEI polyplexes exhibit properties similar to lPEI polyplexes in terms of size, morphology, assembly with pDNA, and polyplex stability while His-lPEI/lPEI polyplexes exhibit properties similar to His-lPEI polyplexes in terms of buffering capacity. Compared to polyplexes consisting only of lPEI or His-lPEI, the transfection profile reveals that His-lPEI/lPEI polyplexes containing 30% to 57% lPEI strongly increase polyfection efficiency of NIH3T3 fibroblasts and murine, as well as human skeletal muscle cell lines without cytotoxicity. Importantly, improved transfection of human dystrophin deficient skeletal muscle cell lines was obtained. These results indicate that His-lPEI/lPEI polyplexes are an improved non-viral vector for efficient transfection of dystrophin deficient skeletal muscle cell lines that should be tested on animals.

Interactions between the Molecular Components of the Cowpea Chlorotic Mottle Virus Investigated by Molecular Dynamics Simulations.

The formation of a viral particle generally involves hundreds of proteins, making the assembly process intricate. Despite its intrinsic complexity, the production of a viral particle begins through the interaction between the basic assembly components. For the cowpea chlorotic mottle virus (CCMV), the first steps of the assembly process involve dimers of the capsid protein. Here, we carried out atomistic molecular dynamics simulations to investigate the initial assembly process of CCMV to get insight into the interactions at the molecular level. We found that salinity not only affects the electrostatic interactions between dimers but also changes the conformation of the positively charged N-terminal tails and can cause a serious steric hindrance for other dimers binding to the hydrophobic domains. An RNA rod was used to mimic a long segment of a viral genome and to study its interaction with dimers. We observed that the dimer with tails prefers to bind on the RNA rod with its positively charged inner side. The dimer-RNA interaction was found to be as strong as the dimer-dimer interaction, whereas the association energies between a dimer and a pentamer or a hexamer of dimers were high but strongly depended on the presence of the tails. Upon heating, the capsid experienced a shrinkage accompanied by a loss of order in the icosahedral crystal structure.

Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte.

The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use time-resolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.

Investigating the thermal dissociation of viral capsid by lattice model.

The dissociation of icosahedral viral capsids was investigated by a homogeneous and a heterogeneous lattice model. In thermal dissociation experiments with cowpea chlorotic mottle virus and probed by small-angle neutron scattering, we observed a slight shrinkage of viral capsids, which can be related to the strengthening of the hydrophobic interaction between subunits at increasing temperature. By considering the temperature dependence of hydrophobic interaction in the homogeneous lattice model, we were able to give a better estimate of the effective charge. In the heterogeneous lattice model, two sets of lattice sites represented different capsid subunits with asymmetric interaction strengths. In that case, the dissociation of capsids was found to shift from a sharp one-step transition to a gradual two-step transition by weakening the hydrophobic interaction between AB and CC subunits. We anticipate that such lattice models will shed further light on the statistical mechanics underlying virus assembly and disassembly.

Identification of a major intermediate along the self-assembly pathway of an icosahedral viral capsid by using an analytical model of a spherical patch.

Viruses are astonishing edifices in which hundreds of molecular building blocks fit into the final structure with pinpoint accuracy. We established a robust kinetic model accounting for the in vitro self-assembly of a capsid shell derived from an icosahedral plant virus by using time-resolved small-angle X-ray scattering (TR-SAXS) data at high spatiotemporal resolution. By implementing an analytical model of a spherical patch into a global fitting algorithm, we managed to identify a major intermediate species along the self-assembly pathway. With a series of data collected at different protein concentrations, we showed that free dimers self-assembled into a capsid through an intermediate resembling a half-capsid. The typical lifetime of the intermediate was a few seconds and yet the presence of so large an oligomer was not reported before. The progress in instrumental detection along with the development of powerful algorithms for data processing contribute to shedding light on nonequilibrium processes in highly complex systems such as viruses.

Reconstruction of the Disassembly Pathway of an Icosahedral Viral Capsid and Shape Determination of Two Successive Intermediates.

Viral capsids derived from an icosahedral plant virus widely used in physical and nanotechnological investigations were fully dissociated into dimers by a rapid change of pH. The process was probed in vitro at high spatiotemporal resolution by time-resolved small-angle X-ray scattering using a high brilliance synchrotron source. A powerful custom-made global fitting algorithm allowed us to reconstruct the most likely pathway parametrized by a set of stoichiometric coefficients and to determine the shape of two successive intermediates by ab initio calculations. None of these two unexpected intermediates was previously identified in self-assembly experiments, which suggests that the disassembly pathway is not a mirror image of the assembly pathway. These findings shed new light on the mechanisms and the reversibility of the assembly/disassembly of natural and synthetic virus-based systems. They also demonstrate that both the structure and dynamics of an increasing number of intermediate species become accessible to experiments.

Weighing polyelectrolytes packaged in viruslike particles.

This Letter reports on the remarkable selectivity of capsid proteins for packaging synthetic polyelectrolytes in viruslike particles. By applying the contrast variation method in small-angle neutron scattering, we accurately estimated the mean mass of packaged polyelectrolytes ⟨Mp⟩ and that of the surrounding capsid ⟨Mcap⟩. Remarkably, the mass ratio ⟨Mp⟩/⟨Mcap⟩ was invariant for polyelectrolyte molecular weights spanning more than 2 orders of magnitude. To do so, capsids either packaged several chains simultaneously or selectively retained the shortest chains that could fit the capsid interior. Our data are in qualitative agreement with theoretical predictions based on free energy minimization and emphasize the importance of protein self-energy. These findings may give new insights into the nonspecific origin of genome selectivity for a number of viral systems.

On-chip controlled surfactant-DNA coil-globule transition by rapid solvent exchange using hydrodynamic flow focusing.

This paper presents a microfluidic method for precise control of the size and polydispersity of surfactant-DNA nanoparticles. A mixture of surfactant and DNA dispersed in 35% ethanol is focused between two streams of pure water in a microfluidic channel. As a result, a rapid change of solvent quality takes place in the central stream, and the surfactant-bound DNA molecules undergo a fast coil-globule transition. By adjusting the concentrations of DNA and surfactant, fine-tuning of the nanoparticle size, down to a hydrodynamic diameter of 70 nm with a polydispersity index below 0.2, can be achieved with a good reproducibility.