Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely ß conformations, analogous to models of pathological phase separation.

Assembling membraneless organelles from de novo designed proteins.

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological processes and augment natural systems. The bottom-up design of self-assembled objects such as microcompartments and membraneless organelles is one such challenge. Here we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli. To do this, we combine de novo α-helical sequences, intrinsically disordered linkers and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from arrested assemblies to dynamic condensates. The designs are characterized in cells and in vitro using biophysical methods and soft-matter physics. Finally, we use the designed polypeptide to co-compartmentalize a functional enzyme pair in E. coli, improving product formation close to the theoretical limit.

Correction: An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii.

[This corrects the article DOI: 10.1371/journal.ppat.1010321.].

Polymer-induced biofilms for enhanced biocatalysis.

The intrinsic resilience of biofilms to environmental conditions makes them an attractive platform for biocatalysis, bioremediation, agriculture or consumer health. However, one of the main challenges in these areas is that beneficial bacteria are not necessarily good at biofilm formation. Currently, this problem is solved by genetic engineering or experimental evolution, techniques that can be costly and time consuming, require expertise in molecular biology and/or microbiology and, more importantly, are not suitable for all types of microorganisms or applications. Here we show that synthetic polymers can be used as an alternative, working as simple additives to nucleate the formation of biofilms. Using a combination of controlled radical polymerization and dynamic covalent chemistry, we prepare a set of synthetic polymers carrying mildly cationic, aromatic, heteroaromatic or aliphatic moieties. We then demonstrate that hydrophobic polymers induce clustering and promote biofilm formation in MC4100, a strain of Escherichia coli that forms biofilms poorly, with aromatic and heteroaromatic moieties leading to the best performing polymers. Moreover, we compare the effect of the polymers on MC4100 against PHL644, an E. coli strain that forms biofilms well due to a single point mutation which increases expression of the adhesin curli. In the presence of selected polymers, MC4100 can reach levels of biomass production and curli expression similar or higher than PHL644, demonstrating that synthetic polymers promote similar changes in microbial physiology than those introduced following genetic modification. Finally, we demonstrate that these polymers can be used to improve the performance of MC4100 biofilms in the biocatalytic transformation of 5-fluoroindole into 5-fluorotryptophan. Our results show that incubation with these synthetic polymers helps MC4100 match and even outperform PHL644 in this biotransformation, demonstrating that synthetic polymers can underpin the development of beneficial applications of biofilms.

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii.

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus.

Core-shell microneedle patch for six-month controlled-release contraceptive delivery.

There is a tremendous need for simple-to-administer, long-acting contraception, which can increase access to improved family planning. Microneedle (MN) patches enable simple self-administration and have previously been formulated for 1-2 months' controlled release of contraceptive hormone using monolithic polymer/drug MN designs having first-order release kinetics. To achieve zero-order release, we developed a novel core-shell MN patch where the shell acts as a rate-controlling membrane to delay release of a contraceptive hormone, levonorgestrel (LNG), for 6 months. In this approach, LNG was encapsulated in a poly(lactide-co-glycolide) (PLGA) core surrounded by a poly(l-lactide) (PLLA) shell and a poly(D,L-lactide) (PLA) cap that were fabricated by sequential casting into a MN mold. Upon application to skin, the core-shell MNs utilized an effervescent interface to separate from the patch backing within 1 min. The core-shell design limited the initial 24 h burst release of LNG to 5.8 ± 0.5% and achieved roughly zero-order LNG release for 6.2 ± 0.1 months in vitro. A monolithic MN patch formulated with the same LNG and PLGA core, but without the rate-controlling PLLA shell and PLA cap had a larger LNG burst release of 22.6 ± 2.0% and achieved LNG release for just 2.1 ± 0.2 months. This study provides the first core-shell MN patch for controlled months-long drug release and supports the development of long-acting contraception using a simple-to-administer, twice-per-year MN patch.

Skin Vaccination with Ebola Virus Glycoprotein Using a Polyphosphazene-Based Microneedle Patch Protects Mice against Lethal Challenge.

Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (poly[di(carboxylatophenoxy)phosphazene], PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.

Binding of chloroaurate to polytyrosine-PEG micelles leads to an anti-Turkevich pattern of reduction.

Here we report formation of gold nanoparticles (GNPs) in micelles of polytyrosine-PEG copolymers that combine the properties of a reducer and a stabilizer. The size and properties of the GNPs were tailored by the excess chloroaurate over the copolymer. The latter quickly formed non-covalent complexes with HAuCl4 and then slowly reduced it to form GNPs. 3 Tyr residues are consumed by reduction of one mole of chloroaurate. The size of the GNPs was controlled by the [Tyr]/[Au(iii)] molar ratio. Small GNPs with D ≅ 8 nm were formed at [Tyr]/[Au(iii)] = 0.5-1.5. 90% of these small GNPs remained bound to the copolymer and could be stored in a lyophilized state. Such polypeptide-gold hybrid materials produced at [Tyr]/[Au(iii)] = 0.5 demonstrated high activity in the catalytic reduction of 4-nitrophenol by sodium borohydride. [Tyr]/[Au(iii)] = 5 led to the formation of large nanoplates (D ≅ 30-60 nm). Thus, in the polymer-based system the GNP size grew in line with the excess of the reducing agent in contrast to Turkevich synthesis of GNPs with citric acid, which also combines the functions of a stabilizer and a reducer. The difference results from the reduction of HAuCl4 in solution according to the Turkevich method and in the micelles of the amphiphilic polymer where the seed growth is limited by the amount of neighboring reducer.

Cutaneous vaccination ameliorates Zika virus-induced neuro-ocular pathology via reduction of anti-ganglioside antibodies.

Zika virus (ZIKV) causes moderate to severe neuro-ocular sequelae, with symptoms ranging from conjunctivitis to Guillain-Barré Syndrome (GBS). Despite the international threat ZIKV poses, no licensed vaccine exists. As ZIKV and DENV are closely related, antibodies against one virus have demonstrated the ability to enhance the other. To examine if vaccination can confer robust, long-term protection against ZIKV, preventing neuro-ocular pathology and long-term inflammation in immune-privileged compartments, BALB/c mice received two doses of unadjuvanted inactivated whole ZIKV vaccine (ZVIP) intramuscularly (IM) or cutaneously with dissolving microneedle patches (MNP). MNP immunization induced significantly higher B and T cell responses compared to IM vaccination, resulting in increased antibody titers with greater avidity for ZPIV as well as increased numbers of IFN-γ, TNF-α, IL- and IL-4 secreting T cells. When compared to IM vaccination, antibodies generated by cutaneous vaccination demonstrated greater neutralization activity, increased cross-reactivity with Asian and African lineage ZIKV strains (PRVABC59, FLR, and MR766) and Dengue virus (DENV) serotypes, limited ADE, and lower reactivity to GBS-associated gangliosides. MNP vaccination effectively controlled viremia and inflammation, preventing neuro-ocular pathology. Conversely, IM vaccination exacerbated ocular pathology, resulting in uncontrolled, long-term inflammation. Importantly, neuro-ocular pathology correlated with anti-ganglioside antibodies implicated in demyelination and GBS. This study highlights the importance of longevity studies in ZIKV immunization, and the need of exploring alternative vaccination platforms to improve the quality of vaccine-induced immune responses.

STAR particles for enhanced topical drug and vaccine delivery.

Drug delivery to the skin is highly constrained by the stratum corneum barrier layer1. Here, we developed star-shaped particles, termed STAR particles, to dramatically increase skin permeability. STAR particles are millimeter-scale particles made of aluminum oxide or stainless steel with micron-scale projections designed to create microscopic pores across the stratum corneum. After gentle topical application for 10 s to porcine skin ex vivo, delivery of dermatological drugs and macromolecules, including those that cannot be given topically, was increased by 1 to 2 orders of magnitude. In mice treated with topical 5-fluorouracil, use of STAR particles increased the efficacy of the drug in suppressing the growth of subcutaneous melanoma tumors and prolonging survival. Moreover, topical delivery of tetanus toxoid vaccine to mice using STAR particles generated immune responses that were at least as strong as delivery of the vaccine by intramuscular injection, albeit at a higher dose for topical than intramuscular vaccine administration. STAR particles were well tolerated and effective at creating micropores when applied to the skin of human participants. Use of STAR particles provides a simple, low-cost and well-tolerated method for increasing drug and vaccine delivery to the skin and could widen the range of compounds that can be topically administered.

Stable incorporation of GM-CSF into dissolvable microneedle patch improves skin vaccination against influenza.

The widely used influenza subunit vaccine would benefit from increased protection rates in vulnerable populations. Skin immunization by microneedle (MN) patch can increase vaccine immunogenicity, as well as increase vaccination coverage due to simplified administration. To further increase immunogenicity, we used granulocyte-macrophage colony stimulating factor (GM-CSF), an immunomodulatory cytokine already approved for skin cancer therapy and cancer support treatment. GM-CSF has been shown to be upregulated in skin following MN insertion. The GM-CSF-adjuvanted vaccine induced robust and long-lived antibody responses cross-reactive to homosubtypic and heterosubtypic influenza viruses. Addition of GM-CSF resulted in increased memory B cell persistence relative to groups given influenza vaccine alone and led to rapid lung viral clearance following lethal infection with homologous virus in the mouse model. Here we demonstrate that successful incorporation of the thermolabile cytokine GM-CSF into MN resulted in improved vaccine-induced protective immunity holding promise as a novel approach to improved influenza vaccination. To our knowledge, this is the first successful incorporation of a cytokine adjuvant into dissolvable MNs, thus advancing and diversifying the rapidly developing field of MN vaccination technology.

Mammalian amyloidogenic proteins promote prion nucleation in yeast.

Fibrous cross-ß aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-ß (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes.

Peroxyoxalate Chemiluminescent Reaction as a Tool for Elimination of Tumour Cells Under Oxidative Stress.

The overproduction of hydrogen peroxide is an inherent feature of some tumour cells and inflamed tissues. We took advantage of this peculiarity to eliminate cells using chemiluminescent peroxyoxalate reaction. We designed dispersions containing polyoxalate and tetramethylhematoporhyrin (TMHP) in dimethylphthalate droplets stabilized with Pluronic L64. The porphyrin plays the dual role. On the one hand, it serves as an activator of the peroxyoxalate reaction of polyoxalate with intracellular hydrogen peroxide and experiences excitation as a result of the reaction. The light emitted in the reaction in the model system without cells was used to optimize the dispersion's composition. On the other hand, TMHP acts as a photosensitizer (PS) causing cell damage. The formation of singlet oxygen led to cell elimination if the dispersions were used in combination with inducers of oxidative stress: hydrogen peroxide, paraquat, antitumour drug doxorubicin, or a nutritional additive menadione. The PS-induced cytotoxicity correlated with the level of intracellular ROS. The developed approach targeted to endogenous ROS is orthogonal to the classical chemotherapy and can be applied to increase its efficiency.

Yeast Short-Lived Actin-Associated Protein Forms a Metastable Prion in Response to Thermal Stress.

Self-perpetuating ordered protein aggregates (amyloids and prions) are associated with a variety of neurodegenerative disorders. Although environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. We have employed endogenous yeast prions as a model system to study environmental control of amyloid formation. A short-lived actin-associated yeast protein Lsb2 can trigger prion formation by other proteins in a mode regulated by the cytoskeleton and ubiquitin-dependent processes. Here, we show that such a heterologous prion induction is due to the ability of Lsb2 to form a transient prion state, generated in response to thermal stress. Evolutionary acquisition of prion-inducing activity by Lsb2 is traced to a single amino acid change, coinciding with the acquisition of thermotolerance in the Saccharomyces yeast lineage. This raises the intriguing possibility that the transient prion formation could aid in functioning of Lsb2 at higher temperatures.

Collection of analytes from microneedle patches.

Clinical medicine and public health would benefit from simplified acquisition of biological samples from patients that can be easily obtained at point of care, in the field, and by patients themselves. Microneedle patches are designed to serve this need by collecting dermal interstitial fluid containing biomarkers without the dangers, pain, or expertise needed to collect blood. This study presents novel methods to collect biomarker analytes from microneedle patches for analysis by integration into conventional analytical laboratory microtubes and microplates. Microneedle patches were made out of cross-linked hydrogel composed of poly(methyl vinyl ether-alt-maleic acid) and poly(ethylene glycol) prepared by micromolding. Microneedle patches were shown to swell with water up to 50-fold in volume, depending on degree of polymer cross-linking, and to collect interstitial fluid from the skin of rats. To collect analytes from microneedle patches, the patches were mounted within the cap of microcentrifuge tubes or formed the top of V-bottom multiwell microplates, and fluid was collected in the bottom of the tubes under gentle centrifugation. In another method, microneedle patches were attached to form the bottom of multiwell microplates, thereby enabling in situ analysis. The simplicity of biological sample acquisition using microneedle patches coupled with the simplicity of analyte collection from microneedles patches integrated into conventional analytical equipment could broaden the reach of future screening, diagnosis, and monitoring of biomarkers in healthcare and environmental/workplace settings.

Prion induction by the short-lived, stress-induced protein Lsb2 is regulated by ubiquitination and association with the actin cytoskeleton.

Yeast prions are self-perpetuating, QN-rich amyloids that control heritable traits and serve as a model for mammalian amyloidoses. De novo prion formation by overproduced prion protein is facilitated by other aggregated QN-rich protein(s) and is influenced by alterations of protein homeostasis. Here we explore the mechanism by which the Las17-binding protein Lsb2 (Pin3) promotes conversion of the translation termination factor Sup35 into its prion form, [PSI(+)]. We show that Lsb2 localizes with some Sup35 aggregates and that Lsb2 is a short-lived protein whose levels are controlled via the ubiquitin-proteasome system and are dramatically increased by stress. Loss of Lsb2 decreases stability of [PSI(+)] after brief heat shock. Mutations interfering with Lsb2 ubiquitination increase prion induction, while a mutation eliminating association of Lsb2 with the actin cytoskeleton blocks its aggregation and prion-inducing ability. These findings directly implicate the UPS and actin cytoskeleton in regulating prions via a stress-inducible QN-rich protein.

Genetic and epigenetic control of the efficiency and fidelity of cross-species prion transmission.

Self-perpetuating amyloid-based protein isoforms (prions) transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Although mechanisms that control species specificity of prion transmission are poorly understood, studies of closely related orthologues of yeast prion protein Sup35 demonstrate that cross-species prion transmission is modulated by both genetic (specific sequence elements) and epigenetic (prion variants, or 'strains') factors. Depending on the prion variant, the species barrier could be controlled at the level of either heterologous co-aggregation or conversion of the aggregate-associated heterologous protein into a prion polymer. Sequence divergence influences cross-species transmission of different prion variants in opposing ways. The ability of a heterologous prion domain to either faithfully reproduce or irreversibly switch the variant-specific prion patterns depends on both sequence divergence and the prion variant. Sequence variations within different modules of prion domains contribute to transmission barriers in different cross-species combinations. Individual amino acid substitutions within short amyloidogenic stretches drastically alter patterns of cross-species prion conversion, implicating these stretches as major determinants of species specificity.

The Hofmeister effect on amyloid formation using yeast prion protein.

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross-beta aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N-proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to beta-sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid-forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B-viscosity coefficient of the Jones-Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.