Role of the Protein Corona in the Colloidal Behavior of Microplastics.

Microparticles of polyethylene and polypropylene are largely found in aquatic environments because they are the most produced and persistent plastic materials. Once in biological media, they are covered by a layer of molecules, the so-called corona, mostly composed of proteins. A yeast protein extract from Saccharomyces cerevisiae was used as a protein system to observe interactions in complex biological media. Proteins, acting as surfactants and providing hydrophilic surfaces, allow the dispersion of highly hydrophobic particles in water and stabilize them. After 24 h, the microplastic quantity was up to 1 × 1011 particles per liter, whereas without protein, no particles remained in solution. Label-free imaging of the protein corona by synchrotron radiation deep UV fluorescence microscopy (SR-DUV) was performed. In situ images of the protein corona were obtained, and the adsorbed protein quantity, the coverage rate, and the corona heterogeneity were determined. The stability kinetics of the microplastic suspensions were measured by light transmission using a Turbiscan analyzer. Together, the microscopic and kinetics results demonstrate that the protein corona can very efficiently stabilize microplastics in solution provided that the protein corona quality is sufficient. Microplastic stability depends on different parameters such as the particle's intrinsic properties (size, density, hydrophobicity) and the protein corona formation that changes the particle wettability, electrostatic charge, and steric hindrance. By controlling these parameters with proteins, it becomes possible to keep microplastics in and out of solution, paving the way for applications in the field of microplastic pollution control and remediation.

A proteome scale study reveals how plastic surfaces and agitation promote protein aggregation.

Protein aggregation in biotherapeutics can reduce their activity and effectiveness. It may also promote immune reactions responsible for severe adverse effects. The impact of plastic materials on protein destabilization is not totally understood. Here, we propose to deconvolve the effects of material surface, air/liquid interface, and agitation to decipher their respective role in protein destabilization and aggregation. We analyzed the effect of polypropylene, TEFLON, glass and LOBIND surfaces on the stability of purified proteins (bovine serum albumin, hemoglobin and α-synuclein) and on a cell extract composed of 6000 soluble proteins during agitation (P = 0.1-1.2 W/kg). Proteomic analysis revealed that chaperonins, intrinsically disordered proteins and ribosomes were more sensitive to the combined effects of material surfaces and agitation while small metabolic oligomers could be protected in the same conditions. Protein loss observations coupled to Raman microscopy, dynamic light scattering and proteomic allowed us to propose a mechanistic model of protein destabilization by plastics. Our results suggest that protein loss is not primarily due to the nucleation of small aggregates in solution, but to the destabilization of proteins exposed to material surfaces and their subsequent aggregation at the sheared air/liquid interface, an effect that cannot be prevented by using LOBIND tubes. A guidance can be established on how to minimize these adverse effects. Remove one of the components of this combined stress - material, air (even partially), or agitation - and proteins will be preserved.

Protein Corona Composition of Silica Nanoparticles in Complex Media: Nanoparticle Size does not Matter.

Biomolecules, and particularly proteins, bind on nanoparticle (NP) surfaces to form the so-called protein corona. It is accepted that the corona drives the biological distribution and toxicity of NPs. Here, the corona composition and structure were studied using silica nanoparticles (SiNPs) of different sizes interacting with soluble yeast protein extracts. Adsorption isotherms showed that the amount of adsorbed proteins varied greatly upon NP size with large NPs having more adsorbed proteins per surface unit. The protein corona composition was studied using a large-scale label-free proteomic approach, combined with statistical and regression analyses. Most of the proteins adsorbed on the NPs were the same, regardless of the size of the NPs. To go beyond, the protein physicochemical parameters relevant for the adsorption were studied: electrostatic interactions and disordered regions are the main driving forces for the adsorption on SiNPs but polypeptide sequence length seems to be an important factor as well. This article demonstrates that curvature effects exhibited using model proteins are not determining factors for the corona composition on SiNPs, when dealing with complex biological media.

Importance of Post-translational Modifications in the Interaction of Proteins with Mineral Surfaces: The Case of Arginine Methylation and Silica surfaces.

Understanding the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest for both basic research and practical applications involving nanotechnology. From the list of cellular proteins with the highest affinity for silica nanoparticles, we highlighted the group of proteins containing arginine-glycine-glycine (RGG) motifs. Biochemical experiments confirmed that RGG motifs interact strongly with the silica surfaces. The affinity of these motifs is further increased when the R residue is asymmetrically, but not symmetrically, dimethylated. Molecular dynamics simulations show that the asymmetrical dimethylation generates an electrostatic asymmetry in the guanidinium group of the R residue, orientating and stabilizing it on the silica surface. The RGG motifs (methylated or not) systematically target the siloxide groups on the silica surface through an ionic interaction, immediately strengthened by hydrogen bonds with proximal silanol and siloxane groups. Given that, in vivo, RGG motifs are often asymmetrically dimethylated by specific cellular methylases, our data add support to the idea that this type of methylation is a key mechanism for cells to regulate the interaction of the RGG proteins with their cellular partners.

The double life of the ribosome: When its protein folding activity supports prion propagation.

It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

RNA-binding proteins are a major target of silica nanoparticles in cell extracts.

Upon contact with biological fluids, nanoparticles (NPs) are readily coated by cellular compounds, particularly proteins, which are determining factors for the localization and toxicity of NPs in the organism. Here, we improved a methodological approach to identify proteins that adsorb on silica NPs with high affinity. Using large-scale proteomics and mixtures of soluble proteins prepared either from yeast cells or from alveolar human cells, we observed that proteins with large unstructured region(s) are more prone to bind on silica NPs. These disordered regions provide flexibility to proteins, a property that promotes their adsorption. The statistical analyses also pointed to a marked overrepresentation of RNA-binding proteins (RBPs) and of translation initiation factors among the adsorbed proteins. We propose that silica surfaces, which are mainly composed of Si-O- and Si-OH groups, mimic ribose-phosphate molecules (rich in -O- and -OH) and trap the proteins able to interact with ribose-phosphate containing molecules. Finally, using an in vitro assay, we showed that the sequestration of translation initiation factors by silica NPs results in an inhibition of the in vitro translational activity. This result demonstrates that characterizing the protein corona of various NPs would be a relevant approach to predict their potential toxicological effects.

Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation.

6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.

Glutathione is essential to preserve nuclear function and cell survival under oxidative stress.

Glutathione (GSH) is considered the most important redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing various amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear components and activities are protected against oxidative injury. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Structural determinants for protein adsorption/non-adsorption to silica surface.

The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption.

Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A.

Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd(2+)) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd(2+) rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd(2+), but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I-Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag(+) and Hg(2+), which likewise perturb the Pol I-Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I-Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals.

Endoplasmic reticulum is a major target of cadmium toxicity in yeast.

Cadmium (Cd(2+)) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd(2+) cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA. Furthermore, mutant strains (Delta ire1 and Delta hac1) unable to induce the UPR are hypersensitive to Cd(2+), but not to arsenite and mercury. The full functionality of the pathways involved in ER stress response is required for Cd(2+) tolerance. The data also suggest that Cd(2+)-induced ER stress and Cd(2+) toxicity are a direct consequence of Cd(2+) accumulation in the ER. Cd(2+) does not inhibit disulfide bond formation but perturbs calcium metabolism. In particular, Cd(2+) activates the calcium channel Cch1/Mid1, which also contributes to Cd(2+) entry into the cell. The results reinforce the interest of using yeast as a cellular model to study toxicity mechanisms in eukaryotic cells.

Development of a new method for absolute protein quantification on 2-D gels.

With the development of systems biology projects aimed at modeling the cell, accurate and absolute measurements of cellular protein concentrations are increasingly important. However, methods for absolute quantification at the proteomic level remain rare. Using the yeast Saccharomyces cerevisiae, we propose a new method based on the radioactive labeling with an (35)S compound and 2-D PAGE. The principle is simple: cells are grown for more than four generations in the presence of a unique sulfur source labeled at a defined specific radioactivity, ensuring that more than 90% of the proteins are labeled at the same specific radioactivity as the sulfur source. After separation of (35)S-labeled proteins on 2-D gels, each protein is counted. The amount of each protein present in the gel is then calculated, from which is deduced the amount of each protein per cell. The method, limited to soluble and abundant proteins visible on 2-D gels, is simple, precise and reproducible and does not require an internal standard. We use it to compare the amounts of proteins in two growth conditions: 100 microM sulfate or 500 microM methionine. Up to now, we only had transcriptional data on the expression of these proteins in both conditions.

H2O2 activates the nuclear localization of Msn2 and Maf1 through thioredoxins in Saccharomyces cerevisiae.

The cellular response to hydrogen peroxide (H(2)O(2)) is characterized by a repression of growth-related processes and an enhanced expression of genes important for cell defense. In budding yeast, this response requires the activation of a set of transcriptional effectors. Some of them, such as the transcriptional activator Yap1, are specific to oxidative stress, and others, such as the transcriptional activators Msn2/4 and the negative regulator Maf1, are activated by a wide spectrum of stress conditions. How these general effectors are activated in response to oxidative stress remains an open question. In this study, we demonstrate that the two cytoplasmic thioredoxins, Trx1 and Trx2, are essential to trigger the nuclear accumulation of Msn2/4 and Maf1, specifically under H(2)O(2) treatment. Contrary to the case with many stress conditions previously described for yeast, the H(2)O(2)-induced nuclear accumulation of Msn2 and Maf1 does not correlate with the downregulation of PKA kinase activity. Nevertheless, we show that PP2A phosphatase activity is essential for driving Maf1 dephosphorylation and its subsequent nuclear accumulation in response to H(2)O(2) treatment. Interestingly, under this condition, the lack of PP2A activity has no impact on the subcellular localization of Msn2, demonstrating that the H(2)O(2) signaling pathways share a common route through the thioredoxin system and then diverge to activate Msn2 and Maf1, the final integrators of these pathways.

Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs.

BACKGROUND: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. CONCLUSION/SIGNIFICANCE: 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.

Is ribosome synthesis controlled by pol I transcription?

Regulation of growth ultimately depends on the control of synthesis of new ribosomes. Ribosome biogenesis is thus a key element of cell biology, which is tightly regulated in response to environmental conditions. In eukaryotic cells, the supply of ribosomal components involves the activities of the three forms of nuclear RNA polymerase (Pol I, Pol II and Pol III). Recently, we demonstrated that upon rapamycin treatment, a partial derepression of Pol I transcription led to a concomitant derepression of Pol II transcription restricted to a small subset of class II genes encompassing the genes encoding all ribosomal proteins, and 19 additional genes. The products of 14 of these 19 genes are principally involved in rDNA structure, ribosome biogenesis or translation, whereas the five remaining genes code for hypothetical proteins. We demonstrate that the proteins encoded by these five genes are required for optimal pre-rRNA processing. In addition, we show that cells in which regulation of Pol I transcription was specifically impaired are either resistant or hypersensitive to different stresses compared to wild-type cells. These results highlight the critical role of the regulation of Pol I activity for the physiology of the cells.

The transcriptional activity of RNA polymerase I is a key determinant for the level of all ribosome components.

Regulation of ribosome biogenesis is a key element of cell biology, not only because ribosomes are directly required for growth, but also because ribosome production monopolizes nearly 80% of the global transcriptional activity in rapidly growing yeast cells. These observations underscore the need for a tight regulation of ribosome synthesis in response to environmental conditions. In eukaryotic cells, ribosome synthesis involves the activities of the three nuclear RNA polymerases (Pol). Although postulated, there is no clear evidence indicating whether the maintenance of an equimolar supply of ribosomal components reflects communication between the nuclear transcriptional machineries. Here, by constructing a yeast strain expressing a Pol I that remains constitutively competent for the initiation of transcription under stress conditions, we demonstrate that derepression of Pol I transcription leads to a derepression of Pol II transcription that is restricted to the genes encoding ribosomal proteins. Furthermore, we show that the level of 5S rRNA, synthesized by Pol III, is deregulated concomitantly with Pol I transcription. Altogether, these results indicate that a partial derepression of Pol I activity drives an abnormal accumulation of all ribosomal components, highlighting the critical role of the regulation of Pol I activity within the control of ribosome biogenesis.