An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants.

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that "mitochondrial translation factors" mTRAN1 and mTRAN2 are land plant-specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)-like RNA binding proteins of the mitoribosomal "small" subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5' regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

The effect of natural biomolecules on yttrium oxide nanoparticles from a Daphnia magna survival rate perspective.

The attention to rare earth oxide nanoparticles (NPs), including yttrium oxide (Y2O3), has increased in many fields due to their unique structural characteristics and functional properties. The aim of our study was to investigate the mechanisms by which bio-corona formation on Y2O3 NPs affects their environmental fate and toxicity. The Y2O3 NPs induced toxicity to freshwater filter feeder Daphnia magna at particle concentrations of 1 and 10 mg/L, regardless of particle size. Interactions between naturally excreted biomolecules (e.g. protein, lipids, and polysaccharides) derived from D. magna, and the Y2O3 NPs (30-45 nm) resulted in the formation of an eco-corona, which reduced their toxic effects toward D. magna at a particle concentration of 10 mg/L. No effects were observed at lower concentrations or for the other particle sizes investigated. Copper-zinc (Cu-Zn) superoxide dismutase, apolipophorins, and vitellogenin-1 proteins proved to be the most prominent proteins of the adsorbed corona, and possibly a reason for the reduced toxicity of the 30-45 nm Y2O3 NPs toward D. magna.

Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils.

Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B.

Ganglioside GM3 stimulates lipid-protein co-assembly in α-synuclein amyloid formation.

Parkinson's disease is characterized by the aggregation of the presynaptic protein α-synuclein (αSyn), and its co-assembly with lipids and other cellular matter in the brain. Here we investigated lipid-protein co-assembly in a system composed of αSyn and model membranes containing the glycolipid ganglioside GM3. We quantified the uptake of lipids into the co-assembled aggregates and investigated how lipid molecular dynamics is altered by being present in the co-assemblies using solution 1H- and solid-state 13C NMR spectroscopy. Aggregate morphology was studied using cryo-TEM. The overall lipid uptake in the co-assembled aggregates was found to increase with the molar ratio of GM3 in the vesicles. The lipids present in the co-assembled aggregates have reduced acyl chain and headgroup dynamics compared to the protein-free bilayer system. These findings may improve our understanding of how different types of lipids can influence the composition of αSyn aggregates, which may have consequences for amyloid formation in vivo.

Surface-Catalyzed Secondary Nucleation Dominates the Generation of Toxic IAPP Aggregates.

The aggregation of the human islet amyloid polypeptide (IAPP) is associated with diabetes type II. A quantitative understanding of this connection at the molecular level requires that the aggregation mechanism of IAPP is resolved in terms of the underlying microscopic steps. Here we have systematically studied recombinant IAPP, with amidated C-terminus in oxidised form with a disulphide bond between residues 3 and 7, using thioflavin T fluorescence to monitor the formation of amyloid fibrils as a function of time and IAPP concentration. We used global kinetic analyses to connect the macroscopic measurements of aggregation to the microscopic mechanisms, and show that the generation of new aggregates is dominated by the secondary nucleation of monomers on the fibril surface. We then exposed insulinoma cells to aliquots extracted from different time points of the aggregation process, finding the highest toxicity at the midpoint of the reaction, when the secondary nucleation rate reaches its maximum. These results identify IAPP oligomers as the most cytotoxic species generated during IAPP aggregation, and suggest that compounds that target secondary nucleation of IAPP could be most effective as therapeutic candidates for diabetes type II.

Proliferation of Tau 304-380 Fragment Aggregates through Autocatalytic Secondary Nucleation.

The self-assembly of the protein tau into neurofibrillary tangles is one of the hallmarks of Alzheimer's disease and related tauopathies. Still, the molecular mechanism of tau aggregation is largely unknown. This problem may be addressed by systematically obtaining reproducible in vitro kinetics measurements under quiescent conditions in the absence of triggering substances. Here, we implement this strategy by developing protocols for obtaining an ultrapure tau fragment (residues 304-380 of tau441) and for performing spontaneous aggregation assays with reproducible kinetics under quiescent conditions. We are thus able to identify the mechanism of fibril formation of the tau 304-380 fragment at physiological pH using fluorescence spectroscopy and mass spectrometry. We find that primary nucleation is slow, and that secondary processes dominate the aggregation process once the initial aggregates are formed. Moreover, our results further show that secondary nucleation of monomers on fibril surfaces dominates over fragmentation of fibrils. Using separate isotopes in monomers and fibrils, through mass spectroscopy measurements, we verify the isotope composition of the intermediate oligomeric species, which reveals that these small aggregates are generated from monomer through secondary nucleation. Our results provide a framework for understanding the processes leading to tau aggregation in disease and for selecting possible tau forms as targets in the development of therapeutic interventions in Alzheimer's disease.

The Bacterial Amyloids Phenol Soluble Modulins from Staphylococcus aureus Catalyze Alpha-Synuclein Aggregation.

Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson's disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak's seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD. Staphylococcus aureus is a bacterium carried by 30-70% of the general population. It has been shown to produce functional amyloids, called phenol soluble modulins (PSMαs). Here, we studied the kinetics of α-syn aggregation under quiescent conditions in the presence or absence of four different PSMα peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure α-syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSMα peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSMα peptides displayed distinct effects on the kinetics of α-syn aggregation. Kinetic analyses of the data suggest that all four peptides catalyze α-syn aggregation through heterogeneous primary nucleation. The immunogold electron microscopic analyses showed that the aggregates were fibrillar and composed of α-syn. In addition of the co-aggregated materials to a cell model expressing the A53T α-syn variant fused to GFP was found to catalyze α-syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.

Purification and HDL-like particle formation of apolipoprotein A-I after co-expression with the EDDIE mutant of Npro autoprotease.

Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in E. coli. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus intact. ApoA-I was then purified using a five-step procedure composed of anion exchange chromatography, immobilized metal ion affinity chromatography, hydrophobic interaction chromatography, boiling and size exclusion chromatography. This led to protein of high purity as confirmed with SDS-PAGE and mass spectrometry. The purified ApoA-I formed discoidal objects in the presence of zwitterionic phospholipid DMPC, showing its retained function of interacting with lipids. The protocol was also tested by expression and purification of two ApoA-I mutants, both of which could be purified in the same manner as the wildtype, showing the robustness of the protocol.

Expression, purification and characterisation of large quantities of recombinant human IAPP for mechanistic studies.

Malfunction and amyloid formation of the Islet Amyloid Polypeptide (IAPP) are factors contributing to Type 2 diabetes. Unravelling the mechanism of IAPP aggregate formation may forward our understanding of this process and its effect on pancreatic ß-islet cell. Such mechanistic studies require access to sequence homogeneous and highly pure IAPP. Here we present a new facile protocol for the production of pure recombinant human IAPP at relatively high yield. The protocol uses a His-tagged version of the Npro mutant EDDIE, which drives expression to inclusion bodies, from which the peptide is purified using sonication, refolding and auto-cleavage, removal of EDDIE using Ni-NTA chromatography and reverse-phase HPLC. The purified material is used at multiple concentrations in aggregation kinetics measurements monitored by thioflavin-T fluorescence. Global analysis of the data implies a double nucleation aggregation mechanism including both primary and secondary nucleation.

Dual topography of laminin corona on gallium arsenide nanowires.

Nanowires (NWs) are novel nanomaterials with applications in everything from medical implants to solar cells. With increasing number of applications, it is increasingly likely that organisms are exposed to these materials either intentionally or by accident. It is, therefore, important to study their interactions with biological systems and biomolecules. Upon exposure to biological fluids, nanostructure surfaces are quickly covered by a biomolecule corona. The composition of the corona determines the nanostructure's biological fate. Furthermore, upon adsorption, the protein structure can be affected. In order to study the corona morphology, we used two model proteins, laminin of the extracellular matrix and the immune system enzyme myeloperoxidase. We image the protein corona directly by cryo-TEM and enhance resolution by labeling the corona with activated gold nanoparticles. Three-dimensional imaging of the protein corona further increases the resolution and reveals irregularities in corona topography. By doing so, we identified bimodal distribution of spacing between gold nanoparticles and the NW surface for laminin corona at 58 and 85 nm distance from the NWs' surface. The dual topography of the corona is adding a new complexity of the protein corona surface and its interactions with the surrounding biology.

Kinetic fingerprints differentiate the mechanisms of action of anti-Aß antibodies.

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-ß peptide (Aß) is a causative process in Alzheimer's disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating Aß-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. Here, we combine kinetic analyses with quantitative binding measurements to address the mechanism of action of four clinical stage anti-Aß antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We quantify the influence of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aß. Our results reveal that, uniquely among these four antibodies, aducanumab dramatically reduces the flux of Aß oligomers.

Author Correction: Dynamics of oligomer populations formed during the aggregation of Alzheimer's Aß42 peptide.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dynamics of oligomer populations formed during the aggregation of Alzheimer's Aß42 peptide.

Oligomeric species populated during the aggregation of the Aß42 peptide have been identified as potent cytotoxins linked to Alzheimer's disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach that combines theory, experiment and simulation, we reveal, in molecular detail, the mechanisms of Aß42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we found that most Aß42 oligomers dissociate into their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar structures. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to those of aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

Understanding the Lipid and Protein Corona Formation on Different Sized Polymeric Nanoparticles.

When in contact with biological fluids, nanoparticles dynamically absorb biomolecules like proteins and lipids onto their surface, forming a "corona". This biocorona is a dynamic and complex structure that determines how host cells respond to nanoparticles. Despite the common use of mouse models in pre-clinical and toxicological experiments, the impact of corona formed in mouse serum on the biophysical and biological properties of different size NP has not been thoroughly explored. Furthering the knowledge on the corona formed on NP exposed to mouse serum proteins can help in understanding what role it might have in in vivo studies at systemic, tissue, and cellular levels. To investigate biocorona formation, different sized polystyrene NP were exposed to mouse serum. Our data show a size- and time-dependent protein and lipid corona formation. Several proteins were identified and apolipoproteins were by far the most common group on the NPs surfaces. Moreover, we observed that cholesterol and triglycerides effectively bind to NP emphasizing that proteins are not the only biomolecules with high-affinity binding to nanomaterial surfaces. These results highlight that further knowledge on NP interactions with mouse serum is necessary regarding the common use of this model to predict the in vivo efficiency of NP.

Analysis of complexes formed by small gold nanoparticles in low concentration in cell culture media.

New nanomaterials are constantly developed with applications in everything from cosmetics to high tech electronics. Assessing their biological impact has been done by analysis of their adsorbed protein corona, in vitro cell assays, and larger scale ecotoxicological studies. This has proved to be a huge challenge due to the wide range of available nanomaterials and their unpredictable behaviour in different environments. Furthermore, the enormous number of experimental variables make comparisons difficult. Concentration is one of these variables and can vary greatly depending on the aim of the study. When analysing the protein corona, concentrations are often higher than in cell assays. Using a combination of complementary techniques, we have characterised 20 nm gold nanoparticles in a concentration level commonly used in cell studies. We compare their behaviour in a commonly used, protein rich medium and one protein poor medium over 24 hours. Under these conditions, the NPs were stable in protein rich environment but underwent gradual aggregation in protein poor medium. We characterise the biomolecular corona in both media. In protein poor medium, we can describe the often overlooked aggregation. The aggregates' morphology is confirmed by cryo-TEM. Finally, in the protein poor medium, by infrared spectroscopy, we have identified the amino acid arginine in the biomolecular corona which drives the aggregation.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Disaggregation of gold nanoparticles by Daphnia magna.

The use of manufactured nanomaterials is rapidly increasing, while our understanding of the consequences of releasing these materials into the environment is still limited and many questions remain, for example, how do nanoparticles affect living organisms in the wild? How do organisms adapt and protect themselves from exposure to foreign materials? How does the environment affect the performance of nanoparticles, including their surface properties? In an effort to address these crucial questions, our main aim has been to probe the effects of aquatic organisms on nanoparticle aggregation. We have, therefore, carried out a systematic study with the purpose to disentangle the effects of the freshwater zooplankter, Daphnia magna, on the surface properties, stability, and aggregation properties of gold (Au) nanoparticles under different aqueous conditions as well as identified the proteins bound to the nanoparticle surface. We show that Au nanoparticles aggregate in pure tap water, but to a lesser extent in water that either contains Daphnia or has been pre-conditioned with Daphnia. Moreover, we show that proteins generated by Daphnia bind to the Au nanoparticles and create a modified surface that renders them less prone to aggregation. We conclude that the surrounding milieu, as well as the surface properties of the original Au particles, are important factors in determining how the nanoparticles are affected by biological metabolism. In a broader context, our results show how nanoparticles released into a natural ecosystem become chemically and physically altered through the dynamic interactions between particles and organisms, either through biological metabolism or through the interactions with biomolecules excreted by organisms into the environment.

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism.

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated. ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion. Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.

Structural modelling of the DNAJB6 oligomeric chaperone shows a peptide-binding cleft lined with conserved S/T-residues at the dimer interface.

The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface. Mixed isotope crosslinking showed that the oligomers are dynamic entities that exchange subunits. The purified protein is well folded, soluble and composed of oligomers with a varying number of subunits according to small-angle X-ray scattering (SAXS). Elongated particles (160 × 120 Å) were detected by electron microscopy and single particle reconstruction resulted in a density map of 20 Å resolution into which the DNAJB6 dimers fit. The structure of the oligomer and the S/T-rich region is of great importance for the understanding of the function of DNAJB6 and how it can bind aggregation-prone peptides and prevent amyloid diseases.

Analysis of nanoparticle biomolecule complexes.

Nanoparticles exposed to biological fluids adsorb biomolecules on their surface forming a biomolecular corona. This corona determines, on a molecular level, the interactions and impact the newly formed complex has on cells and organisms. The corona formation as well as the physiological and toxicological relevance are commonly investigated. However, an acknowledged but rarely addressed problem in many fields of nanobiotechnology is aggregation and broadened size distribution of nanoparticles following their interactions with the molecules of biological fluids. In blood serum, TiO2 nanoparticles form complexes with a size distribution from 30 nm to more than 500 nm. In this study we have separated these complexes, with good resolution, using preparative centrifugation in a sucrose gradient. Two main apparent size populations were obtained, a fast sedimenting population of complexes that formed a pellet in the preparative centrifugation tube, and a slow sedimenting complex population still suspended in the gradient after centrifugation. Concentration and surface area dependent differences are found in the biomolecular corona between the slow and fast sedimenting fractions. There are more immunoglobulins, lipid binding proteins, and lipid-rich complexes at higher serum concentrations. Sedimentation rate and the biomolecular corona are important factors for evaluating any experiment including nanoparticle exposure. Our results show that traditional description of nanoparticles in biological fluids is an oversimplification and that more thorough characterisations are needed.