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Small soluble oligomers of the protein α-synuclein (αSO) have been linked to disruptions in neuronal homeostasis, contributing to the development of Parkinson's Disease (PD). While this makes αSO an obvious drug target, the development of effective therapeutics against αSO is challenged by its low abundance and structural and morphological complexity. Here, we employ two different approaches to neutralize toxic interactions made by αSOs with different cellular components. First, we use available data to identify four neuronal proteins as likely candidates for αSO interactions, namely Cfl1, Uchl1, Sirt2 and SerRS. However, despite promising results when immobilized, all 4 proteins only bind weakly to αSO in solution in microfluidic assays, making them inappropriate for screening. In contrast, the formation of stable contacts formed between αSO and vesicles consisting of anionic lipids not only mimics a likely biological role of αSO but also provided a platform to screen two small molecule libraries for disruptors of these contacts. Of the 7 best leads obtained in this way, 2 significantly impaired αSO contacts with other proteins in a sandwich ELISA assay using αSO-binding monoclonal antibodies and nanobodies. In addition, 5 of these leads suppressed α-synuclein amyloid formation. Thus, a repurposing screening that directly targets a key culprit in PD pathogenesis shows therapeutic potential.

Structural biology describes biological processes at the molecular level and is an integral part of undergraduate study programs in molecular biosciences. Students are often fascinated by the visualizations created by molecular graphics software, which allow them to see the molecular world for the first time. Today, molecular visualization and structural analysis do not require expensive high-end computers but can be performed on the students' own laptops and are therefore highly suited for active learning approaches. We have designed a semester-long learning path that integrates molecular graphics and structural analysis using PyMOL into an undergraduate course in biomolecular structure and function. Compared to stand-alone PyMOL introductions, the semester-long learning path allows for an improved pedagogical design. The path progressively introduces more advanced functions in relevant scientific contexts and allows for spaced repetition. Advanced analysis functions in PyMOL are available only via the command line, so the learning path also teaches basic scripting and serves as an accessible introduction to computational thinking because a few lines of code can produce stunning results. Student surveys carried out at the end of the course suggest that the learning path supported the ability to perform structural analysis to a high degree. Moreover, a simulated exam showed that almost all students were able to carry out basic visualization tasks using PyMOL scripts, while three-quarters could undertake advanced structural analysis after following the course. In summary, integration of molecular graphics software with teaching of structural biochemistry allows a hands-on approach to analyzing molecular mechanisms and introduces biologically oriented students to computational thinking.

HYPOTHESIS: Lipases are widely used in the detergent industry and must withstand harsh conditions involving both anionic and zwitterionic surfactants at alkaline pH. Thermomyces lanuginosus lipase (TlL) is often used and stays active at high concentrations of the anionic surfactant sodium dodecyl sulfate (SDS) at pH 8.0, but is sensitive to SDS at pH 6.0 and below. We propose that enhanced stability at pH 8.0 results from a structurally distinct complex formation with SDS.EXPERIMENTS: We use small-angle X-ray scattering (SAXS) to elucidate structures of TlL:SDS at pH 4.0, 6.0, and 8.0 and further investigate the complexes at pH 8.0 using hydrogen/deuterium exchange mass spectrometry (HDX-MS).FINDINGS: At pH 4.0, large dense aggregates are formed at low [SDS], which become gradually less dense at higher [SDS], resulting in a core-shell structure. At pH 6.0, SDS induces a TlL dimer and forms a hemi-micelle along the side of the dimer. At higher [SDS], TlL adopts a core-shell structure. At pH 8.0, TlL forms a dimer with a SDS hemi-micelle but avoids a core-shell structure and maintains activity. Three helices are identified as SDS anchor points. This study provides important structural insight into the stability of TlL towards SDS under alkaline conditions.

Nanoparticles (NPs) can modulate protein aggregation and fibril formation in the context of amyloid diseases. Understanding the mechanism of this action remains a critical next step in developing nanomedicines for the treatment or prevention of Parkinson's disease. α-Synuclein (α-Syn) can undergo interactions of different strength with nanoparticles, and these interactions can be prevented by the presence of a protein corona (PC) acquired during the exposure of NPs to serum proteins. Here, we develop a method to attach the PC irreversibly to the NPs, which enables us to study in detail the interaction of α-Syn and polyethylenimine-coated carboxyl-modified polystyrene NPs (PsNPs-PEI) and the role of the dynamics of the interactions. Analysis of the kinetics of fibril formation reveals that the NPs surface promotes the primary nucleation step of amyloid fibril formation without significantly affecting the elongation and fragmentation steps or the final equilibrium. Furthermore, the results show that even though α-Syn can access the surface of NPs that are precoated with a PC, due to the dynamic nature of the PC proteins, the PC nevertheless reduces the acceleratoring effect of the NPs. This effect is likely to be caused by reducing the overall amount of weakly interacting α-Syn molecules on the NP surface and the access of further α-Syn required for fibril elongation. Our experimental approach provides microscopic insight into how serum proteins can modulate the complex interplay between NPs and amyloid proteins.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson’s disease. However, their structure and the molecular determinants driving their conversion to fibrils remain elusive. In this work, we determined the symmetry and architecture of α-synuclein oligomers, dissecting the conformational properties of individual chains within these toxic assemblies. We demonstrate that the NAC domain is insufficient to promote oligomer to fibril conversion; instead, this transition is controlled by a short α-synuclein N-terminal motif. A missense mutation causing early-onset Parkinson’s disease remodels this N-terminal region conformation, which results in a population of long-lived oligomers less susceptible to disaggregation by the human Hsp70 machinery. Our results provide a structural understanding of oligomer to amyloid conversion and identify targets for therapeutic intervention.One sentence summaryα-Synuclein oligomers are symmetric and well-organized particles with a short N-terminal region controlling fibril conversion.

Multidrug resistance of bacteria and persistent infections related to biofilms, as well as the low availability of new antibacterial drugs, make it urgent to develop new antibiotics. Here, we evaluate the antibacterial and anti-biofilm properties of ticlopidine (TP), an anti-platelet aggregation drug, TP showed antibacterial activity against both gram-positive (MRSA) and gram-negative (E. coli, and P. aeruginosa) bacteria over a long treatment period. TP significantly reduced the survival of gram-negative bacteria in human blood though impact on gram-positives was more limited. TP may cause death in MRSA by inhibiting staphyloxanthin pigment synthesis, leading to oxidative stress, while scanning electron microscopy imaging indicate a loss of membrane integrity, damage, and consequent death due to lysis in gram-negative bacteria. TP showed good anti-biofilm activity against P. aeruginosa and MRSA, and a stronger biofilm degradation activity on P. aeruginosa compared to MRSA. Measuring fluorescence of the amyloid-reporter Thioflavin T (ThT) in biofilm implicated inhibition of amyloid formation as part of TP activity. This was confirmed by assays on the purified protein in P. aeruginosa, FapC, whose fibrillation kinetics was inhibited by TP. TP prolonged the lag phase of aggregation and reduced the subsequent growth rate and prolonging the lag phase to very long times provides ample opportunity to exert TP's antibacterial effect. We conclude that TP shows activity as an antibiotic against both gram-positive and gram-negative bacteria thanks to a broad range of activities, targeting bacterial metabolic processes, cellular structures and the biofilm matrix.

Amyloid fibrils are protein aggregates formed by protein assembly through cross β structures. Inhibition of amyloid fibril formation may contribute to therapy against amyloid-related disorders like Parkinson's, Alzheimer's, and type 2 diabetes. Here we report that several fluorinated sulfonamide compounds, previously shown to inhibit human carbonic anhydrase, also inhibit the fibrillation of different proteins. Using a range of spectroscopic, microscopic and chromatographic techniques, we found that the two fluorinated sulfonamide compounds completely inhibit insulin fibrillation over a period of 16 h and moderately suppress α-synuclein and Aβ fibrillation. In addition, these compounds decreased cell toxicity of insulin incubated under fibrillation-inducing conditions. We ascribe these effects to their ability to maintain insulin in the native monomeric state. Molecular dynamic simulations suggest that these compounds inhibit insulin self-association by interacting with residues at the dimer interface. This highlights the general anti-aggregative properties of aromatic sulfonamides and suggests that sulfonamide compounds which inhibit carbonic anhydrase activity may have potential as therapeutic agents against amyloid-related disorders.

The role of the blood-brain barrier (BBB) is to control trafficking of biomolecules and protect the brain. This function can be compromised by pathological conditions. Parkinson’s disease (PD) is characterized by the accumulation of α-synuclein aggregates (αSN-AGs) such as oligomers and fibrils, which contribute to disease progression and severity. Here we study how αSN-AGs affect the BBB in in vitro co-culturing models consisting of human brain endothelial hCMEC/D3 cells alone and co-cultured with astrocytes and neurons/glial cells. When cultivated on their own, hCMEC/D3 cells were compromised by αSN-AGs, which decreased cellular viability, mitochondrial membrane potential, wound healing activity, TEER and permeability parameters, as well as increased the levels of ROS and NO. Co-culturing of these cells with activated microglia also increased BBB impairment according to TEER and systemic immune cell transmigration assays. In contrast, hCMEC/D3 cells co-cultured with astrocytes or dopaminergic neurons or simultaneously treated with their conditioned media showed increased resistance against αSN-AGs. Our work demonstrates the complex relationship between members of the neurovascular unit (NVU) (perivascular astrocytes, neurons, microglia, and endothelial cells), αSN-AGs and BBB.Graphical Abstract

Microfluidic systems under laminar flow conditions provide in-solution information about species size and binding affinities at very modest sample costs. Flow-induced dispersion analysis directly measures the spread of the analyte profile using Taylor dispersion analysis, whereas microfluidic diffusional sizing quantifies the transfer of analyte from one phase to another. Species of sizes between 0.5 and 1000 nm can be analyzed, and different populations resolved. Both techniques also allow analysis in complex media and medium throughput analysis. These properties make them valuable complements to existing approaches to measure biomolecular interactions.

Empirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MF SDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labeling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for seven single-Cys GlpG variants over most of the explored MF SDS range, along with exchange from the native state at low MF SDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labeling rates decline with MF SDS at 0–0.2 MF SDS (i.e., native conditions). Thus, an increase in MF SDS seems to be a protective factor for these two positions, but not for the five others. We propose different scenarios to explain this and find the most plausible ones to involve preferential binding of SDS monomers to the site of CL (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MF SDS (based on SAXS studies). These nonlinear impacts on protein stability highlights a multifaceted role for SDS in membrane protein denaturation, involving both direct interactions of monomeric SDS and changes in micelle size and shape along with the general effects on protein stability of changes in micelle composition.

Functional amyloids are highly organized protein/peptide structures that inter alia promote biofilm formation in different bacteria. One such example is provided by a family of 20-45 residue-long peptides called phenol-soluble modulins (PSMs) from Staphylococcus aureus. External components such as eukaryotic host proteins, which alter self-assembly of bacterial amyloids, can affect the biofilm matrix. Here, we studied the effect of the highly prevalent human plasma protein fibrinogen (Fg) on fibrillation of PSMs. Fg inhibits or suppresses fibrillation of most PSMs tested (PSMα1, PSMβ1, and PSMβ2) except for PSMα3, whose already rapid aggregation is accelerated even further by Fg but leads to amorphous β-rich aggregates rather than fibrils. Fg also induces PSMβ2 to form amorphous aggregates and diverts PSMα1 into off-pathway oligomers which consist of both Fg and PSMα1 and cannot seed fibrillation. Peptide arrays showed that Fg bound to the N-terminus of PSMα1, while it bound to the entire length of PSMα3 (except the C terminus) and to the C-termini of PSMβ1 and PSMβ2. The latter peptides are all positively charged, while Fg is negatively charged at physiological pH. The positive charges complement Fg's net negative charge of -7.6 at pH 7.4. Fg's ability to inhibit PSM fibrillation reveals a potential host-defense mechanism to prevent bacterial biofilm growth and infections in the human body.

The anionic surfactant sodium dodecyl sulfate (SDS) interacts strongly with most globular proteins and denatures and unfolds them. While scattering studies using X-rays and neutrons have shown that this denaturation generally leads to protein-decorated SDS micelles, a different SDS-decorated polypeptide model has recently been suggested for complexes between SDS and Ubiquitin (UBI), in which individual SDS molecules are distributed on a partially stretched protein. To resolve this apparent discrepancy, we have investigated the SDS-UBI system by a number of complementary techniques. Small-angle X-ray scattering (SAXS) provides the overall structure of the SDS-UBI complexes, Tyr fluorescence and circular dichroism follow changes in tertiary and secondary structure, and isothermal titration calorimetry determines the stoichiometries of complexes and the amount of free SDS as a function of [SDS]. At low [SDS], UBI preserves its folded structure but dimerizes to a small extent. At 4 SDS per UBI, a complex is formed with two UBI and a small shared SDS cluster with 8 SDS molecules. In these complexes UBI preserves most of its native fold. At 10-12 SDS per UBI, which remains below the critical micelle concentration under our conditions, UBI-covered SDS micelles form with four UBIs around a core of 40 SDSs. This implies a protein-assisted micellization and an associated unfolding of UBI involving a change from mainly β-strands to mainly α-helical secondary structure. As [SDS] is increased, the complex gradually changes so that finally only one UBI covers one micelle with a similar number of SDS molecules at SDS saturation. Thus, we conclude that SDS unfolds UBI by mechanisms very similar to those observed for other globular proteins, leading to a protein-decorated SDS micelle rather than an SDS-decorated unfolded polypeptide chain.

Chemical glycosylation of proteins is a powerful tool applied widely in biomedicine and biotechnology. However, it is a challenging undertaking and typically relies on recombinant proteins and site-specific conjugations. The scope and utility of this nature-inspired methodology would be broadened tremendously by the advent of facile, scalable techniques in glycosylation, which are currently missing. In this work, we investigated a one-pot aqueous protocol to achieve indiscriminate, surface-wide glycosylation of the surface accessible amines (lysines and/or N-terminus). We reveal that this approach afforded minimal if any change in the protein activity and recognition events in biochemical and cell culture assays, but at the same time provided a significant benefit of stabilizing proteins against aggregation and fibrillation - as demonstrated on serum proteins (albumins and immunoglobulin G, IgG), an enzyme (uricase), and proteins involved in neurodegenerative disease (α-synuclein) and diabetes (insulin). Most importantly, this highly advantageous result was achieved via a one-pot aqueous protocol performed on native proteins, bypassing the use of complex chemical methodologies and recombinant proteins.

Parkinson’s disease (PD) is the second most common neurodegenerative disease. The presence of lewy bodies, primarily consisting of α-synuclein (α-syn) aggregates is one of the common features seen in the substantia nigra region of the brain in PD patients. The disease remains incurable and only symptomatic relief is available. We screened various cell-penetrating peptides and reveal that penetratin is a potent inhibitor of α-syn aggregation in-vitro, and significantly improved locomotor coordination in mice models of PD in-vivo. The peptide inhibits α-syn aggregation in vitro as well as in yeast, and C.elegans models. We further made a cyclic derivative of penetratin by disulfide coupling of N- and C-terminal cysteine residues. Both penetratin and its cyclized derivative interact with α-syn. NMR studies show that both linear as well as cyclic derivative interact at the acidic C-terminal tail of the protein. Similar to penetratin, its cyclic derivative inhibited α-syn aggregation in the C.elegans model of Parkinson’s disease, and also improved worm motility. Molecular Dynamics studies show that penetratin interacts with α-synuclein and prevents its conformational transition from disordered into β-sheet rich structure. The therapeutic efficacy of penetratin was further confirmed in a transgenic mice model of the disease, wherein penetratin treatment over a period of 90 days improved locomotor coordination, and halted disease progression. Overall, the present work provides a potent therapeutic agent that could be further explored in the management of PD.

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a “tropism” for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-021-02316-0.

JBC papers in press 298(7), 102142 (2022). doi:10.1016/j.jbc.2022.102142, The bacterial stringent response involves wide-ranging metabolic reprogramming aimed at increasing long-term survivability during stress conditions. One of the hallmarks of the stringent response is the production of a set of modified nucleotides, known as alarmones, which affect a multitude of cellular pathways in diverse ways. Production and degradation of these molecules depend on the activity of enzymes from the RelA/SpoT homologous family, which come in both bifunctional (containing domains to both synthesize and hydrolyze alarmones) and monofunctional (consisting of only synthetase or hydrolase domain) variants, of which the structure, activity, and regulation of the bifunctional RelA/SpoT homologs have been studied most intensely. Despite playing an important role in guanosine nucleotide homeostasis in particular, mechanisms of regulation of the small alarmone hydrolases (SAHs) are still rather unclear. Here, we present crystal structures of SAH enzymes from Corynebacterium glutamicum (RelH$_{Cg}$) and Leptospira levettii (RelH$_{Ll}$) and show that while being highly similar, structural differences in substrate access and dimer conformations might be important for regulating their activity. We propose that a varied dimer form is a general property of the SAH family, based on current structural information as well as prediction models for this class of enzymes. Finally, subtle structural variations between monofunctional and bifunctional enzymes point to how these different classes of enzymes are regulated., Published by American Soc. for Biochemistry and Molecular Biology, Bethesda, MD.

There is an intense search for natural compounds that can inhibit the oligomerization and fibrillation of α-Synuclein (α-Syn), whose aggregation is key to the development of Parkinson's disease (PD). Rosa damascena is a medicinal herb widely used in Middle Eastern food, ceremonies, and perfumes. The herb is known to contain many different polyphenols. Here we investigated the existence of α-Syn fibrillation inhibitors in R. damascena extract. Different HPLC fractions of the extract were assessed in α-Syn fibrillation and toxicity assays. The most active fractions led to the formation of more α-Syn oligomers but with less toxicity to SH-SY5Y cells, according to MTT and LDH assays. LC-MS analysis identified gallic acid, kaempferol 3-glucoside, kaempferol-3-O-β-rutinoside, and quercetin which were subsequently shown to be strong α-Syn fibrillation inhibitors. Our results highlight the benefits of R. damascena extract to combat PD at the population level.

Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype, and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria, such as E. coli, Salmonella, Bacillus, and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three β-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers, and cylindrical, elliptical cross-section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα–Cβ chemical shifts, indicative of a predominant β-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e., predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation is inhibited by chaperones targeting pathological misfolding and if so by what mechanism. Here we analyze how four entirely different human chaperones or protein modulators (transthyretin, S100A9, Bri2 BRICHOS and DNAJB6) and bacterial CsgC affect CsgA and FapC fibrillation. CsgA is more susceptible to inhibition than FapC and the chaperones vary considerably in the efficiency of their inhibition. However, mechanistic analysis reveals that all predominantly target primary nucleation rather than elongation or secondary nucleation, while stoichiometric considerations suggest that DNAJB6 and CsgC target nuclei rather than monomers. Inhibition efficiency broadly scales with the chaperones' affinity for monomeric CsgA and FapC. The chaperones tend to target the most aggregation-prone regions of CsgA, but do not display such tendencies towards the more complex FapC sequence. Importantly, the most efficient inhibitors (Bri2 BRICHOS and DNAJB6) significantly reduce bacterial biofilm formation. This commonality of chaperone action may reflect the simplicity of functional amyloid formation, driven largely by primary nucleation, as well as the ability of non-bacterial chaperones to deploy their proteostatic capacities across biological kingdoms., Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated.

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.

High-resolution structural information on membrane proteins is essential for understanding cell biology and for the structure-based design of new medical drugs and drug delivery strategies. X-ray diffraction (XRD) can provide angstrom-level information about the structure of membrane proteins, yet for XRD experiments, proteins are removed from their native membrane environment, chemically stabilized, and crystallized, all of which can compromise the conformation. Here, we describe how a combination of surface-sensitive vibrational spectroscopy and molecular dynamics simulations can account for the native membrane environment. We observe the structure of a glycerol facilitator channel (GlpF), an aquaporin membrane channel finely tuned to selectively transport water and glycerol molecules across the membrane barrier. We find subtle but significant differences between the XRD structure and the inferred in situ structure of GlpF.

Biofilms enable bacteria to colonize numerous ecological niches. Bacteria within a biofilm are protected by the extracellular matrix (ECM), of which the fibril-forming amyloid protein curli and polysaccharide cellulose are major components in members of Salmonella, Eschericha and Mycobacterium genus. A shortage of real-time detection methods has limited our understanding of how ECM production contributes to biofilm formation and pathogenicity. Here we present optotracing as a new semi-high throughput method for dynamic monitoring of Salmonella biofilm growth on air-solid interfaces. We show how an optotracer with binding-induced fluorescence acts as a dynamic fluorescent reporter of curli expression during biofilm formation on agar. Using spectrophotometry and microscopic imaging of fluorescence, we analyse in real-time the development of the curli architecture in relation to bacterial cells. With exceptional spatial and temporal precision, this revealed a well-structured, non-uniform distribution of curli organised in distally projecting radial channel patterns. Dynamic monitoring of the biofilm also showed defined regions undergoing different growth phases. ECM structures were found to assemble in regions of late exponential growth phase, suggesting that ECM forms on site after bacteria colonize the surface. As the optotracer biofilm method expedites screening of curli production, providing exceptional spatial-temporal understanding of the surface-associated biofilm lifestyle, this method adds a new technique to further our understanding of bacterial biofilms., Highlights • Design and evaluation of a method for real-time biofilm experimentation. • Optotracing enables real-time monitoring of biofilm formation on solid supports. • Definitive biofilm monitoring by selective tracking of ECM components. • A method reducing the inherent biases of morphotyping. • A semi-high throughput method increasing the ease and efficiency of biofilm detection.

Cross-seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation-prone proteins such as αSN, which is central in the development of Parkinson's disease. This is particularly pertinent in view of the growing number of functional (i.e. benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from Parkinson's disease transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid. Both proteins show robust aggregation into ThT-positive aggregates that contain higher-order β-sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against formic acid, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross-seed fibrillation of αSN. Our results support the use of proteomics and formic acid to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes. This article is protected by copyright. All rights reserved.

Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein separation and size estimation, there is still controversy about its mode of interaction with proteins. The term "rod-like" structures for the complexes that form between SDS and protein, originally introduced by Tanford, is not sufficiently descriptive and does not distinguish between the two current vying models, namely protein-decorated micelles a.k.a. the core-shell model (in which denatured protein covers the surface of micelles) versus beads-on-a-string model (where unfolded proteins are surrounded by surfactant micelles). Thanks to a combination of structural, kinetic and computational work particularly within the last 5-10 years, it is now possible to rule decisively in favor of the core-shell model. This is supported unambiguously by a combination of calorimetric and small-angle X-ray scattering (SAXS) techniques and confirmed by increasingly sophisticated molecular dynamics simulations. Depending on the SDS:protein ratio and the protein molecular mass, the formed structures can range from multiple partly unfolded protein molecules surrounding a single shared micelle to a single polypeptide chain decorating multiple micelles. We also have much new insight into how this species forms. It is preceded by the binding of small numbers of SDS molecules which subsequently grow by accretion. Time-resolved SAXS analysis reveals an asymmetric attack by SDS micelles followed by distribution of the increasingly unfolded protein around the micelle. The compactness of the protein chain continues to evolve at higher SDS concentrations according to single-molecule studies, though the protein remains completely denatured on the tertiary structural level. SDS denaturation can be reversed by addition of nonionic surfactants that absorb SDS forming mixed micelles, leaving the protein free to refold. Refolding can occur in parallel tracks if only a fraction of the protein is initially stripped of SDS. SDS unfolding is nearly always reversible unless carried out at low pH, where charge neutralization can lead to superclusters of protein-surfactant complexes. With the general mechanism of SDS denaturation now firmly established, it largely remains to explore how other ionic surfactants (including biosurfactants) may diverge from this path.

Phenol-soluble modulins (PSMs), such as α-PSMs, β-PSMs, and δ-toxin, are virulence peptides secreted by different Staphylococcus aureus strains. PSMs are able to form amyloid fibrils, which may strengthen the biofilm matrix that promotes bacterial colonization of and extended growth on surfaces (e.g., cell tissue) and increases antibiotic resistance. Many components contribute to biofilm formation, including the human-produced highly sulfated glycosaminoglycan heparin. Although heparin promotes S. aureus infection, the molecular basis for this is unclear. Given that heparin is known to induce fibrillation of a wide range of proteins, we hypothesized that heparin aids bacterial colonization by promoting PSM fibrillation. Here, we address this hypothesis using a combination of thioflavin T-fluorescence kinetic studies, CD, FTIR, electron microscopy, and peptide microarrays to investigate the mechanism of aggregation, the structure of the fibrils, and identify possible binding regions. We found that heparin accelerates fibrillation of all α-PSMs (except PSMα2) and δ-toxin but inhibits β-PSM fibrillation by blocking nucleation or reducing fibrillation levels. Given that S. aureus secretes higher levels of α-PSM than β-PSM peptides, heparin is therefore likely to promote fibrillation overall. Heparin binding is driven by multiple positively charged lysine residues in α-PSMs and δ-toxins, the removal of which strongly reduced binding affinity. Binding of heparin did not affect the structure of the resulting fibrils, that is, the outcome of the aggregation process. Rather, heparin provided a scaffold to catalyze or inhibit fibrillation. Based on our findings, we speculate that heparin may strengthen the bacterial biofilm and therefore enhance colonization via increased PSM fibrillation.

Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The conversion from IDP to amyloid is evolutionarily optimized and likely couples folding to association. Many FuBA contain several imperfect repeat sequences which contribute to the stability of mature FuBA fibrils. Aggregation can be considered an intermolecular extension of the process of intramolecular protein folding which has traditionally been studied using chemical denaturants. Here we employ denaturants to investigate folding steps during fibrillation of CsgA and FapC. We quantify protein compactification (i.e. the extent of burial of otherwise exposed surface area upon association of proteins) during different stages of fibrillation based on the dependence of fibrillation rate constants on the denaturant concentration (m-values) determined from fibrillation curves. For both proteins, urea mainly affects nucleation and elongation (not fragmentation), consistent with the fact that these steps involve both intra- and intermolecular association. The two steps have similar m-values, indicating that activation steps in nucleation and elongation involve the same level of folding. Surprisingly, deletion of two or three repeats from FapC leads to larger m-values (i.e. higher compactification) during the activation step of fibril growth. This observation is extended by SAXS analysis of the fibrils which indicates that weakening of the amyloidogenic core caused by repeat deletions causes a larger portion of normally unstructured regions of the protein to be included into the amyloid backbone. We conclude that the sensitivity of fibrillation to denaturants can provide useful insight into molecular mechanisms of aggregation.

Formation of amyloid structures is originally linked to human disease. However, amyloid materials are found extensively in the animal and bacterial world where they stabilize intra- and extra-cellular environments like biofilms or cell envelopes. To date, functional amyloids have largely been studied using optical microscopy techniques in vivo, or after removal from their biological context for higher-resolution studies in vitro. Furthermore, conventional microscopies only indirectly identify amyloids based on morphology or unspecific amyloid dyes. Here, the high chemical and spatial (≈20 nm) resolution of Infrared Nanospectroscopy (AFM-IR) to investigate functional amyloid from Escherichia coli (curli), Pseudomonas (Fap), and the Archaea Methanosaeta (MspA) in situ is exploited. It is demonstrated that AFM-IR identifies amyloid protein within single intact cells through their cross β-sheet secondary structure, which has a unique spectroscopic signature in the amide I band of protein. Using this approach, nanoscale-resolved chemical images and spectra of purified curli and Methanosaeta cell wall sheaths are provided. The results highlight significant differences in secondary structure between E. coli cells with and without curli. Taken together, these results suggest that AFM-IR is a new and powerful label-free tool for in situ investigations of the biophysical state of functional amyloid and biomolecules in general.

Amyloid proteins are found in a wide range of organisms owing to the high stability of the β-sheet core of the amyloid fibrils. There are both pathological amyloids involved in various diseases and functional amyloids that play a beneficial role for the organism. The aggregation process is complex and often involves many different species. Full understanding of this process requires parallel acquisition of data by complementary techniques monitoring the time course of aggregation. This is not an easy task, given the often-stochastic nature of aggregation, which can lead to significant variations in lag time. Here, we investigate the aggregation process of the functional amyloid FapC by simultaneous use of four different techniques, namely dynamic light scattering, small-angle x-ray scattering (SAXS), circular dichroism, and Thioflavin T fluorescence. All these approaches are applied to the same FapC sample just after desalting. Our data allow us to construct a master time-course graph showing the same time-course of aggregation by all techniques. This allows us to integrate insights from approaches that report on different structural and length scales. During the lag phase, loosely aggregated oligomers with random-coil structure are formed, which subsequently transform to fibrils without accumulation of additional significant species. Subsequently, the loosely associated protofilaments/subfilaments, which form side by side, mature to more compact fibrils. Furthermore, we determine the mass per length of the mature fibrils, obtaining very similar results by SAXS (33 kDa/nm) and tilted-beam transmission electron microscopy (31 kDa/nm). Transmission electron microscopy showed that the fibrils consist of primarily two protofilaments and similar dimensions of the cross section of the fibrils as revealed by SAXS modeling when the number of protofilaments per fibril was taken into account. Mass per length information underscores the general usefulness of SAXS in fibrillation analysis and provides an important constraint for further modeling the fibril structures.

BACKGROUND: PD is a multisystem disease where both central and peripheral nervous systems are affected. This systemic involvement also includes the immune response in PD, which implicates not only microglia in the brain, but also peripheral immune cells, such as monocytes; however, this aspect has been understudied.OBJECTIVES: The purpose of this study was to investigate the PD-related changes in peripheral immune cells, their responsiveness to stimulation, and their ability to release immunomodulatory molecules that might have consequences for the disease progression.METHODS: Using flow cytometry, we investigated the monocytic population in peripheral blood mononuclear cells from PD patients and healthy individuals. We also evaluated the in vitro response to inflammogen lipopolysaccharides and to fibrillar α-synuclein by measuring the expression of CD14, CD163, and HLA-DR and by analysis of soluble immune-related molecules in the supernatant.RESULTS: Peripheral blood immune cells from PD patients had lower survival in culture, but showed a higher monocytic proliferative ability than control cells, which was correlated with shorter disease duration and late disease onset. In addition, PD patients' cells were less responsive to stimulation, as shown by the lack of changes in CD163 and CD14 expression, and by the absence of significant upregulation of anti-inflammatory cytokines in culture. Moreover, PD peripheral immune cells shed lower in vitro levels of soluble CD163, which suggests a less responsive monocytic population and/or an activation status different from control cells. Interestingly, some of the results were sex associated, supporting a differential immune response in females versus males.CONCLUSIONS: Our data suggest that PD involves monocytic changes in blood. These cells show reduced viability and are unresponsive to specific stimuli, which might have a relevant consequence for disease progression. © 2019 International Parkinson and Movement Disorder Society.