Folding Steps in the Fibrillation of Functional Amyloid: Denaturant Sensitivity Reveals Common Features in Nucleation and Elongation.

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.

Plant Polyphenols Inhibit Functional Amyloid and Biofilm Formation in Pseudomonas Strains by Directing Monomers to Off-Pathway Oligomers.

Self-assembly of proteins to ß-sheet rich amyloid fibrils is commonly observed in various neurodegenerative diseases. However, amyloid also occurs in the extracellular matrix of bacterial biofilm, which protects bacteria from environmental stress and antibiotics. Many Pseudomonas strains produce functional amyloid where the main component is the highly fibrillation-prone protein FapC. FapC fibrillation may be inhibited by small molecules such as plant polyphenols, which are already known to inhibit formation of pathogenic amyloid, but the mechanism and biological impact of inhibition is unclear. Here, we elucidate how polyphenols modify the self-assembly of functional amyloid, with particular focus on epigallocatechin gallate (EGCG), penta-O-galloyl-ß-d-glucose (PGG), baicalein, oleuropein, and procyanidin B2. We find EGCG and PGG to be the best inhibitors. These compounds inhibit amyloid formation by redirecting the aggregation of FapC monomers into oligomeric species, which according to small-angle X-ray scattering (SAXS) measurements organize into core-shell complexes of short axis diameters 25-26 nm consisting of ~7 monomers. Using peptide arrays, we identify EGCG-binding sites in FapC's linker regions, C and N-terminal parts, and high amyloidogenic sequences located in the R2 and R3 repeats. We correlate our biophysical observations to biological impact by demonstrating that the extent of amyloid inhibition by the different inhibitors correlated with their ability to reduce biofilm, highlighting the potential of anti-amyloid polyphenols as therapeutic agents against biofilm infections.

Lipidoid-polymer hybrid nanoparticles loaded with TNF siRNA suppress inflammation after intra-articular administration in a murine experimental arthritis model.

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.

Insight into the Structure and Activity of Surface-Engineered Lipase Biofluids.

Despite a successful application of solvent-free liquid protein (biofluids) concept to a number of commercial enzymes, the technical advantages of enzyme biofluids as hyperthermal stable biocatalysts cannot be fully utilized as up to 90-99% of native activities are lost when enzymes were made into biofluids. With a two-step strategy (site-directed mutagenesis and synthesis of variant biofluids) on Bacillus subtilis lipase A (BsLA), we elucidated a strong dependency of structure and activity on the number and distribution of polymer surfactant binding sites on BsLA surface. Here, it is demonstrated that improved BsLA variants can be engineered via site-mutagenesis by a rational design, either with enhanced activity in aqueous solution in native form, or with improved physical property and increased activity in solvent-free system in the form of a protein liquid. This work answered some fundamental questions about the surface characteristics for construction of biofluids, useful for identifying new strategies for developing advantageous biocatalysts.

Mesoporous silica nanoparticles carrying multiple antibiotics provide enhanced synergistic effect and improved biocompatibility.

Treatment of polymicrobial infections requires combination therapy with drugs that have different antimicrobial spectra and possibly work in synergy. However, the different pharmacokinetics and adverse side effects challenge the simultaneous delivery of multiple drugs at the appropriate concentrations to the site of infection. Formulation of multiple drugs in nano-carrier systems may improve therapeutic efficacy by increasing the local concentration and lowering the systemic concentration, leading to fewer side effects. In this study, we loaded polymyxin B and vancomycin on bare and carboxyl-modified mesoporous silica nanoparticles (B-MSNs and C-MSNs, respectively) to achieve simulataneous local delivery of antibiotics against Gram-positive and -negative bacteria. Polymyxin B adsorbed preferentially to nanoparticles compared to vancomycin. The total antibiotic loading was 563 µg and 453 µg per mg B-MSNs or C-MSNs, respectively. Both B-MSNs and C-MSNs loaded with antibiotics were effective against Gram-negative and Gram-positive bacteria. The antibiotics had synergistic interactions against Gram-negative bacteria, and the antimicrobial efficacy was higher for antibiotic-loaded C-MSNs compared to free antibiotics at the same concentration even though the cytotoxicity was lower. Our study shows that formulations of existing antibiotics in nanocarrier systems can improve their therapeutic efficiency, indicating that combination therapy with drug-loaded silica nanoparticles may provide a better treatment outcome for infections that require high concentrations of multiple drugs.

Role of Charge and Hydrophobicity in Liprotide Formation: A Molecular Dynamics Study with Experimental Constraints.

Bovine α-lactalbumin (aLA) and oleate (OA) form a complex that has been intensively studied for its tumoricidal activity. Small-angle X-ray scattering (SAXS) has revealed that this complex consists of a lipid core surrounded by partially unfolded protein. We call this type of complex a liprotide. Little is known of the molecular interactions between OA and aLA, and no technique has so far provided any high-resolution structure of a liprotide. Here we have used coarse-grained (CG) molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC) and SAXS to investigate the interactions between aLA and OA during the process of liprotide formation. With ITC we found that the strongest enthalpic interactions occurred at a molar ratio of 12.0±1.4:1 OA/aLA. Liprotides formed between OA and aLA at several OA/aLA ratios in silico were stable both in CG and in all-atom simulations. From the simulated structures we calculated SAXS spectra that show good agreement with experimentally measured patterns of matching liprotides. The simulations showed that aLA assumes a molten globular (MG) state, exposing several hydrophobic patches involved in interactions with OA. Initial binding of aLA to OA occurs in an area of aLA in which a high amount of positive charge is located, and only later do hydrophobic interactions become important. The results reveal how unfolding of aLA to expose hydrophobic residues is important for complex formation between aLA and OA. Our findings suggest a general mechanism for liprotide formation and might explain the ability of a large number of proteins to form liprotides with OA.

Liprotides assist in folding of outer membrane proteins.

Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α-lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α-lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non-ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8-9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding-inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins.

Nanostructure of the neurocentral growth plate: Insight from scanning small angle X-ray scattering, atomic force microscopy and scanning electron microscopy.

In this study, the experimental techniques scanning electron microscopy (SEM) including energy-dispersive X-ray analysis, atomic force microscopy (AFM) and scanning small angle X-ray scattering (SAXS) have been exploited to characterize the organization of large molecules and nanocrystallites in and around the neurocentral growth plate (NGP) of a pig vertebrae L4. The techniques offer unique complementary information on the nano- to micrometer length scale and provide new insight in the changes in the matrix structure during endochondral bone formation. AFM and SEM imaging of the NGP reveal a fibrous network likely to consist of collagen type II and proteoglycans. High-resolution AFM imaging shows that the fibers have a diameter of approximately 100 nm and periodic features along the fibers with a periodicity of 50-70 nm. This is consistent with the SAXS analysis that yields a cross-sectional diameter of the fibers in the range of 90 to 112 nm and a predominant orientation in the longitudinal direction of the NGP. Furthermore, we find inhomogeneities around 7 nm in the NGP by SAXS analysis. Moving towards the bone in the direction perpendicular to the growth plate, a systematic change in apparent thickness is observed, while the large-scale structural features remain constant. In the region of bone, the apparent thickness equals the mean mineral thickness and increases from 2 nm to approximately 3.5 nm as a function distance from the NGP. The mineral particles are organized as plates in a rather compact network structure. We have demonstrated that SEM, AFM and SAXS are valuable tools for the investigation of the organization of large molecules and nanocrystallites in the NGP and adjacent trabecular bone. Our findings will be an important basis for future work into identifying the defects on nanometer length scale responsible for idiopathic scoliosis and other growth-plate-related diseases.