Anti-amyloid treatment is broadly effective in neuronopathic mucopolysaccharidoses and synergizes with gene therapy in MPS-IIIA.

Mucopolysaccharidoses (MPSs) are childhood diseases caused by inherited deficiencies in glycosaminoglycan degradation. Most MPSs involve neurodegeneration, which to date is untreatable. Currently, most therapeutic strategies aim at correcting the primary genetic defect. Among these strategies, gene therapy has shown great potential, although its clinical application is challenging. We have shown previously in an MPS-IIIA mouse model that the molecular tweezer (MT) CLR01, a potent, broad-spectrum anti-amyloid small molecule, inhibits secondary amyloid storage, facilitates amyloid clearance, and protects against neurodegeneration. Here, we demonstrate that combining CLR01 with adeno-associated virus (AAV)-mediated gene therapy, targeting both the primary and secondary pathologic storage in MPS-IIIA mice, results in a synergistic effect that improves multiple therapeutic outcomes compared to each monotherapy. Moreover, we demonstrate that CLR01 is effective therapeutically in mouse models of other forms of neuronopathic MPS, MPS-I, and MPS-IIIC. These strongly support developing MTs as an effective treatment option for neuronopathic MPSs, both on their own and in combination with gene therapy, to improve therapeutic efficacy and translation into clinical application.

Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity.

Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Poration of mitochondrial membranes by amyloidogenic peptides and other biological toxins.

Mitochondria are essential organelles known to serve broad functions, including in cellular metabolism, calcium buffering, signaling pathways and the regulation of apoptotic cell death. Maintaining the integrity of the outer (OMM) and inner mitochondrial membranes (IMM) is vital for mitochondrial health. Cardiolipin (CL), a unique dimeric glycerophospholipid, is the signature lipid of energy-converting membranes. It plays a significant role in maintaining mitochondrial architecture and function, stabilizing protein complexes and facilitating efficient oxidative phosphorylation (OXPHOS) whilst regulating cytochrome c release from mitochondria. CL is especially enriched in the IMM and at sites of contact between the OMM and IMM. Disorders of protein misfolding, such as Alzheimer's and Parkinson's diseases, involve amyloidogenic peptides like amyloid-ß, tau and α-synuclein, which form metastable toxic oligomeric species that interact with biological membranes. Electrophysiological studies have shown that these oligomers form ion-conducting nanopores in membranes mimicking the IMM's phospholipid composition. Poration of mitochondrial membranes disrupts the ionic balance, causing osmotic swelling, loss of the voltage potential across the IMM, release of pro-apoptogenic factors, and leads to cell death. The interaction between CL and amyloid oligomers appears to favour their membrane insertion and pore formation, directly implicating CL in amyloid toxicity. Additionally, pore formation in mitochondrial membranes is not limited to amyloid proteins and peptides; other biological peptides, as diverse as the pro-apoptotic Bcl-2 family members, gasdermin proteins, cobra venom cardiotoxins and bacterial pathogenic toxins, have all been described to punch holes in mitochondria, contributing to cell death processes. Collectively, these findings underscore the vulnerability of mitochondria and the involvement of CL in various pathogenic mechanisms, emphasizing the need for further research on targeting CL-amyloid interactions to mitigate mitochondrial dysfunction.

HSF1 protects cells from cadmium toxicity by governing proteome integrity.

BACKGROUND: Cadmium toxicity has been associated with disruption of protein homeostasis by interfering with protein folding processes. Heat shock factor 1 (HSF1) coordinates the rapid and extensive cellular response to maintain proteomic balance facing the challenges from many environmental stressors. Thus, we suspect that HSF1 may shield cells from cadmium toxicity by conserving proteome integrity. RESULTS: Here, we demonstrate that cadmium, a highly poisonous metal, induces aggregation of cytosolic proteins in human cells, which disrupts protein homeostasis and activates HSF1. Cadmium exposure increases HSF1's phosphorylation, nuclear translocation and DNA bindings. Aside from this, HSF1 goes through liquid-liquid phase separation to form small nuclear condensates upon cadmium exposure. A specific regulatory domain of HSF1 is critical for HSF1's phase separation capability. Most importantly, human cells with impaired HSF1 are sensitized to cadmium, however, cells with overexpressed HSF1 are protected from cadmium toxicity. Overexpression of HSF1 in human cells reduces protein aggregates, amyloid fibrils and DNA damages to antagonize cadmium toxicity. CONCLUSIONS: HSF1 protects cells from cadmium toxicity by governing the integrity of both proteome and genome. Similar mechanisms may enable HSF1 to alleviate cellular toxicity caused by other heavy metals. HSF1's role in cadmium exposure may provide important insights into the toxic effects of heavy metals on human cells and body organs, allowing us to better manage heavy metal poisoning.

Metal Nanomaterials and Hydrolytic Enzyme-Based Formulations for Improved Antifungal Activity.

Active research of metal-containing compounds and enzymes as effective antifungal agents is currently being conducted due to the growing antifungal resistance problem. Metals are attracting special attention due to the wide variety of ligands that can be used for them, including chemically synthesized and naturally obtained variants as a result of the so-called "green synthesis". The main mechanism of the antifungal action of metals is the triggering of the generation and accumulation of reactive oxygen species (ROS). Further action of ROS on various biomolecules is nonspecific. Various hydrolytic enzymes (glucanases and proteases), in turn, exhibit antifungal properties by affecting the structural elements of fungal cells (cell walls, membranes), fungal quorum sensing molecules, fungal own protective agents (mycotoxins and antibiotics), and proteins responsible for the adhesion and formation of stable, highly concentrated populations in the form of biofilms. A wide substrate range of enzymes allows the use of various mechanisms of their antifungal actions. In this review, we discuss the prospects of combining two different types of antifungal agents (metals and enzymes) against mycelial fungi and yeast cells. Special attention is paid to the possible influence of metals on the activity of the enzymes and the possible effects of proteins on the antifungal activity of metal-containing compounds.

Protein Oligomer Engineering: A New Frontier for Studying Protein Structure, Function, and Toxicity.

Prevalent in nature, protein oligomers play critical roles both physiologically and pathologically. The multimeric nature and conformational transiency of protein oligomers greatly complicate a more detailed glimpse into the molecular structure as well as function. In this minireview, the oligomers are classified and described on the basis of biological function, toxicity, and application. We also define the bottlenecks in recent oligomer studies and further review numerous frontier methods for engineering protein oligomers. Progress is being made on many fronts for a wide variety of applications, and protein grafting is highlighted as a promising and robust method for oligomer engineering. These advances collectively allow the engineering and design of stabilized oligomers that bring us one step closer to understanding their biological functions, toxicity, and a wide range of applications.

J-domain proteins interaction with neurodegenerative disease-related proteins.

HSP70 chaperones, J-domain proteins (JDPs) and nucleotide exchange factors (NEF) form functional networks that have the ability to prevent and reverse the aggregation of proteins associated with neurodegenerative diseases. JDPs can interact with specific substrate proteins, hold them in a refolding-competent conformation and target them to specific HSP70 chaperones for remodeling. Thereby, JDPs select specific substrates and constitute an attractive target for pharmacological intervention of neurodegenerative diseases. This, under the condition that the exact mechanism of JDPs interaction with specific substrates is unveiled. In this review, we provide an overview of the structural and functional variety of JDPs that interact with neurodegenerative disease-associated proteins and we highlight those studies that identified specific residues, domains or regions of JDPs that are crucial for substrate binding.

Nanomechanical properties of steric zipper globular structures.

The term amyloid defines a group of proteins that aggregate into plaques or fibers. Amyloid fibers gained their fame mostly due to their relation with neurodegenerative diseases in humans. However, secreted by lower organisms, such as bacteria and fungi, amyloid fibers play a functional role: for example, when they serve as cement in the extracellular matrix of biofilms. Originating either in humans or in microorganisms, the sequence of amyloid proteins is decorated with hexapeptides with high propensity to form fibers, known as steric zippers. We have found that steric zippers form globular structures on route to making fibers and exhibit a characteristic force-distance (F-D) fingerprint when pulled with an atomic force microscope (AFM) tip. Particularly, the F-D pulling curves showed force plateau steps, suggesting that the globular structures were composed of chains that were unwound like a yarn ball. Force plateau analysis showed that the F-D characteristic parameters were sequence sensitive, representing differences in the packing of the hexapeptides within the globules. These unprecedented findings show that steric zippers exhibit a characteristic nanomechanical signature in solution in addition to previously observed characteristic crystallographic structure. Getting to the fundamental interactions that govern the unzipping of full-length amyloid fibers may initiate the development of antiamyloid methods that target the physical in addition to the structural properties of steric zippers.

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases.

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.

Interactions between S100A9 and Alpha-Synuclein: Insight from NMR Spectroscopy.

S100A9 is a pro-inflammatory protein that co-aggregates with other proteins in amyloid fibril plaques. S100A9 can influence the aggregation kinetics and amyloid fibril structure of alpha-synuclein (α-syn), which is involved in Parkinson's disease. Currently, there are limited data regarding their cross-interaction and how it influences the aggregation process. In this work, we analyzed this interaction using solution 19F and 2D 15N-1H HSQC NMR spectroscopy and studied the aggregation properties of these two proteins. Here, we show that α-syn interacts with S100A9 at specific regions, which are also essential in the first step of aggregation. We also demonstrate that the 4-fluorophenylalanine label in alpha-synuclein is a sensitive probe to study interaction and aggregation using 19F NMR spectroscopy.

Amyloid Cross-Seeding: Mechanism, Implication, and Inhibition.

Most neurodegenerative diseases such as Alzheimer's disease, type 2 diabetes, Parkinson's disease, etc. are caused by inclusions and plaques containing misfolded protein aggregates. These protein aggregates are essentially formed by the interactions of either the same (homologous) or different (heterologous) sequences. Several experimental pieces of evidence have revealed the presence of cross-seeding in amyloid proteins, which results in a multicomponent assembly; however, the molecular and structural details remain less explored. Here, we discuss the amyloid proteins and the cross-seeding phenomena in detail. Data suggest that targeting the common epitope of the interacting amyloid proteins may be a better therapeutic option than targeting only one species. We also examine the dual inhibitors that target the amyloid proteins participating in the cross-seeding events. The future scopes and major challenges in understanding the mechanism and developing therapeutics are also considered. Detailed knowledge of the amyloid cross-seeding will stimulate further research in the practical aspects and better designing anti-amyloid therapeutics.

Comparative analysis of 13C chemical shifts of ß-sheet amyloid proteins and outer membrane proteins.

Cross-ß amyloid fibrils and membrane-bound ß-barrels are two important classes of ß-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the 13C chemical shifts of 17 amyloid proteins and 7 ß-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 ß-sheet residues in amyloid fibrils and 521 ß-sheet residues in ß-barrel membrane proteins. The 13C chemical shifts are shown in 2D 13C-13C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in ß-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and ß-barrels and compare the sidechain rotameric populations. The 13C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in ß-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in ß-barrels. These trends can be explained by steric zipper interactions between ß-sheet planes in cross-ß fibrils, and by the interactions of ß-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and ß-barrels based principally on NMR chemical shifts.

Gene Therapy Strategy for Alzheimer's and Parkinson's Diseases Aimed at Preventing the Formation of Neurotoxic Oligomers in SH-SY5Y Cells.

We present here a gene therapy approach aimed at preventing the formation of Ca2+-permeable amyloid pore oligomers that are considered as the most neurotoxic structures in both Alzheimer's and Parkinson's diseases. Our study is based on the design of a small peptide inhibitor (AmyP53) that combines the ganglioside recognition properties of the ß-amyloid peptide (Aß, Alzheimer) and α-synuclein (α-syn, Parkinson). As gangliosides mediate the initial binding step of these amyloid proteins to lipid rafts of the brain cell membranes, AmyP53 blocks, at the earliest step, the Ca2+ cascade that leads to neurodegeneration. Using a lentivirus vector, we genetically modified brain cells to express the therapeutic coding sequence of AmyP53 in a secreted form, rendering these cells totally resistant to oligomer formation by either Aß or α-syn. This protection was specific, as control mCherry-transfected cells remained fully sensitive to these oligomers. AmyP53 was secreted at therapeutic concentrations in the supernatant of cultured cells, so that the therapy was effective for both transfected cells and their neighbors. This study is the first to demonstrate that a unique gene therapy approach aimed at preventing the formation of neurotoxic oligomers by targeting brain gangliosides may be considered for the treatment of two major neurodegenerative disorders, Alzheimer's and Parkinson's diseases.

S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation.

The formation of amyloid fibril plaques in the brain creates inflammation and neuron death. This process is observed in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Alpha-synuclein is the main protein found in neuronal inclusions of patients who have suffered from Parkinson's disease. S100A9 is a calcium-binding, pro-inflammation protein, which is also found in such amyloid plaques. To understand the influence of S100A9 on the aggregation of α-synuclein, we analyzed their co-aggregation kinetics and the resulting amyloid fibril structure by Fourier-transform infrared spectroscopy and atomic force microscopy. We found that the presence of S100A9 alters the aggregation kinetics of α-synuclein and stabilizes the formation of a particular amyloid fibril structure. We also show that the solution's ionic strength influences the interplay between S100A9 and α-synuclein, stabilizing a different structure of α-synuclein fibrils.

Low-Resolution Models for the Interaction Dynamics of Coated Gold Nanoparticles with ß2-microglobulin.

A large number of low-resolution models have been proposed in the last decades to reduce the computational cost of molecular dynamics simulations for bio-nano systems, such as those involving the interactions of proteins with functionalized nanoparticles (NPs). For the proteins, "minimalist" models at the one-bead-per residue (Cα-based) level and with implicit solvent are well established. For the gold NPs, widely explored for biotechnological applications, mesoscale (MS) models treating the NP core with a single spheroidal object are commonly proposed. In this representation, the surface details (coating, roughness, etc.) are lost. These, however, and the specificity of the functionalization, have been shown to have fundamental roles for the interaction with proteins. We presented a mixed-resolution coarse-grained (CG) model for gold NPs in which the surface chemistry is reintroduced as superficial smaller beads. We compared molecular dynamics simulations of the amyloid ß2-microglobulin represented at the minimalist level interacting with NPs represented with this model or at the MS level. Our finding highlights the importance of describing the surface of the NP at a finer level as the chemical-physical properties of the surface of the NP are crucial to correctly understand the protein-nanoparticle association.

Multicomponent Synthesis of Polyphenols and their in vitro Evaluation as Potential ß-Amyloid Aggregation Inhibitors.

While plant polyphenols possess a variety of biological properties, exploration of chemical diversity around them is still problematic. Here, an example of application of the Ugi multicomponent reaction to the combinatorial assembly of artificial, yet "natural-like", polyphenols is presented. The synthesized compounds represent a second-generation library directed to the inhibition of ß-amyloid protein aggregation. Chiral enantiopure compounds, and polyphenol-ß-lactam hybrids have been prepared too. The biochemical assays have highlighted the importance of the key pharmacophores in these compounds. A lead for inhibition of aggregation of truncated protein AßpE3-42 was selected.

Epibiont growth on filamentous bacteria found in activated sludge: a morphological approach.

Occurrence of epibiont attachment on filamentous bacteria is a common phenomenon in activated sludge. In this study, an attempt has been made to elucidate the intrinsic nature of the attachment between the epibionts and filamentous bacteria based on microscopic observations. Characterization of the epiflora based on fluorescence in situ hybridization using group level probes revealed that the epibionts colonizing these filamentous bacteria largely belongs to the class Alphaproteobacteria, followed by Beta and Gammaproteobacteria. The ultrastructural examination using transmission electron microscopy pointed to the existence of a possible cell-to-cell interaction between epibionts and the selected filaments. Common bacterial appendages such as pili and fimbria were absent at the interface and further noted was the presence of cell membrane extensions on epibiont bacteria protruding towards the targeted filamentous cell. Fibrillar structures resembling amyloid-like proteins were observed within the filament cells targeted by the epibionts. An interaction was apparent between amyloid such as proteins and epibionts with regards to the direction of fibrillar structures and the distance of approaching epibiont bacteria. Due to the lack of visual evidence in support of penetration, the role of these amyloid-like fibrils as potential attachment sites for the epibionts was taken into consideration, and required further validation using conformational antibodies.

Hsp90 directly interacts, in vitro, with amyloid structures and modulates their assembly and disassembly.

BACKGROUND: The 90kDa heat shock protein (Hsp90) participates in regulating the homeostasis of cellular proteins and was considered one of the key chaperones involved in the control and regulation of amyloid deposits. Hsp90 interacts with the amyloid protein tau through tau aggregation-prone regions, including the VQIVYK hexapeptide motif. This hexapeptide, which self-aggregates, forming amyloid fibrils, is widely used to model amyloid formation mechanisms. Despite evidence showing that Hsp90 interacts directly with Ac-VQIVYK-NH2, its role in the hexapeptide fibrillation process and its binding to peptide structures have not yet been determined. METHODS: Various biochemical and biophysical techniques, including ultracentrifugation, spectrophotometry, spectrofluorimetry, and electron microscopy, were employed to assess the effects of Hsp90 on Ac-VQIVYK-NH2 assembly and disassembly processes. RESULTS: At sub-stoichiometric concentrations, Hsp90 bound directly to Ac-VQIVYK-NH2 amyloid structures in vitro, with each Hsp90 dimer interacting with an amyloid structure made of around 50 hexapeptide subunits. Hsp90 inhibited Ac-VQIVYK-NH2 assembly by increasing the critical concentrations of Ac-VQIVYK-NH2 required for assembly. Hsp90 also inhibited the disassembly of Ac-VQIVYK-NH2 amyloid fibrils and promoted their rescue. CONCLUSIONS: A model explaining the dual effect of Hsp90 on the Ac-VQIVYK-NH2 amyloid fibrillation process has been proposed. GENERAL SIGNIFICANCE: These in vitro results provide new insights into the possible roles of molecular chaperones in modulating amyloid structures by limiting the spread of toxic species.

Photoluminescence of Diphenylalanine Peptide Nano/Microstructures: From Mechanisms to Applications.

Diphenylalanine (Phe-Phe, FF) molecules, which can self-assemble into highly ordered nano/microstructures, have increasingly aroused intense interests due to their special optical properties. In this review, recent advances in photoluminescence (PL) of supramolecular architectures of FF-based peptide and the underlying mechanisms are highlighted. Mainly deep ultraviolet emission at around 285 nm and/or blue emission at ≈450 nm are observed in various FF peptide structures and its derivatives, which are primarily interpreted by quantum confinement effects, shallow radiative traps, and electron delocalization via hydrogen bonds in ß-sheet structures. Furthermore, current applications of such fluorescent peptide nano/microstructures are also reviewed here, e.g., probing the number of water molecules confined in FF, temperature sensing, and visualization of deep ultraviolet beam. Yet, the PL mechanism is still under fierce debate and the application based on fluorescence is constantly under exploration. Thus, this review is endeavored to boost future explorations on the PL of the bioinspired FF peptide nano/microstructures.

Chemical Methods to Knock Down the Amyloid Proteins.

Amyloid proteins are closely related with amyloid diseases and do tremendous harm to human health. However, there is still a lack of effective strategies to treat these amyloid diseases, so it is important to develop novel methods. Accelerating the clearance of amyloid proteins is a favorable method for amyloid disease treatment. Recently, chemical methods for protein reduction have been developed and have attracted much attention. In this review, we focus on the latest progress of chemical methods that knock down amyloid proteins, including the proteolysis-targeting chimera (PROTAC) strategy, the "recognition-cleavage" strategy, the chaperone-mediated autophagy (CMA) strategy, the selectively light-activatable organic and inorganic molecules strategy and other chemical strategies.