Search Results (230)

The misfolding and aggregation of proteins into amyloidogenic assemblies are key features of several metabolic and neurodegenerative diseases. Human insulin has long been known to form amyloid fibrils under various conditions, which affects its bioavailability and function. Clinically, insulin aggregation at recurrent injection sites poses a challenge for diabetic patients who rely on insulin therapy. Furthermore, decreased responsiveness to insulin in type 2 diabetic (T2D) patients may lead to its overproduction and accumulation as aggregates. Earlier reports have reported that various factors such as pH, temperature, agitation, and the presence of lipids or other proteins influence insulin aggregation. Our present study aims to elucidate the effects of non–micellar anionic DMPG (1,2–dimyristoyl–sn–glycero–3–phosphoglycerol) lipids on insulin aggregation. Distinct pathways of insulin aggregation and intermediate formation were observed in the presence of DMPG using a ThT fluorescence assay. The formation of soluble intermediates alongside large insulin fibrils was observed in insulin incubated with DMPG via TEM, DLS, and NMR as opposed to insulin aggregates generated without lipids. ¹³C magic angle spinning solid–state NMR and FTIR experiments indicated that lipids do not alter the conformation of insulin fibrils but do alter the time scale of motion of aromatic and aliphatic side chains. Furthermore, the soluble intermediates were found to be more cytotoxic than fibrils generated with or without lipids. Overall, our study elucidates the importance of anionic lipids in dictating the pathways and intermediates associated with insulin aggregation. These findings could be useful in determining various approaches to avoid toxicity and enhance the effectiveness of insulin in therapeutic applications. Full article

Peptide-induced disruption of lipid membranes is central to both amyloid diseases and the activity of antimicrobial peptides. Here, we combine all-atom molecular dynamics simulations with biophysical experiments to investigate how four amphipathic peptides interact with lipid bilayers. All peptides adsorb on the membrane surface. Peptide M01 [Ac-(FKFE)₂-NH₂] self-assembles into $\beta$-sheet nanofibrils that span both leaflets of the membrane, creating water-permeable channels. The other three peptides adopt $\alpha$-helical structures at the water–lipid interface. Peptide M02 [Ac-FFKKFFEE-NH₂], a sequence isomer of M01, does not form $\beta$-sheet aggregates and is too short to span the bilayer, resulting in no observable water permeation across the membrane. Peptides M03 and M04 are $\alpha$-helical isomers long enough to span the bilayer, with a polar face that allows the penetration of water deep inside the membrane. For the M03 peptide [Ac-(FFKKFFEE)₂-NH₂], insertion into the bilayer starts with the nonpolar N-terminal amino acids penetrating the hydrophobic core of the bilayer, while electrostatic interactions hold negative residues at the C-terminus on the membrane surface. The M04 peptide, [Ac-FFKKFFEEFKKFFEEF-NH₂], is made by relocating a single nonpolar residue from the central region of M03 to the C-terminus. This nonpolar residue becomes unfavorably exposed to the solvent upon insertion of the N-terminal region of the peptide into the membrane. Consequently, higher concentrations of M04 peptides are required to induce water permeation compared to M03. Overall, our comparative analysis reveals how subtle rearrangements of polar and nonpolar residues modulate peptide-induced water permeation. This provides mechanistic insights relevant to amyloid pathology and antimicrobial peptide design. Full article

Over 150 transthyretin (TTR) mutations have been identified in hereditary transthyretin (ATTRv) amyloidosis, and new TTR variants have recently emerged. However, the pathogenicity of several new variants remains unclear, making it important to elucidate the differences between amyloidogenic and wild-type TTR. In this study, we report a novel TTR variant (V121A) identified in two unrelated amyloidosis patients aged > 60 years who developed cardiomyopathy. We evaluated the detailed biochemical features of this TTR variant to confirm its amyloidogenicity using plasma samples from these patients and recombinant TTR proteins. While the V121A TTR variant has a similar ability to assemble into a tetramer as wild-type TTR, it aggregates more readily over a wide potential hydrogen range than wild-type TTR. Additionally, the V121A variant is highly prone to dissociation and resistant to binding with known TTR tetramer stabilizers. Clinical and biochemical data suggest that this novel variant is clearly pathogenic, is highly prone to dissociation and aggregation, and is associated with the development of late-onset amyloid cardiomyopathy. Interestingly, amyloid fibril formation due to this variant may not be affected by known TTR stabilizers. Full article

Postoperative peritoneal adhesion and high recurrence rates are critical challenges in the clinical treatment of colorectal cancer. In this study, based on amyloid-like protein self-assembly technology, a novel Janus protein film was developed. The protein film encapsulates glucose oxidase (GOx) and catalase (CAT), which is named PTL@GC. Through a one-step method involving cysteine-reduced lysozyme-induced amyloid-like self-assembly, the film was co-loaded with GOx and CAT to achieve synergistic anti-adhesion and anti-tumor recurrence effects. The Janus film features a hydrophobic side that stably adheres to the intestinal surface without exogenous chemical modification and a hydrophilic side that prevents adhesion. The loaded GOx selectively induces disulfidptosis in SLC7A11-overexpressing tumor cells, while CAT degrades H₂O₂ to alleviate hypoxia and inhibit oxidative stress, significantly reducing adhesion-related fibrosis. The experimental results demonstrate that PTL@GC exhibited excellent mechanical properties, high enzyme activity retention (>90%), and controllable degradability (complete metabolism within 50 days). In animal models, PTL@GC reduced postoperative adhesion area by 22.77%, decreased local tumor burden to 28.42% of the control group, and achieved an inhibition rate of 58.49%, without inducing systemic toxicity. This study presents a biologically safe and functionally synergistic approach to addressing dual complications following colorectal cancer surgery, offering potential insights for future research on multifunctional Janus materials. Full article

α-Synuclein (αS) interacts with lipid membranes in neurons to form amyloid fibrils that contribute to Parkinson’s disease, and its non-amyloid-β component domain is critical in the fibrillation. In this study, the salt (NaCl) effect on the membrane interaction and fibril formation of αS_57–102 peptide (containing the non-amyloid-β component domain) was characterized at the molecular level because the αS_57–102 fibrils exhibited structural polymorphism with two morphologies (thin and thick) in the presence of NaCl but showed one morphology (thin) in the absence of NaCl. The membrane-bound conformation (before fibrillation) of αS_57–102 had two helical regions (first and second) on the membrane regardless of salt, but the length of the first region largely shortened when NaCl was present, exposing its hydrophobic area to the solvent. The exposed region induced two distinct pathways of fibril nucleation, depending on the molar ratios of free and membrane-bound αS_57–102: one from the association of free αS_57–102 with membrane-bound αS_57–102 and the other from the assembly among membrane-bound αS_57–102. The differences mainly affected the β-strand orientation and helical content within the fibril conformations, probably contributing to the thickness degree, leading to structural polymorphism. Full article

The assembly of amyloidogenic proteins and peptides into toxic oligomeric and fibrillar aggregates is closely connected to the onset and progression of more than 50 protein diseases, such as Alzheimer’s disease, Parkinson’s disease, prion disease, and type 2 diabetes, to name only a few. Considerable research efforts at identifying the therapeutic strategies against these maladies are currently focused on preventing and inhibiting pathogenic protein aggregation by various agents. Plant-based extracts and compounds have emerged as promising sources of potential inhibitors due to their dual role as nutraceuticals as part of healthy diets and as specific pharmaceuticals when administered at higher concentrations. In recent decades, several plant extracts and plant-extracted compounds have shown potential to modulate protein aggregation. An ever-growing body of research on plant-based amyloid inhibitors requires a detail analysis of existing data to identify potential knowledge gaps. This review summarizes the recent progress in amyloid inhibition using 17 flavonoids, 11 polyphenolic non-flavonoid compounds, 23 non-phenolic inhibitors, and 59 plant extracts, with the main emphasis on directly modulating the fibrillation of four amyloid proteins, namely amyloid-β peptide, microtubule-associated protein tau, α-synuclein, and human islet amyloid polypeptide. Full article

Amyloid fibrils are one of the important forms of protein aggregates, first discovered in the pathological brain tissues of patients with various neurodegenerative diseases. They are considered the core pathological markers of different neurodegenerative diseases. In recent years, research has found that multiple proteins or peptides dynamically assemble to form functional amyloid-like nanofibrils under physiological conditions, exhibiting excellent mechanical properties, high environmental stability, and self-healing ability. Therefore, they have become a class of functional biological nanomaterials with important development potential. This article systematically reviews the latest progress in the preparation, functionalization, and application of amyloid-like nanofibrils in engineering and provides an outlook on possible future development directions. Full article

The misfolding and amyloid aggregation of proteins have been attracting scientific interest for a few decades, due to their link with several diseases, particularly neurodegenerative diseases. Proteins can assemble and result in insoluble aggregates that, together with intermediate oligomeric species, modify the extracellular environment. Many efforts have been and are devoted to the search for cosolvents and cosolutes able to interfere with amyloid aggregation. In this work, we intensively study the effect of saponins, bioactive compounds, on human insulin aggregation. To monitor the kinetic of amyloid aggregation following secondary structure changes, we perform fluorescence and UV-Visible absorption spectroscopies, using Thioflavin T and Congo Red as amyloid specific probes, and Circular Dichroism. To study the overall structural features and size of aggregates, we perform Synchrotron Small-Angle X-ray Scattering and Dynamic Light Scattering experiments. The morphology of the aggregates was assessed by Atomic Force Microscopy. To deepen the understanding of the saponins interaction with insulin, a Molecular Dynamics investigation is performed, too. The reported data demonstrate that saponins interfere with the amyloid aggregation by inducing a strong inhibition on the formation of insulin fibrils, likely through specific interactions with insulin monomers. A dose-dependent effect is evident, and amyloid inhibition is already clear when saponins are just 0.01% w/w in solution. We suggest that saponins, which are natural metabolites present in a wide range of foods ranging from grains, pulses, and green leaves to sea stars and cucumbers, can be promising metabolites to inhibit human insulin aggregation. This basic research work can pave the way to further investigations concerning insulin amyloidosis, suggesting the use of saponins as amyloid inhibitors and/or stabilizing agents in solution. Full article

Background/Objectives: This study aimed to design and evaluate Chol-PEG₂₀₀₀ micelles and Chol-PEG₅₀₀ vesicles as drug delivery system (DDS) carriers and inhibitors of amyloid-β (Aβ) aggregation, a key factor in Alzheimer’s disease (AD). Methods: The physical properties of Chol-PEG assemblies were characterized using dynamic light scattering (DLS), electrophoretic light scattering (ELS), and transmission electron microscopy (TEM). Inhibitory effects on Aβ aggregation were assessed via thioflavin T (ThT) assay, circular dichroism (CD) spectroscopy, and native polyacrylamide gel electrophoresis (native-PAGE). Results: Chol-PEG₂₀₀₀ micelles and Chol-PEG₅₀₀ vesicles were found to exhibit diameters of 20–30 nm and 70–80 nm, respectively, with neutral surface charges and those physical properties indicated the high affinity for Aβ. At a 10-fold molar ratio, thioflavin T (ThT) assay revealed that Chol-PEG₂₀₀₀ delayed Aβ fibril elongation by 20 hours, while Chol-PEG₅₀₀ delayed it by 40 hours against Aβ peptide. At a 50-fold molar ratio, both Chol-PEG₂₀₀₀ and Chol-PEG₅₀₀ significantly inhibited Aβ aggregation, as indicated by minimal fluorescence intensity increases over 48 hours. CD spectroscopy indicated that Aβ maintained its random coil structure in the presence of Chol-PEG assemblies at a 50-fold molar ratio. Native-PAGE analysis demonstrated a retardation in Aβ migration immediately after mixing with Chol-PEG assemblies, suggesting complex formation. However, this retardation disappeared within 5 min, implying rapid dissociation of the complexes. Conclusions: This study demonstrated that Chol-PEG₅₀₀ vesicles more effectively inhibit Aβ aggregation than Chol-PEG₂₀₀₀ micelles. Chol-PEG assemblies perform as DDS carriers to be capable of inhibiting Aβ aggregation. Chol-PEG assemblies can deliver additional therapeutics targeting other aspects of AD pathology. This dual-function platform shows promise as both a DDS carrier and a therapeutic agent, potentially contributing to a fundamental cure for AD. Full article

Background: At present, the complexity that governs the associations between different biological entities is understood better than ever before, owing to high-throughput techniques and systems biology. Networks of interactions are necessary not only for the visualization of these complex relationships but also because their analysis tends to be valuable for the extraction of novel biological knowledge. Methods: For this reason, we constructed a disease–protein–drug network, focusing on a category of rare protein-misfolding diseases, known as amyloidoses, and on other pathological conditions also associated with amyloid deposition. Apart from the amyloidogenic proteins that self-assemble into fibrils, we also included other co-deposited proteins found in amyloid deposits. Results: In this work, protein–protein, protein–drug, and disease–drug associations were collected to create a heterogenous network. Through disease-based and drug-based analyses, we highlighted commonalities between diseases and proposed an approved drug with prospects of repurposing. Conclusions: The identified disease associations and drug candidates are proposed for further study that will potentially help treat diseases associated with amyloid deposition. Full article

Alzheimer’s disease (AD) is primarily driven by the formation of toxic amyloid-β (Aβ) aggregates, with Aβ42 being a pivotal contributor to disease pathology. This study investigates a novel agent to mitigate Aβ42-induced toxicity by co-assembling Aβ42 with its G37V variant (Aβ42(G37V)), where Gly at position 37 is substituted with valine. Using a combination of Thioflavin-T (Th-T) fluorescence assays, Western blot analysis, atomic force microscopy (AFM)/transmission electron microscopy (TEM), and biochemical assays, we demonstrated that adding Aβ42(G37V) significantly accelerates Aβ42 aggregation rate and mass while altering the morphology of the resulting aggregates. Consequently, adding Aβ42(G37V) reduces the Aβ42 aggregates-induced cytotoxicity, as evidenced by improved cell viability assays. The possible mechanism for this effect is that adding Aβ42(G37V) reduces the production of reactive oxygen species (ROS) and lipid peroxidation, typically elevated in response to Aβ42, indicating its protective effects against oxidative stress. These findings suggest that Aβ42(G37V) could be a promising candidate for modulating Aβ42 aggregation dynamics and reducing its neurotoxic effects, providing a new avenue for potential therapeutic interventions in AD. Full article

Alzheimer’s disease (AD) is a complex neurodegenerative disorder influenced by various genetic factors. In addition to the well-established amyloid precursor protein (APP), Presenilin-1 (PSEN1), Presenilin-2 (PSEN2), and apolipoprotein E (APOE), several other genes such as Sortilin-related receptor 1 (SORL1), Phospholipid-transporting ATPase ABCA7 (ABCA7), Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), Phosphatidylinositol-binding clathrin assembly protein (PICALM), and clusterin (CLU) were implicated. These genes contribute to neurodegeneration through both gain-of-function and loss-of-function mechanisms. While it was traditionally thought that heterozygosity in autosomal recessive mutations does not lead to disease, haploinsufficiency was linked to several conditions, including cancer, autism, and intellectual disabilities, indicating that a single functional gene copy may be insufficient for normal cellular functions. In AD, the haploinsufficiency of genes such as ABCA7 and SORL1 may play significant yet under-explored roles. Paradoxically, heterozygous knockouts of PSEN1 or PSEN2 can impair synaptic plasticity and alter the expression of genes involved in oxidative phosphorylation and cell adhesion. Animal studies examining haploinsufficient AD risk genes, such as vacuolar protein sorting-associated protein 35 (VPS35), sirtuin-3 (SIRT3), and PICALM, have shown that their knockout can exacerbate neurodegenerative processes by promoting amyloid production, accumulation, and inflammation. Conversely, haploinsufficiency in APOE, beta-secretase 1 (BACE1), and transmembrane protein 59 (TMEM59) was reported to confer neuroprotection by potentially slowing amyloid deposition and reducing microglial activation. Given its implications for other neurodegenerative diseases, the role of haploinsufficiency in AD requires further exploration. Modeling the mechanisms of gene knockout and monitoring their expression patterns is a promising approach to uncover AD-related pathways. However, challenges such as identifying susceptible genes, gene–environment interactions, phenotypic variability, and biomarker analysis must be addressed. Enhancing model systems through humanized animal or cell models, utilizing advanced research technologies, and integrating multi-omics data will be crucial for understanding disease pathways and developing new therapeutic strategies. Full article

Functional amyloids (protein nanofibrils, PNF) synthesized from plant sources exhibit unique physicochemical and nanomechanical properties that could improve food texture. While environmental factors affecting PNFs are well-known, scientific evidence on how cells (focus on the oral cavity) respond to them under physiological conditions is lacking. Self-assembled PNFs synthesized from fava bean whole protein isolate show a strong pH- and solvent-dependent morphology and elasticity modification measured by atomic force microscopy (AFM). After incubation of PNFs with an oral mechanosensitive model cell line at pH 7.3, difference in cell-surface roughness without significant changes in the overall cell elasticity were measured. The role of cell membrane composition on supported lipid bilayers was also tested, showing an increase in membrane elasticity with increasing fibril concentration and the possible impact of annular phospholipids in binding. Genetic responses of membrane proteins involved in texture and fat perception were detected at the mRNA level by RT-qPCR assay and both mechano- and chemosensing proteins displayed responses highlighting an interface dependent interaction. The outcomes of this study provide a basis for understanding the changing physicochemical properties of PNFs and their effect on flavor perception by altering mouthfeel and fat properties. This knowledge is important in the development of plant-based texture enhancers for sensory-appealing foods that require consumer acceptance and further promote healthy diets. Full article

Tau is an intrinsically disordered protein involved in several neurodegenerative diseases where a common hallmark is the appearance of tau aggregates in the brain. One common approach to elucidate the mechanisms behind the aggregation of tau has been to recapitulate in vitro the self-assembly process in a fast and reproducible manner. While the seeding of tau aggregation is prompted by negatively charged cofactors, the obtained fibrils are morphologically distinct from those found in vivo. The Tau AD core fragment (TADC, tau 306–378) has emerged as a new model and potential solution for the cofactor-free in vitro aggregation of tau. Here, we use TADC to further study this process combining multiple amyloid-detecting fluorophores and fibril bioimaging. We confirmed by transmission electron microscopy that this fragment forms fibrils after quiescent incubation at 37 °C. We then employed a panel of eight amyloid-binding fluorophores to query the formed species by acquiring their emission spectra. The results obtained showed that nearly all dyes detect TADC self-assembled species. However, the successful monitoring of TADC aggregation kinetics was limited to three fluorophores (X-34, Bis-ANS, and pFTAA) which yielded sigmoidal curves but different aggregation half-times, hinting to different species being detected. Altogether, this study highlights the potential of using multiple extrinsic fluorescent probes, alone or in combination, as tools to further clarify mechanisms behind the aggregation of amyloidogenic proteins. Full article

Atomic force microscopy (AFM) imaging enables the visualization of protein molecules with high resolution, providing insights into their shape, size, and surface topography. Here, we use AFM to study the aggregation process of protein S100A9 in physiological conditions, in the presence of calcium at a molar ratio 4Ca²⁺:S100A9. We find that S100A9 readily assembles into a worm-like fibril, with a period dimension along the fibril axis of 11.5 nm. The fibril’s chain length extends up to 136 periods after an incubation time of 144 h. At room temperature, the fibril’s bending stiffness was found to be $2.95\times {10}^{-28}$ Nm², indicating that the fibrils are relatively flexible. Additionally, the values obtained for the Young’s modulus ($E_x=6.96\times {10}^5$ Pa and $E_y=3.37\times {10}^5$ Pa) are four orders of magnitude lower than those typically reported for canonical amyloid fibrils. Our findings suggest that, under the investigated conditions, a distinct aggregation mechanism may be in place in the presence of calcium. Therefore, the findings reported here could have implications for the field of biomedicine, particularly with regard to Alzheimer’s disease. Full article