Abnormal aggregation of microtubule-associated protein tau into β-sheet-rich fibrils is a hallmark feature of Alzheimer's disease and other tauopathies. The pathogenic P301L mutation within the microtubule-binding domain of tau promotes tau filament formation; however, the molecular mechanisms by which intracellular RNAs regulate this aggregation process remain not fully understood. Here, we investigated the mechanistic effects of RNA homopolymers on the aggregation of a tau fragment peptide (residues 298-317) derived from the microtubule-binding region and its P301L mutant. The results showed that while the wild-type peptide remained resistant to aggregation in the presence of RNA, pyrimidine-rich RNAs (poly(C) and poly(U)) significantly accelerated fibrillation of the P301L mutant. The mutation likely disrupts the local conformational constraints, leading to a more flexible conformation and exposure of hydrophobic residues. This facilitates intermolecular interactions to form β-sheet-rich aggregates after RNA-induced local condensation and alignment of the peptide as a nucleation scaffold for aggregation. In contrast, purine-rich RNAs (poly(A) and poly(G)) had negligible effects on P301L mutant aggregation, suggesting that the specific chemical and conformational properties of RNA, such as base structures, geometrical arrangement, and base stacking, in addition to its polyanionic nature, are critical determinants of its ability to modulate tau peptide amyloid formation. Furthermore, the polycationic molecules spermine and polyarginine effectively delayed or inhibited RNA-induced aggregation, indicating that rationally designed polycations could serve as valuable modulators of RNA-mediated fibrillation within the crucial tau aggregation-prone region.

Aberrant proteolytic processing of amyloid precursor protein (APP) can alter amyloid-β (Aβ) peptide trafficking, with recent studies implicating MUC1-type O-glycosylation as a modulatory factor. In this study, we synthesized native and Swedish-mutated (Lys670Asn/Met671Leu) APP glycopeptides spanning the Aβ(1-23) region, including the β- and α-secretase cleavage sites, and introduced O-GalNAc moieties at Thr663, Ser667, and/or Tyr681. Circular dichroism (CD) revealed conformational changes governed by the glycosylation site and glycan density. Increased glycan valency favored the stabilization of β-turn-rich structures typically associated with oligomeric and prefibrillar intermediates. The Swedish mutation enhanced β-secretase (BACE1) cleavage, especially when Ser667 was glycosylated, while additional glycans favored α-secretase (ADAM10) processing. However, this shift was not sufficient to counterbalance the amyloidogenic pathway. Similarly, Ser667 glycosylation promoted fibril formation in coincubation assays with Aβ40, while di- and triglycosylated peptides disrupted fibril architecture and favored oligomer formation, as confirmed by ThT kinetics, AFM/TEM imaging, and dynamic light scattering. These findings highlight the critical role of mutation and site-specific glycosylation in shaping APP proteolytic processing, secondary structure, and aggregation behavior, underscoring their importance for understanding APP function in both healthy and diseased states.

Prion diseases are characterized by the self-association and amyloid formation of misfolded prion proteins. Developing effective inhibitors of protein aggregation is critical for therapeutic intervention. In this study, we systematically evaluated a range of polyphenolic compounds as potential inhibitors of amyloid fibril formation of PrP(106-128), a prion fragment crucially involved in prion aggregation and propagation. Our findings demonstrate that the basic aromatic backbone structure of flavone alone is insufficient to inhibit PrP(106-128) amyloid formation. Remarkably, flavone molecules containing adjacent hydroxyl groups on the phenolic B or A ring efficiently inhibited PrP(106-128) fibrillization, whereas compounds lacking vicinal hydroxyl groups were less effective in inhibiting amyloid formation. Epigallocatechin-3-gallate (EGCG) was one of the most potent inhibitors found in this study, with the gallate moiety playing an active role in the inhibitory function. Our findings indicate a structure-dependent inhibition activity of the phenolic small molecules, where the number and positioning of hydroxyl groups on the phenyl ring play a pivotal role in inhibiting the aggregation of the peptide. The auto-oxidation of the catechol or pyrogallol moieties to form quinone structures, followed by their reaction with amino acid side chains of the peptide to form covalent adducts, likely account for the inhibitory activity of these phenolic compounds on PrP(106-128) amyloidogenesis. These results will help the design of novel polyphenolic molecules with optimized structural features as potent inhibitors of amyloid formation of both PrP(106-128) and the full-length prion proteins.

Aggregation of tau protein is a hallmark feature of tauopathies such as Alzheimer’s disease. The microtubule-binding domain of tau plays a crucial role in the tau aggregation process. In this study, we investigated the dual effects of membrane interactions of tau298–317, a fragment peptide from the microtubule-binding domain, on peptide-induced membrane disruption and membrane-mediated peptide self-assembly. Our results show that neither wild-type tau298–317 nor its P301L or Ser305-phosphorylated mutants aggregate in the presence of zwitterionic POPC vesicles or cause lipid vesicle leakage, indicating weak peptide–membrane interactions. In contrast, tau298–317 strongly interacts with negatively charged POPG liposomes, leading to a rapid transition of the peptide conformation from random coils to α-helical intermediate conformation upon membrane adsorption, which may further promote peptide self-association to form oligomers and β-sheet-rich fibrillar structures. Tau298–317-induced rapid POPG membrane leakage indicates a synergistic process of the peptide self-assembly at the membrane interface and the aggregation-induced membrane disruption. Notably, phosphorylation at Ser305 disrupts favorable electrostatic interactions between the peptide and POPG membrane surface, thus preventing peptide aggregation and membrane leakage. In contrast, the P301L mutation significantly enhances membrane-mediated peptide aggregation and peptide-induced membrane disruption, likely due to alleviation of local conformational constraints and enhancement of local hydrophobicity, which facilitates fast conformational conversion to β-sheet structures. These findings provide mechanistic insights into the molecular mechanisms underlying membrane-mediated aggregation of crucial regions of tau and peptide-induced membrane damage, indicating potential strategies to prevent tau aggregation and membrane rupture by targeting critical electrostatic interactions between membranes and key local regions of tau.

Misfolding and aggregation of cellular prion protein (PrPc) is a major molecular process involved in the pathogenesis of prion diseases. Here, we studied the aggregation properties of a prion fragment peptide PrP (106-128). The results show that the peptide aggregates in a concentration-dependent manner in an aqueous solution and that the aggregation is sensitive to pH and the preformed amyloid seeds. Furthermore, we show that the zwitterionic POPC liposomes moderately inhibit the aggregation of PrP(106-128), whereas POPC/cholesterol (8:2) vesicles facilitate peptide aggregation likely due to the increase of the lipid packing order and membrane rigidity in the presence of cholesterol. In addition, anionic lipid vesicles of POPG and POPG/cholesterol above a certain concentration accelerate the aggregation of the peptide remarkably. The strong electrostatic interactions between the N-terminal region of the peptide and POPG may constrain the conformational plasticity of the peptide, preventing insertion of the peptide into the inner side of the membrane and thus promoting fibrillation on the membrane surface. The results suggest that the charge properties of the membrane, the composition of the liposomes, and the rigidity of lipid packing are critical in determining peptide adsorption on the membrane surface and the efficiency of the membrane in catalyzing peptide oligomeric nucleation and amyloid formation. The peptide could be used as an improved model molecule to investigate the mechanistic role of the crucial regions of PrP in aggregation in a membrane-rich environment and to screen effective inhibitors to block key interactions between these regions and membranes for preventing PrP aggregation.

Tauopathies are neurodegenerative diseases characterized by the deposition of abnormal tau in the brain. To date, there are no disease-modifying therapies approved by the U.S. Food and Drug Administration (US FDA) for the treatment of tauopathies. In the past decades, extensive efforts have been provided to develop disease-modifying therapies to treat tauopathies. Specifically, exploring existing drugs with the intent of repurposing for the treatment of tauopathies affords a reasonable alternative to discover potent drugs for treating these formidable diseases. Drug repurposing will not only reduce formulation and development stage effort and cost but will also take a key advantage of the established toxicological studies, which is one of the main causes of clinical trial failure of new molecules. In this review, we provide an overview of the current treatment strategies for tauopathies and the recent progress in drug repurposing as an alternative approach to treat tauopathies.

Amyloid fibril formation is associated with protein-misfolding disorders and is a key pathway for the pathogenesis of Alzheimer’s disease (AD). The fibrillar structure of amyloid beta-protein (Aβ) is the hallmark of amyloid plaques in the brains of patients with Alzheimer’s disease (AD). The interaction between the cell membrane and the Aβ40 peptide may induce membrane disruption and membrane leakage and may be related to the toxicity in Alzheimer’s disease (AD). In this regard, we evaluated the competitive effects of different polyelectrolytes such as chitosan (CHT), N-trimethyl chitosan (TMC), heparin and dextran on peptide-phospholipid membrane interactions and Aβ40 fibrillation. The impact of polyelectrolytes on AB aggregation at membrane interface and fibrillation of Aβ40 peptide was evaluated by utilizing biophysical techniques such as ThT binding assays and atomic force microscopy. The polyelectrolytes and lipid membranes co-exist extensively in the cellular environment, we demonstrate that the interactions of polyelectrolytes and Aβ may substantially influence the role of the N-terminal charged region of Aβ in initiating and guiding adsorption and aggregation of the peptide on the membrane surface. Hence, there is a great deal of interest in polyelectrolytes that can bind to the Aβ40 peptide and interfere with the Aβ40 peptide-membrane interactions and manipulate amyloid formation in Alzheimer’s disease (AD).

The microtubule-associated protein tau is involved in more than 20 different neurological disorders characterized by aberrant intracellular aggregation of tau in the brain. Here, we investigated the aggregation of a novel 20-residue model peptide, tau298-317, which is derived from the key microtubule binding domain of the full sequence tau. Our results show that tau298-317 highly mimics the physical and aggregation properties of tau. Under normal physiological conditions, the peptide maintains a disordered random coil without aggregation. The presence of polyanionic heparin (Hep) significantly promotes the aggregation of this peptide to form amyloid fibrils. The Hep-induced aggregation is sensitive to the ionic strength of the solution and the introduction of the negatively charged phosphate group on a serine (Ser305) residue in the sequence, suggesting an important role of electrostatic interactions in the mechanism of Hep-mediated aggregation. In addition, two positively charged polysaccharides, chitosan (CHT) and its quaternary derivative N-trimethyl chitosan (TMC), were found to effectively inhibit Hep-induced aggregation of tau298-317 in a concentration-dependent manner. Attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues of Hep play a critical role in inhibiting Hep-peptide interactions and suppressing peptide aggregation. Our results suggest that positively charged polyelectrolytes with optimized charged groups and charge distribution patterns can serve as effective molecular candidates to block tau-Hep interactions and prevent aggregation of tau induced by Hep and other polyanions.

The amyloid-β precursor protein (APP) undergoes proteolysis by β- and γ-secretases to form amyloid-β peptides (Aβ), which is a hallmark of Alzheimer's disease (AD). Recent findings suggest a possible role of O-glycosylation on APP's proteolytic processing and subsequent fate for AD-related pathology. We have previously reported that Tyr681-O-glycosylation and the Swedish mutation accelerate cleavage of APP model glycopeptides by β-secretase (amyloidogenic pathway) more than α-secretase (non-amyloidogenic pathway). Therefore, to further our studies, we have synthesized additional native and Swedish-mutated (glyco)peptides with O-GalNAc moiety on Thr663 and/or Ser667 to explore the role of glycosylation on conformation, secretase activity, and aggregation kinetics of Aβ40. Our results show that conformation is strongly dependent on external conditions such as buffer ions and solvent polarity as well as internal modifications of (glyco)peptides such as length, O-glycosylation, and Swedish mutation. Furthermore, the level of β-secretase activity significantly increases for the glycopeptides containing the Swedish mutation compared to their nonglycosylated and native counterparts. Lastly, the glycopeptides impact the kinetics of Aβ40 aggregation by significantly increasing the lag phase and delaying aggregation onset, however, this effect is less pronounced for its Swedish-mutated counterparts. In conclusion, our results confirm that the Swedish mutation and/or O-glycosylation can render APP model glycopeptides more susceptible to cleavage by β-secretase. In addition, this study sheds new light on the possible role of glycosylation and/or glycan density on the rate of Aβ40 aggregation.

Abnormal deposition of tau in neurons is a hallmark of Alzheimer's disease and several other neurodegenerative disorders. In the past decades, extensive efforts have been made to explore the mechanistic pathways underlying the development of tauopathies. Recently, the discovery of tau droplet formation by liquid-liquid phase separation (LLPS) has received a great deal of attention. It has been reported that tau condensates have a biological role in promoting and stabilizing microtubule (MT) assembly. Furthermore, it has been hypothesized that the transition of phase-separated tau droplets to a gel-like state and then to fibrils is associated with the pathology of neurodegenerative diseases. In this review, we outline LLPS, the structural disorder that facilitates tau droplet formation, the effects of posttranslational modification of tau on condensate formation, the physiological function of tau droplets, the pathways from droplet to toxic fibrils, and the therapeutic strategies for tauopathies that might evolve from toxic droplets. We expect a deeper understanding of tau LLPS will provide additional insights into tau physiology and tauopathies.

Amyloidogenesis of amyloid-β (Aβ) peptides is intimately related to pathological neurodegeneration in Alzheimer’s disease. Here, we investigated the membrane damage activity of Aβ40 and its derivatives that contain mutation at the N-terminal charged residues using a membrane leakage assay. A model 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipid vesicle encapsulating the fluorescent dye carboxyfluorescein was used in the study. Our results show that the mutations of the N-terminal charged residues of Aβ40 significantly affect the peptide-induced membrane leakage. The results suggest that favorable electrostatic interactions of the N-terminal charged residues and the phosphatidylcholine membrane surface are crucial in Aβ-mediated membrane permeation. The flexible and charge-rich N-terminal region may play a critical role in directing Aβ self-association on the membrane surface and in anchoring and stabilizing the peptide aggregates inserted in the phospholipid membrane, which are closely related with membrane disruption activity of Aβ. The results provide new mechanistic insight into the Aβ-mediated membrane damage process, which may be critical for understanding the mechanism of Aβ neurotoxicity in Alzheimer’s disease.

Alzheimer's disease (AD) has been consistently related to the formation of senile amyloid plaques mainly composed of amyloid β (Aβ) peptides. The toxicity of Aβ aggregates has been indicated to be responsible for AD pathology. One scenario to decrease Aβ toxicity is the development of effective inhibitors against Aβ amyloid formation. In this study, we investigate the effect of gallium nitride nanoparticles (GaN NPs) as inhibitors of Aβ40 amyloid formation using a combination of biophysical approaches. Our results show that the lag phase of Aβ40 aggregation kinetics is significantly retarded by GaN NPs in a concentration dependent manner, implying the activity of GaN NPs in interfering with the formation of the crucial nucleus during Aβ aggregation. Our results also show that GaN NPs can reduce the amyloid fibril elongation rate in the course of the aggregation kinetics. It is speculated that the high polarization characteristics of GaN NPs may provoke a strong interaction between the particles and Aβ40 peptide and in this way decrease self-association of the peptide monomers to form amyloids.

Human calcitonin (hCT) is a 32-residue peptide hormone that can aggregate into amyloid fibrils and cause cellular toxicity. In this study, we investigated the inhibition effects of a group of polyphenolic molecules on hCT amyloid formation. Our results suggest that the gallate moiety in epigallocatechin-3-gallate (EGCG), a well-recognized amyloid inhibitor, is not critical for its inhibition function in the hCT amyloid formation. Our results demonstrate that flavonoid compounds, such as myricetin, quercetin, and baicalein, that contain vicinal hydroxyl groups on the phenyl ring effectively prevent hCT fibrillization. This structural feature may also be applied to non-flavonoid polyphenolic inhibitors. Moreover, our results indicate a plausible mechanistic role of these vicinal hydroxyl groups which might include the oxidation to form a quinone and the subsequent covalent linkage with amino acid residues such as lysine or histidine in hCT. This may further disrupt the crucial electrostatic and aromatic interactions involved in the process of hCT amyloid fibril formation. The inhibition activity of the polyphenolic compounds against hCT fibril formation may likely be attributed to a combination of factors such as covalent linkage formation, aromatic stacking, and hydrogen bonding interactions.

ObjectiveTo synthesize both O‐glycosylated and non‐glycosylated APP peptide analogs bearing the Tn antigen on Thr663, Ser667, and Tyr681 in and around the Aβ42 region, to understand the role of O‐glycosylation on APP’s conformation, proteolytic stability and aggregation kinetics.AbstractThe amyloid‐β precursor protein (APP) is a transmembrane protein that can undergo proteolytic cleavage by three proteases, α‐, β‐ and γ‐secretases, to determine its fate in Alzheimer’s disease (AD) pathogenesis. The protein undergoes proteolysis by β‐ and γ‐secretases to produce amyloid‐β peptides, which is a hallmark of AD. However, only ten‐percent of the protein will follow this amyloidogenic pathway, whereas the remaining ninety‐percent will undergo proteolysis by α‐secretase and γ‐secretases, resulting in the non‐amyloidogenic pathway. Recently, it was found that the four threonine residues Thr633, Thr651, Thr652, and Thr659, in the vicinity of the β‐secretase cleavage site (Met671~Asp672) of APP, are modified by complex mucin‐type O‐glycans. Moreover, an increase of up to 2.5 times in Tyr681 glycosylation, located within the Aβ42 region of APP, was found in AD patients in comparison to the non‐AD patients. These findings suggest the possible role of O‐glycosylation in APP proteolytic processing. Therefore, we synthesized native and Swedish‐double‐mutated (Met671Asn and Asp672Leu) APP (glyco)peptides with O‐GalNAc and studying conformational changes, secretase activity, and aggregation kinetics using circular dichroism, enzyme kinetic and Thioflavin T assays, respectively. Our results show that the non‐glycosylated peptide analogs in water show characteristics of β‐sheet conformation, however, when there is a site‐specific O‐glycosylation on Thr, Ser or Tyr residues of their counterparts, there is a change in conformation that resembles a mixture of α‐helix and random coil. All peptides adopt an α‐helix in 50/50 (v/v) trifluoroethanol/water mixture. Furthermore, the level of β‐secretase activity significantly increases for the glycosylated analogs containing the Swedish mutation compared to their nonglycosylated counterparts. Lastly, the glycosylated analogs impact the protein’s aggregation kinetics by decreasing its lag phase, and this effect seems to be more pronounced in the Swedish mutation analogs. In conclusion, our results suggest that APP’s site‐specific O‐glycosylation can induce a conformational change in the protein and subsequently affect its proteolytic processing fate towards either amyloidogenic or non‐amyloidogenic pathway. Our long‐term goals are to assess the role of certain patterns of multiple glycosylation sites on Ser, Thr, and Tyr that are in close proximity to the relevant secretases cleavage sites to develop specific inhibitors of amyloid pathway‐stimulating secretases (β‐secretase) or activators of non‐amyloid pathway (α‐secretase).Support or Funding InformationNational Institute of Health Grant CA242351 to M. C.

Human calcitonin (hCT) is a 32-residue peptide that aggregates to form amyloid fibrils under appropriate conditions. In this study, we investigated the effect of the intramolecular disulfide bond formed at the N-terminal region of the peptide in the aggregation kinetics of hCT. Our results indicate that the presence of the disulfide bond in hCT plays a crucial role in forming the critical nucleus needed for fibril formation, facilitating the rate of hCT amyloidogenesis. Furthermore, we reported for the first time the effects of cholesterol, cholesterol sulfate, and 3β-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol) on the amyloid formation of oxidized hCT. Our results show that while cholesterol does not affect amyloidogenesis of oxidized hCT, high concentrations of cholesterol sulfate exhibits a moderate inhibiting activity on hCT amyloid formation. In particular, our results show that DC-cholesterol strongly inhibits amyloidogenesis of oxidized hCT in a dose-dependent manner. Further studies at different pH conditions imply the crucial impact of electrostatic and hydrogen bonding interactions in mediating the interplay of hCT and the surface of DC-cholesterol vesicles and the inhibiting function of DC-cholesterol on hCT fibrillization.

Amyloid diseases, including neurodegenerative diseases such as Alzheimer's and Parkinson's, are linked to a poorly understood progression of protein misfolding and aggregation events that culminate in tissue-selective deposition and human pathology. Elucidation of the mechanistic details of protein aggregation and the structural features of the aggregates is critical for a comprehensive understanding of the mechanisms of protein oligomerization and fibrillization. Vibrational spectroscopies, such as Fourier transform infrared (FTIR) and Raman, are powerful tools that are sensitive to the secondary structure of proteins and have been widely used to investigate protein misfolding and aggregation. We address the application of the vibrational approaches in recent studies of conformational dynamics and structural characteristics of protein oligomers and amyloid fibrils. In particular, introduction of isotope labelled carbonyl into a peptide backbone, and incorporation of the extrinsic unnatural amino acids with vibrational moieties on the side chain, have greatly expanded the ability of vibrational spectroscopy to obtain site-specific structural and dynamic information. The applications of these methods in recent studies of protein aggregation are also reviewed.

In this study, zinc (II) 1, 8(11), 15(18), 22(25)-tetrakis(4-carboxylphenoxy) phthalocyanine (alpha-ZnTcPc) was synthesized as sensitizer to construct a novel photocatalyst alpha-ZnTcPc/g-C3N4 by a polycondensation strategy. The FT-IR and XPS spectra demonstrated that the a-ZnTcPc molecules were covalently coupled on g-C3N4. The 10% alpha-ZnTcPc/g-C3N4 composites exhibit excellent extended spectral response with intense broad near-infrared absorption at 695 nm and with significantly fascinated charge carrier separation. As expected, the 10% alpha-ZnTcPc/g-C3N4 displayed a remarkable improved photocatalytic performance in decomposition of methylene blue (MB) and antibiotic tetracycline with degradation rates of 94.49% and 91.43% under visible light irradiation, respectively. The hydroxyl radicals (center dot OH) were observed as the main reactive oxidizing species in tetracycline decomposition. Moreover, the possible mechanism for enhanced visible light photocatalytic capability might be ascribed to the synergetic effect between alpha-ZnTcPc and g-C3N4, which creates large surface areas, decreases the recombination rate of photogenerated charge carries, as well as improves the visible light harvesting significantly. This work not only presents a practical method to conquer the drawback of pure g-C3N4, but also provides useful insights to construct other g-C3N4-based composites to take full advantage of solar light towards practical applications for eliminating recalcitrant organic pollutants.