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The Lewy bodies and Lewy neurites are key pathological hallmarks of Parkinson’s disease (PD). Single-point mutations associated with familial PD cause α-synuclein (α-Syn) aggregation, leading to the formation of Lewy bodies and Lewy neurites. Recent studies suggest α-Syn nucleates through liquid–liquid phase separation (LLPS) to form amyloid aggregates in a condensate pathway. How PD-associated mutations affect α-Syn LLPS and its correlation with amyloid aggregation remains incompletely understood. Here, we examined the effects of five mutations identified in PD, A30P, E46K, H50Q, A53T, and A53E, on the phase separation of α-Syn. All other α-Syn mutants behave LLPS similarly to wild-type (WT) α-Syn, except that the E46K mutation substantially promotes the formation of α-Syn condensates. The mutant α-Syn droplets fuse to WT α-Syn droplets and recruit α-Syn monomers into their droplets. Our studies showed that α-Syn A30P, E46K, H50Q, and A53T mutations accelerated the formation of amyloid aggregates in the condensates. In contrast, the α-Syn A53E mutant retarded the aggregation during the liquid-to-solid phase transition. Finally, we observed that WT and mutant α-Syn formed condensates in the cells, whereas the E46K mutation apparently promoted the formation of condensates. These findings reveal that familial PD-associated mutations have divergent effects on α-Syn LLPS and amyloid aggregation in the phase-separated condensates, providing new insights into the pathogenesis of PD-associated α-Syn mutations.

Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, “holding-like” mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differential scanning calorimetry (DSC), light scattering, and circular dichroism. The results showed that the protein in monomeric form is less stable, but more effective against amyloid fibrillization. This greater functionality is attributed to the disordered nature of the domains involved in subunit contacts.

The aggregation of amyloid beta (Aβ) into fibrillar aggregates is a key feature of Alzheimer’s disease (AD) pathology. β-carotene and related compounds have been shown to associate with amyloid aggregates and have direct impact on the formation of amyloid fibrils. However, the precise effect of β-carotene on the structure of amyloid aggregates is not known, which poses a limitation towards developing it as a potential AD therapeutic. In this report, we use nanoscale AFM-IR spectroscopy to probe the structure of Aβ oligomers and fibrils at the single aggregate level and demonstrate that the main effect of β-carotene towards modulating Aβ aggregation is not to inhibit fibril formation but to alter the secondary structure of the fibrils and promote fibrils that lack the characteristic ordered beta structure.

Human γD-crystallin protein is abundant in the lens and is essential for preserving lens transparency. With age the protein may lose its native structure resulting in the formation of cataract. We recently reported an aggregative peptide, 41Gly-Cys-Trp-Met-Leu-Tyr46 from the human γD-crystallin, termed GDC6, exhibiting amyloidogenic properties in vitro. Here, we aimed to determine the contribution of each residue of the GDC6 to its amyloidogenicity. Molecular dynamic (MD) simulations revealed that the residues Trp, Leu, and Tyr played an important role in the amyloidogenicity of GDC6 by facilitating inter-peptide main-chain hydrogen bonds, and π-π interactions. MD predictions were further validated using single-, double- and triple-alanine-substituted GDC6 peptides in which their amyloidogenic propensity was individually evaluated using complementary biophysical techniques including Thioflavin T assay, turbidity assay, CD spectroscopy, and TEM imaging. Results revealed that the substitution of Trp, Leu, and Tyr together by Ala completely abolished aggregation of GDC6 in vitro, highlighting their importance in the amyloidogenicity of GDC6. • Trp, Leu and Tyr from the GDC6 peptide form both inter-peptide main-chain hydrogen bonds and π-π interactions. • Alanine-substituted GDC6 peptides exhibit slower aggregation kinetics and less fibrils formation than the wt. • Substitution of Trp, Leu and Tyr together completely abolishes GDC6 amyloidogenicity. [ABSTRACT FROM AUTHOR]

Insulin is a hormone that attends to energy metabolism by regulating glucose levels in the bloodstream. It is synthesised within pancreas beta-cells where, before being released into the serum, it is stored in granules as hexamers coordinated by Zn2+ and further packaged in microcrystalline structures. The group I chaperonin cpn60, known for its assembly-assisting function, is present, together with its cochaperonin cpn10, at each step of the insulin secretory pathway. However, the exact function of the heat shock protein in insulin biosynthesis and processing is still far from being understood. Here we explore the possibility that the molecular machine cpn60/cpn10 could have a role in insulin hexameric assembly and its further crystallization. Moreover, we also evaluate their potential protective effect in pathological insulin aggregation. The experiments performed with the cpn60 bacterial homologue, GroEL, in complex with its cochaperonin GroES, by using spectroscopic methods, microscopy and hydrodynamic techniques, reveal that the chaperonins in vitro favour insulin hexameric organisation and inhibit its aberrant aggregation. These results provide new details in the field of insulin assembly and its related disorders. [ABSTRACT FROM AUTHOR]

The anti-oncogenic protein p53 is a transcription factor that prevents tumorigenesis by inducing gene repair proteins or apoptosis under DNA damage. Since the DNA-binding domain of p53 (p53C) is aggregation-prone, the anti-oncogenic function of p53 is often lost in cancer cells. This tendency is rather severe in some tumor-related p53 mutants, such as R175H. In this study, we examined the effect of salts, including KCl and sugars, on the aggregation of p53C by monitoring two distinct aggregates: amorphous-like and amyloid-like. The amorphous aggregates are detectable with 8-(phenylamino)-1-naphthalenesulfonic acid (ANS) fluorescence, whereas the amyloid aggregates are sensitive to thioflavin-T (ThT) fluorescence. We found that KCl inhibited the formation of amorphous aggregates but promoted the formation of amyloid aggregates in a p53C R175H mutant. The salts exhibited different effects against the wild-type and R175H mutants of p53C. However, the ratio of ANS/ThT fluorescence for the wild-type and R175H mutant remained constant. KCl also suppressed the structural transition and loss of the DNA-binding function of p53C. These observations indicate the existence of multiple steps of p53C aggregation, probably coupled with the dissociation of Zn. Notably, amorphous aggregates and amyloid aggregates have distinct properties that could be discriminated by various small additives upon aggregation.

The islet amyloid polypeptide (IAPP) is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The aggregation of IAPP is known to cause cell death, where the cell membrane plays a dual role: being a catalyst of IAPP aggregation and being the target of IAPP toxicity. Using ATR-FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Distinct IAPP-membrane interaction modes for the various IAPP variants are revealed. Membrane binding causes IAPP to fold into an amphipathic α-helix, which in the case of H18K-, and H18R-IAPP readily moves beyond the headgroup region. For all IAPP variants but H18E-IAPP, the membrane-bound helix is an intermediate on the way to amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel β-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel β-sheets as well as amorphous aggregates. Our results emphasize the decisive role of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good therapeutic target for destabilizing membrane-bound IAPP fibrils to inhibit their toxic actions.

Type 2 diabetes mellitus and Alzheimer's disease (AD), both are chronic and progressive diseases. Many cardiovascular and genetic risk factors are considered responsible for the development of AD and diabetes mellitus (DM). Genetic risk factor such as apolipoprotein E (APOE) plays a critical role in the progression of AD. Specifically, APOEε4 is genetically the strongest isoform associated with neuronal insulin deficiency, altered lipid homeostasis, and metabolism, decreased glucose uptake, impaired gray matter volume, and cerebrovascular functions. In this article, we have summarized the mechanisms of cardiovascular disturbances associated with AD and DM, impact of amyloid‐β aggregation, and neurofibrillary tangles formation in AD. Moreover, cardiovascular risk factors leading to insulin resistance (IR) and amyloid‐β aggregation are highlighted along with the effects of APOE risk alleles on cerebral, lipid, and cholesterol metabolism leading to CVD‐mediated IR. Correspondingly, the contribution of IR, genetic and cardiovascular risk factors in amyloid‐β aggregation, which may lead to the late onset of AD and DM, has been also discussed. In short, IR is related to significantly lower cerebral glucose metabolism, which sequentially forecasts poorer memory performance. Hence, there will be more chances for neural glucose intolerance and impairment of cognitive function in cardiac patients, particularly APOEε4 carriers having IR. Hence, this review provides a better understanding of the corresponding crosstalk among different pathways. This will help to investigate the rational application of preventive measures against IR and cognitive dysfunction, specifically in APOEε4 carriers' cardio‐metabolic patients. [ABSTRACT FROM AUTHOR]

Amyloid cross‐seeding and amyloid inhibition are two different research subjects being studied separately for different pathological purposes, in which amyloid cross‐seeding targets to study the co‐aggregation of different amyloid proteins and potential molecular links between different neurodegenerative diseases, while amyloid inhibition aims to design different molecules for preventing amyloid aggregation. While both amyloid cross‐seeding and amyloid inhibition are critical for better understanding the pathological causes of different neurodegenerative diseases including Parkinson disease (PD) and Type 2 diabetes (T2D), less efforts have been made to reconcile the two phenomena. Herein, we proposed a new preventive strategy to demonstrate (a) the cross‐seeding of octapeptide TKEQVTNV from α‐synuclein (associated with PD) with hIAPP (associated with T2D) and (b) the cross‐seeding‐promoted hIAPP fibrillization and cross‐seeding‐reduced hIAPP toxicity. Collective results confirmed that TKEQVTNV can indeed cross‐seed with hIAPP monomers and oligomers, not protofibrils, to form β‐structure‐rich fibrils and to accelerate hIAPP fibrillization. Moreover, such cross‐seeding‐induced promotion effect by TKEQVTNV also rescued the pancreatic cells from hIAPP‐induced cytotoxicity by increasing cell viability and reducing cell apoptosis simultaneously. This work provides a new angle to discover amyloid fragments and use them as amyloid modulators (inhibitors or promotors) to interfere with amyloid aggregation of other amyloid proteins, as well as sequence/structure basis to explore the amyloid cross‐seeding between different amyloid proteins that may help explain a potential molecular talk between different neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid‐β 40 and α‐synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well‐known polyphenol fibril inhibitors, (‐)‐epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off‐pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development. [ABSTRACT FROM AUTHOR]

Pathological aggregation of amyloid polypeptides is associated with numerous degenerative diseases. Preventing aggregation and clearing amyloid deposits are considered as promising strategies against amyloidosis. With the capacity of crossing the blood-brain barrier and good biocompatibility, the hydroxylated single-walled carbon nanotube (SWCNT-OH) has been shown with excellent anti-amyloid properties. Here, we systematically studied the SWCNT-OH effects on the fibrillization of the β2m 21 – 31 peptides utilizing all-atom discrete molecular dynamics (DMD) simulation. Our results demonstrated the isolated β2m 21 – 31 peptides first nucleated into unstructured oligomers followed by coil-to-sheet conformational conversions in oligomers with at least six peptides. The elongation and lateral surfaces of the preformed β-sheet could catalyze the other unstructured monomers and small oligomers converted into β-sheet formations via dock-lock fibril growth and secondary nucleation processes. Eventually, the β2m 21 – 31 peptides would self-assemble into well-ordered cross-β structures. Regardless of isolated monomers or well-defined cross-β assemblies, the β2m 21 – 31 would attach on the surfaces of SWCNT-OH adopting unstructured formations indicating the SWCNT-OH not only inhibited the fibrillization of β2m 21 – 31 but also destroyed pre-formed proto-fibrils. Overall, our study displays a complete picture of the fibrillization mechanism of β2m 21 – 31 and the amyloid inhibitory mechanism of SWCNT-OH, offering new insight into the de-novo design of anti-amyloid inhibitors. [Display omitted] • The fibrillization of β2m 21-31 involves the primary and secondary nucleations of β-sheet aggregates and β-sheet growth. • The conformational conversion of β2m 21-31 from unstructured to β-sheet starts from hexamers. • The SWCNT-OH not only inhibits the aggregation of β2m 21-31 but also destroys the pre-formed cross-β aggregates of β2m 21-31. [ABSTRACT FROM AUTHOR]

Abstract Background Aggregation of amyloid β into plaques in the brain is one of the earliest pathological events in Alzheimer’s disease (AD). The exact pathophysiology leading to dementia is still uncertain, but the apolipoprotein E (APOE) ε4 genotype plays a major role. We aimed to identify the molecular pathways associated with amyloid β aggregation using cerebrospinal fluid (CSF) proteomics and to study the potential modifying effects of APOE ε4 genotype. Methods We tested 243 proteins and protein fragments in CSF comparing 193 subjects with AD across the cognitive spectrum (65% APOE ε4 carriers, average age 75 ± 7 years) against 60 controls with normal CSF amyloid β, normal cognition, and no APOE ε4 allele (average age 75 ± 6 years). Results One hundred twenty-nine proteins (53%) were associated with aggregated amyloid β. APOE ε4 carriers with AD showed altered concentrations of proteins involved in the complement pathway and glycolysis when cognition was normal and lower concentrations of proteins involved in synapse structure and function when cognitive impairment was moderately severe. APOE ε4 non-carriers with AD showed lower expression of proteins involved in synapse structure and function when cognition was normal and lower concentrations of proteins that were associated with complement and other inflammatory processes when cognitive impairment was mild. Repeating analyses for 114 proteins that were available in an independent EMIF-AD MBD dataset (n = 275) showed that 80% of the proteins showed group differences in a similar direction, but overall, 28% effects reached statistical significance (ranging between 6 and 87% depending on the disease stage and genotype), suggesting variable reproducibility. Conclusions These results imply that AD pathophysiology depends on APOE genotype and that treatment for AD may need to be tailored according to APOE genotype and severity of the cognitive impairment.

Interactions of key amyloidogenic proteins with SARS-CoV-2 proteins may be one of the causes of expanding and delayed post-COVID-19 neurodegenerative processes. Furthermore, such abnormal effects can be caused by proteins and their fragments circulating in the body during vaccination. The aim of our work was to analyze the effect of the receptor-binding domain of the coronavirus S-protein domain (RBD) on alpha-synuclein amyloid aggregation. Molecular modeling showed that the predicted RBD complex with monomeric alpha-synuclein is stable over 100 ns of molecular dynamics. Analysis of the interactions of RBD with the amyloid form of alpha-synuclein showed that during molecular dynamics for 200 ns the number of contacts is markedly higher than that for the monomeric form. The formation of the RBD complex with the alpha-synuclein monomer was confirmed immunochemically by immobilization of RBD on its specific receptor ACE2. Changes in the spectral characteristics of the intrinsic tryptophans of RBD and hydrophobic dye ANS indicate an interaction between the monomeric proteins, but according to the data of circular dichroism spectra, this interaction does not lead to a change in their secondary structure. Data on the kinetics of amyloid fibril formation using several spectral approaches strongly suggest that RBD prevents the amyloid transformation of alpha-synuclein. Moreover, the fibrils obtained in the presence of RBD showed significantly less cytotoxicity on SH-SY5Y neuroblastoma cells.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500–1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure—unlike other amyloidogenic proteins—formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

Amyloid-β (Aβ) is a peptide that undergoes self-assembly into amyloid fibrils, which compose the hallmark plaques observed in Alzheimer's disease (AD). TAR DNA-binding protein 43 (TDP-43) is a protein with mislocalization and aggregation implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases. Recent work suggests that TDP-43 may interact with Aβ, inhibiting the formation of amyloid fibrils and worsening AD pathology, but the molecular details of their interaction remain unknown. Using all-atom discrete molecular dynamics simulations, we systematically investigated the direct molecular interaction between Aβ and TDP-43. We found that Aβ monomers were able to bind near the flexible nuclear localization sequence of the N-terminal domain (NTD) of TDP-43, adopting β-sheet rich conformations that were promoted by the interaction. Furthermore, Aβ associated with the nucleic acid binding interface of the tandem RNA recognition motifs of TDP-43 via electrostatic interactions. Using the computational peptide array method, we found the strongest C-terminal domain interaction with Aβ to be within the amyloidogenic core region of TDP-43. With experimental evidence suggesting that the NTD is necessary for inhibiting Aβ fibril growth, we also simulated the NTD with an Aβ40 fibril seed. We found that the NTD was able to strongly bind the elongation surface of the fibril seed via extensive hydrogen bonding and could also diffuse along the lateral surface via electrostatic interactions. Our results suggest that TDP-43 binding to the elongation surface, thereby sterically blocking Aβ monomer addition, is responsible for the experimentally observed inhibition of fibril growth. We conclude that TDP-43 may promote Aβ toxicity by stabilizing the oligomeric state and kinetically delaying fibril maturation.

In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in Drosophila melanogaster and Saccharomyces cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sublethal, stress response pathway that is tailored to the environmental norms of an organism. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species highlights the physiological significance of this protein conformation, and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events. [ABSTRACT FROM AUTHOR]

Several prediction algorithms and tools have been developed in the last two decades to predict protein and peptide aggregation. These in silico tools aid to predict the aggregation propensity and amyloidogenicity as well as the identification of aggregation-prone regions. Despite the immense interest in the field, it is of prime importance to systematically compare these algorithms for their performance. In this review, we have provided a rigorous performance analysis of nine prediction tools using a variety of assessments. The assessments were carried out on several non-redundant datasets ranging from hexapeptides to protein sequences as well as amyloidogenic antibody light chains to soluble protein sequences. Our analysis reveals the robustness of the current prediction tools and the scope for improvement in their predictive performances. Insights gained from this work provide critical guidance to the scientific community on advantages and limitations of different aggregation prediction methods and make informed decisions about their research needs. [ABSTRACT FROM AUTHOR]

A series of novel thiophene pyrazolines were designed as potential bioactive molecules against Alzheimer’s disease. The probable binding modes of these molecules in AChE were evaluated using in silico techniques, and promising molecules were then synthesized and characterized. Their biological activities were profiled to confirm their potential as multifunctional molecules for the treatment of Alzheimer’s disease by addressing multiple pathological mechanisms. While the anti-oxidant activities of the synthesized molecules needed further optimization, the series showed excellent potential in mitigating the multiple causative factors via Aβ aggregation inhibition, AChE inhibition, metal chelation, and inhibition of advanced glycation products.

Misfolding and aggregation of tau protein, into pathological amyloids, are hallmarks of a group of neurodegenerative diseases collectively termed tauopathies and their modulation may be therapeutically valuable. Herein, we describe the synthesis and characterization of a dopamine‐based hybrid molecule, naphthoquinone–dopamine (NQDA). Using thioflavin S assay, CD, transmission electron microscopy, dynamic light scattering, Congo Red birefringence, and large unilamellar vesicle leakage assays, we demonstrated its efficacy in inhibiting the in vitro aggregation of key tau‐derived amyloidogenic fragments, PHF6 (VQIVYK) and PHF6* (VQIINK), prime drivers of aggregation of full‐length tau in disease pathology. Isothermal titration calorimetry analysis revealed that the interaction between NQDA and PHF6 is spontaneous and has significant binding efficiency driven by both entropic and enthalpic processes. Furthermore, NQDA efficiently disassembled preformed fibrils of PHF6 and PHF6* into nontoxic species. Molecular dynamic simulations supported the in vitro results and provided a plausible mode of binding of NQDA with PHF6 fibril. NQDA was also capable of inhibiting the aggregation of full‐length tau protein and disrupting its preformed fibrils in vitro in a dose‐dependent manner. In a comparative study, the IC50 value (50% inhibition of fibril formation) of NQDA in inhibiting the aggregation of PHF6 (25 µm) was ~ 17 µm, which is lower than for other bona fide amyloid inhibitors, naphthoquinone‐tryptophan, rosmarinic acid, epigallocatechin gallate, ~ 21, ~ 77, or ~ 19 µm, respectively. Comparable superiority of NQDA was observed for inhibition of PHF6*. These findings suggest that NQDA can be a useful scaffold for designing new therapeutics for Alzheimer's disease and other tauopathies. [ABSTRACT FROM AUTHOR]

Aberrant protein aggregation and the formation of amyloid deposits are associated with numerous neuro- and non-neurodegenerative disorders. Thus, one potential strategy is to eliminate these deposits by halting amyloid aggregation. Selenium nanoparticles (Se-NPs) have great potential in biomedicine for various therapeutic and diagnostic purposes and also have the ability to inhibit amyloid fibrillation. Herein, Hen Egg White Lysozyme (HEWL) was chosen as a protein model, and rod-like Se-NPs with diameters ranging from 90 to 120 nm were synthesized and the influence of shape and concentration of the particles on HEWL fibrillation was investigated. The effect of the nanoparticles on HEWL amyloid formation was analyzed using thioflavin T and Congo red binding assays, atomic force microscopy, and cytotoxicity assays. In the present study, it has been observed that these particles have a dual function in various concentrations. Using lower concentrations of Se-NPs ranging from 3-30 µg/ml, the Thioflavin T (ThT) fluorescence intensity decreased significantly by 60%, with an increased lag time compared to that of the control. While HEWL fibrillation substantially increased upon co-incubation with a higher concentration of these particles (300-2400µg/ml), and these results were verified by AFM, Congo red, and MTT assay. We showed that inhibitory or inductive influences of Se-NPs on the hen egg-white lysozyme (HEWL) amyloid aggregation are achieved via different independent mechanisms. These results demonstrate that dual-activity of Se-NPs might be a valuable targeting system for inhibiting amyloid aggregation, and thus, may play a useful role in new therapeutic and diagnostic strategies for amyloid-related disorders. [ABSTRACT FROM AUTHOR]

α-Synuclein (α-syn), a disordered cytoplasmatic protein, plays a fundamental role in the pathogenesis of Parkinson’s disease (PD). Here, we have shown, using photophysical measurements, that addition of FKBP12 to α-syn solutions, dramatically accelerates protein aggregation, leading to an explosion of dendritic structures revealed by fluorescence and phase-contrast microscopy. We have further demonstrated that this aberrant α-syn aggregation can be blocked using a recently discovered non-immunosuppressive synthetic inhibitor of FKBP12, ElteN378. The role of FKBP12 and of ElteN378 in the α-syn aggregation mechanism has been elucidated using molecular dynamics simulations based on an effective coarse-grained model. The reported data not only reveal a new potent synthetic drug as a candidate for early stage treatment of α-syn dependent neurodegenerations but also pave the way to a deeper understanding of the mechanism of action of FKBP12 on α-syn oligomeric aggregation, a topic which is still controversial.

Alzheimer’s disease (AD) is the most serious and prevalent neurodegenerative disorder still without cure. Since its aetiology is diverse, recent research on anti-AD drugs has been focused on multi-target compounds. In this work, seven novel hybrids (RIV–BIM) conjugating the active moiety of the drug rivastigmine (RIV) with 2 isomeric hydroxyphenylbenzimidazole (BIM) units were developed and studied. While RIV assures the inhibition of cholinesterases, BIM provides further appropriate properties, such as inhibition of amyloid β-peptide (Aβ) aggregation, antioxidation and metal chelation. The evaluated biological properties of these hybrids included antioxidant activity; inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and Aβ42 aggregation; as well as promotion of cell viability and neuroprotection. All the compounds are better inhibitors of AChE than rivastigmine (IC50 = 32.1 µM), but compounds of series 5 are better inhibitors of BChE (IC50 = 0.9−1.7 µM) than those of series 4. Series 5 also showed good capacity to inhibit self- (42.1−58.7%) and Cu(II)-induced (40.3−60.8%) Aβ aggregation and also to narrow (22.4−42.6%) amyloid fibrils, the relevant compounds being 5b and 5d. Some of these compounds can also prevent the toxicity induced in SH-SY5Y cells by Aβ42 and oxidative stress. Therefore, RIV–BIM hybrids seem to be potential drug candidates for AD with multi-target abilities.

Nanoplastics (NPs) are becoming an emerging pollutant of global concern. A potential risk of NPs is that they can serve as carriers and synergistically function with other contaminants to cause diseases. A variety of diseases such as Alzheimer’s disease are related to the generation of amyloid fibrils, and insulin is typically used as a model to study the fibrillation process. In this study, we examined the fibrillation of insulin promoted by polystyrene nanoplastics (PSNPs) alone and synergistically with organic contaminants (denoted as X, X = pyrene, bisphenol A, 2,2′,4,4′-tetrabromodiphenyl ether, 4,4′-dihydroxydiphenylmethane, or 4-nonylphenol) having different polarities using thioflavin T fluorescence assays, dynamic light scattering, and circular dichroism spectroscopy. The presence of PSNPs and small organic contaminants decreased the lag phase time (tlag) for insulin fibrillation from 54.6 h to 35-51 h and their combination (PS-X) enhanced this process (tlag = 21-30 h). Notably, the lag phase time for insulin fibrillation with PS-nonpolar contaminants, PS-weakly polar contaminants, and PS-polar contaminants is around 20.8, 26.7, and 30.1 h, respectively, indicating the synergistic effect of PS-nonpolar contaminants or PS-weakly polar contaminants was more obvious than that of PS-polar contaminants. Moreover, molecular dynamic simulation reveal the interactions between insulin and PSs or small organic contaminants are primarily driven by van der Waals forces and hydrophobic interactions. Overall, the findings of this study underscore the potentially significant environmental impact of small organic contaminants assisting NPs in promoting insulin fibrillation.

Amyloid aggregation and neurofibrillary pathology is the characteristic feature of Alzheimer's disease (AD). Streptozotocin (STZ) is a glucosamine nitrosourea compound that is toxic to the cells, impairs insulin signaling in the brain. STZ induces DNA damage and oxidative stress, which leads to cognitive impairments. In this experimental study, STZ treatment induces Alzheimer's pathology in mouse neuroblastoma (N2A) cells. The study also explored the protection of cellular damage by pre-treatment with test drugs Apple cider vinegar (ACV), chrysin and rivastigmine. This study had been concentrated mainly on the cellular mechanism of neuromodulation and anti –oxidant potency of test drugs to attenuate cellular toxicity induced by STZ treatment. The 100 μM concentration of STZ was used for treatment in N2A cells for 24 h and 48 h and multiple studies were performed. The STZ causes tau phosphorylation, amyloid aggregation and increased acetyl cholinesterase (AChE) activity. Along with STZ at a concentration of 100 μM, the cells were pre-treated with ACV, chrysin and rivastigmine at a concentration of 2, 10, 50 μM each. The results show that 2 μM test drug concentration presented considerable protection, against STZ generated neurocytotoxicity via. restoration of anti-oxidant enzymes, MDA and AChE level, compared to the other two concentrations. [ABSTRACT FROM AUTHOR]

The aggregation of proteins is considered to be the main cause of several neurodegenerative diseases. Despite much progress in amyloid research, the process of fibrillization is still not fully understood, which is one of the main reasons why there are still very few effective treatments available. When the aggregation of insulin, a model amyloidogenic protein, is tracked using thioflavin-T (ThT), an amyloid specific dye, there is an anomalous occurrence of double-sigmoidal aggregation kinetics. Such an event is likely related to the formation of ThT-positive intermediates, which may affect the outcome of both aggregation kinetic data, as well as final fibril structure. In this work we explore insulin fibrillization under conditions, where both normal and double-sigmoidal kinetics are observed and show that, despite their dye-binding properties and random occurrence, the ThT-positive intermediates do not significantly alter the overall aggregation process.

Alzheimer disease is an age-linked neurodegenerative disorder representing one of the greatest medical care challenges of our century. Several drugs are useful in ameliorating the symptoms, even if none could stop or reverse disease progression. The standard approach is represented by the cholinesterase inhibitors (ChEIs) that restore the levels of acetylcholine (ACh) by inhibiting the acetylcholinesterase (AChE). Still, their limited efficacy has prompted researchers to develop new ChEIs that could also reduce the oxidative stress by exhibiting antioxidant properties and by chelating the main metals involved in the disease. Recently, we developed some derivatives constituted by a 2-amino-pyrimidine or a 2-amino-pyridine moiety connected to various aromatic groups by a flexible amino-alkyl linker as new dual inhibitors of AChE and butyrylcholinesterase (BChE). Following our previous studies, in this work we explored the role of the flexible linker by replacing the amino group with an amide or a carbamic group. The most potent compounds showed higher selectivity against BChE in respect to AChE, proving also to possess a weak anti-aggregating activity toward Aβ42 and tau and to be able to chelate Cu2+ and Fe3+ ions. Molecular docking and molecular dynamic studies proposed possible binding modes with the enzymes. It is noteworthy that these compounds were predicted as BBB-permeable and showed low cytotoxicity on the human brain cell line.

The effects of membranes on the early-stage aggregation of amyloid β (Aβ) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer’s disease. We have shown that direct Aβ-membrane interactions dramatically enhance the Aβ aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aβ monomers and significantly changes the conformational sampling of Aβ monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aβ-lipid interaction is an important factor in the disease prone amyloid assembly process.

Insulin is a hormone that attends to energy metabolism by regulating glucose levels in the bloodstream. It is synthesised within pancreas beta-cells where, before being released into the serum, it is stored in granules as hexamers coordinated by Zn2+ and further packaged in microcrystalline structures. The group I chaperonin cpn60, known for its assembly-assisting function, is present, together with its cochaperonin cpn10, at each step of the insulin secretory pathway. However, the exact function of the heat shock protein in insulin biosynthesis and processing is still far from being understood. Here we explore the possibility that the molecular machine cpn60/cpn10 could have a role in insulin hexameric assembly and its further crystallization. Moreover, we also evaluate their potential protective effect in pathological insulin aggregation. The experiments performed with the cpn60 bacterial homologue, GroEL, in complex with its cochaperonin GroES, by using spectroscopic methods, microscopy and hydrodynamic techniques, reveal that the chaperonins in vitro favour insulin hexameric organisation and inhibit its aberrant aggregation. These results provide new details in the field of insulin assembly and its related disorders.

The aggregation of proteins is considered to be the main cause of several neurodegen-erative diseases. Despite much progress in amyloid research, the process of fibrillization is still not fully understood, which is one of the main reasons why there are still very few effective treatments available. When the aggregation of insulin, a model amyloidogenic protein, is tracked using thioflavin-T (ThT), an amyloid specific dye, there is an anomalous occurrence of double-sigmoidal aggregation kinetics. Such an event is likely related to the formation of ThT-positive intermediates, which may affect the outcome of both aggregation kinetic data, as well as final fibril structure. In this work we explore insulin fibrillization under conditions, where both normal and double-sigmoidal kinetics are observed and show that, despite their dye-binding properties and random occurrence, the ThT-positive intermediates do not significantly alter the overall aggregation process. [ABSTRACT FROM AUTHOR]

In lysozyme amyloidosis, fibrillar aggregates of lysozyme are associated with severe renal, hepatic, and gastrointestinal manifestations, with no definite therapy. Current drugs are now being tested in amyloidosis clinical trials as aggregation inhibitors to mitigate disease progression. The tetracycline group among antimicrobials in use is in phase II of clinical trials, whereas some macrolides and cephalosporins have shown neuroprotection. In the present study, two cephalosporins, ceftazidime (CZD) and cefotaxime (CXM), and a glycopeptide, vancomycin (VNC), are evaluated for inhibition of amyloid aggregation of hen egg white lysozyme (HEWL) under two conditions (i) 4 M guanidine hydrochloride (GuHCl) at pH 6.5 and 37° C, (ii) At pH 1.5 and 65 °C. Fluorescence quench titration and molecular docking methods report that CZD, CXM, and VNC interact more strongly with the partially folded intermediates (PFI) in comparison to the protein's natural state (N). However, only CZD and CXM proficiently inhibit the aggregation. Transmission electron microscopy, tinctorial assessments, and aggregation kinetics all support oligomer-level inhibition. Transition structures in CZD-HEWL and CXM-HEWL aggregation are shown by circular dichroism (CD). On the other hand, kinetic variables and soluble fraction assays point to a localized association of monomers. Intrinsic fluorescence (IF),1-Anilino 8-naphthalene sulphonic acid, and CD demonstrate structural and conformational modifications redesigning the PFI. GuHCl-induced unfolding and differential scanning fluorimetry suggested that the PFI monomers bound to CZD and CXM exhibited partial stability. Our results present two mechanisms that function in both solution conditions, creating a novel avenue for the screening of putative inhibitors for drug repurposing. We extend our proposed mechanisms in the designing of physical inhibitors of amyloid aggregation considering shorter time frames and foolproof methods.Communicated by Ramaswamy H. Sarma.

The role exerted by the nucleus in the regulation of proteostasis in both health and disease is recognized of outmost importance, even though not fully understood. Many recent investigations are focused on its ability to modulate and coordinate protein quality control machineries in mammalian cells. Nucleophosmin 1 (NPM1) is one of the most abundant nucleolar proteins and its gene is mutated in ~30% of Acute Myeloid Leukemia (AML) patients. Mutations are localized in the C-terminal domain of the protein and cause cytoplasmatically delocalized and possibly aggregated forms of NPM1 (NPM1c+). Therapeutic interventions targeted on NPM1c+ are in demand and, to this end, deeper knowledge of NPM1c+ behavior in the blasts' cytosol is required. Here by means of complementary biophysical techniques we compared the conformational and aggregative behavior of the entire C-terminal domains of NPM1wt and type A NPM1c+ (bearing the most common mutation). Overall data show that only Cterm_mutA is able to form amyloid-like assemblies with fibrillar morphology and that the oligomers are toxic in human neuroblastoma SHSY cells. This study adds a novel piece of knowledge to the comprehension of the molecular roles exerted by cytoplasmatic NPM1c+ and suggests the exploitation of the amyloidogenic propensity of NPM1c+ as a new strategy for targeting AML with NPM1 mutations. [ABSTRACT FROM AUTHOR]

Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In post-mitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often associated to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degradation function, thus generating a vicious cycle, which boost neurodegenerative cascades.

Amyloid fibrils draw attention as potential novel biomaterials due to their high stability, strength, elasticity or resistance against degradation. Therefore, the controlled and fast fibrillization process is of great interest, which raises the demand for effective tools capable of regulating amyloid fibrillization. Ionic liquids (ILs) were identified as effective modulators of amyloid aggregation. The present work is focused on the study of the effect of 1-ethyl-3-methyl imidazolium-based ILs with kosmotropic anion acetate (EMIM-ac) and chaotropic cation tetrafluoroborate (EMIM-BF4) on the kinetics of lysozyme amyloid aggregation and morphology of formed fibrils using fluorescence and CD spectroscopy, differential scanning calorimetry, AFM with statistical image analysis and docking calculations. We have found that both ILs decrease the thermal stability of lysozyme and significantly accelerate amyloid fibrillization in a dose-dependent manner at concentrations of 0.5%, 1% and 5% (v/v) in conditions and time-frames when no fibrils are formed in ILs-free solvent. The effect of EMIM-BF4 is more prominent than EMIM-ac due to the different specific interactions of the anionic part with the protein surface. Although both ILs induced formation of amyloid fibrils with typical needle-like morphology, a higher variability of fibril morphology consisting of a different number of intertwining protofilaments was identified for EMIM-BF4.

Proteins and polypeptides containing a high proportion of β-sheets have been recently reported to exhibit, in their amyloid aggregated states, an intrinsic fluorescence in the blue-green range of wavelength where the aromatic residues do not emit. Lately, growing attention has been devoted to the identification of a specific, structure-related fluorophore for the detection of amyloid aggregates due to their implications in the physio-pathology of neurodegenerative diseases and other aggregation-related diseases. Indeed, the appearance of blue-green fluorescence could be used as an alternative method for the investigation and the detection of the aggregation state without using external probes. Several hypotheses have been suggested to explain the molecular bases of this rather unusual intrinsic emission. In particular, it has been related to an expansion of the electronic delocalization of π-electrons of peptide bonds through the backbone-to-backbone hydrogen bonds connecting the β-sheets. Alternatively, the formation of the intrinsic chromophore has been associated to chemical modifications of the aromatic residues or arising from dipolar coupling between excited states of aromatic amino acids densely packed in the fibril structures. More recently, it has been proposed that the blue-green amyloid fluorophore does not require neither the presence of aromatic residues nor multiple bond conjugation. In this study, we critically review the above hypotheses with particular attention to the electronic transitions responsible for the appearance of blue-green fluorescence in amyloid fibrils.

Many neurodegenerative disorders, including Alzheimer's, Parkinson's and the prion diseases, are characterized by a conformational conversion of normally soluble proteins or peptides into pathological species, by a process of misfolding and self-assembly that leads ultimately to the formation of amyloid fibrils. Recent studies support the idea that multiple intermediate species with a wide variety of degrees of neuronal toxicity are generated during such processes. The development of a high level of knowledge of the nature and structure of the pathogenic amyloid species would significantly enhance efforts to underline the molecular origins of these disorders and also to develop both accurate diagnoses and effective therapeutic interventions for these types of conditions. In this review, we discuss recent biophysical and structural information concerning different types of amyloid aggregates and the way in which such information can guide rational therapeutic approaches designed to target specific pathogenic events that occur during the development of these highly debilitating and increasingly common diseases.

The eye lens is rich in proteins called crystallins, whose native conformation is crucial for preserving its transparency. With aging, crystallins may be exposed to environmental changes, which could lead to their aggregation and eventually to cataract development. Human γD-crystallin, among the most abundantly expressed γ-crystallins in the lens, was shown to form amyloid aggregates under denaturing conditions in vitro. However, the exact mechanism of aggregation remains to be clearly defined. Here, using prediction algorithms and biophysical methods, we identified a hexapeptide 41GCWMLY46 as a most aggregative fragment in human γD-crystallin. Two aromatic naphthoquinone-tryptophan hybrid molecules (NQTrp and Cl-NQTrp), inhibited the in vitro aggregation of this hexapeptide as well as full-length γD-crystallin in a dose-dependent manner, plausibly facilitated by hydrogen bonding and aromatic contacts with the hydrophobic residues. The two compounds had no toxic effect toward retinal cell culture and reduced the cytotoxicity induced by aggregates of the hexapeptide. In addition, NQTrp and Cl-NQTrp were able to disassemble pre-formed aggregates of the hexapeptide and the full-length γD-crystallin. Our results indicate that the amyloidogenic hexapeptide is a useful model for screening inhibitors of γD-crystallin and that the NQTrp hybrid scaffolds may serve as leads for developing new drugs for treating cataract. • The 41GCWMLY46 peptide fragment (GDC6), located in the N-terminal domain of the human γD-crystallin, exhibits amyloidogenic characteristics. • The GDC6 peptide could be used as a proxy model for screening aggregation inhibitors of the full-length human γD-crystallin. • NQTrp and Cl-NQTrp inhibited aggregation of the GDC6 peptide and full-length human γD-crystallin and disassembled their pre-formed aggregates in a dose-dependent manner. • NQTrp and Cl-NQTrp reduced the cytotoxicity of GDC6 aggregates toward ARPE-19 cells in culture. [ABSTRACT FROM AUTHOR]

Background: Aggregation of amyloid β into plaques in the brain is one of the earliest pathological events in Alzheimer's disease (AD). The exact pathophysiology leading to dementia is still uncertain, but the apolipoprotein E (APOE) ε4 genotype plays a major role. We aimed to identify the molecular pathways associated with amyloid β aggregation using cerebrospinal fluid (CSF) proteomics and to study the potential modifying effects of APOE ε4 genotype. Methods: We tested 243 proteins and protein fragments in CSF comparing 193 subjects with AD across the cognitive spectrum (65% APOE ε4 carriers, average age 75 ± 7 years) against 60 controls with normal CSF amyloid β, normal cognition, and no APOE ε4 allele (average age 75 ± 6 years). Results: One hundred twenty-nine proteins (53%) were associated with aggregated amyloid β. APOE ε4 carriers with AD showed altered concentrations of proteins involved in the complement pathway and glycolysis when cognition was normal and lower concentrations of proteins involved in synapse structure and function when cognitive impairment was moderately severe. APOE ε4 non-carriers with AD showed lower expression of proteins involved in synapse structure and function when cognition was normal and lower concentrations of proteins that were associated with complement and other inflammatory processes when cognitive impairment was mild. Repeating analyses for 114 proteins that were available in an independent EMIF-AD MBD dataset (n = 275) showed that 80% of the proteins showed group differences in a similar direction, but overall, 28% effects reached statistical significance (ranging between 6 and 87% depending on the disease stage and genotype), suggesting variable reproducibility. Conclusions: These results imply that AD pathophysiology depends on APOE genotype and that treatment for AD may need to be tailored according to APOE genotype and severity of the cognitive impairment. [ABSTRACT FROM AUTHOR]

Aggregation of an amyloid protein, α-synuclein (αS), is a critical step in the neurodegenerative pathway of Parkinson's diseases (PD). Specific detection of amyloid conformers (i.e., monomers, oligomers, and fibrils) produced during αS aggregation is critical in better understanding a molecular basis of PD and developing a diagnostic tool. While various molecular probes are available for detection of αS fibrils, which may serve as a reservoir of toxic αS aggregate forms, these probes suffer from limited conformer-specificity and operational flexibility. In the present study, we explored the potential of non-self-aggregating peptides derived from the highly aggregation-prone KLVFFAE region of an amyloid protein, β-amyloid, as molecular probes for αS aggregates. We show that of the four peptides tested (KLVFWAK, ELVFWAE, and their C-terminal capping variants, all of which were attached with fluorescein isothiocyanate at their respective N-termini), KLVFWAK with C-terminal capping was selectively bound to αS fibrils over monomers and oligomers and readily used for monitoring αS fibrilization. Our analyses suggest that binding of the peptide to αS fibrils is mediated by both electrostatic and hydrophobic interactions. We anticipate that our peptide can readily be optimized for conformer-specificity and operational flexibility. Overall, this study presents the creation of a KLVFFAE-based molecular probe for αS fibrils and demonstrates fine-tuning of its conformer-specificity by terminal mutations and capping. [ABSTRACT FROM AUTHOR]

Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In post-mitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often associated to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degradation function, thus generating a vicious cycle, which boost neurodegenerative cascades. [ABSTRACT FROM AUTHOR]

Protein (mis)folding, stability and aggregation are of interest in numerous fields, such as food sciences, biotechnology, and health sciences, and efforts are directed towards the elucidation of the underlying molecular mechanisms. Through an integrative approach, we show that a subtle balance between hydrogen bond formation and hydrophobic interactions defines protein self-assembly pathways. Hydrophobic co-solvents, such as monohydric alcohols, modulate these two forces through a combination of direct solvent-protein and solvent-mediated interactions, depending on the size of the alcohol. This affects the initial conformation of the model protein α-lactalbumin, which can be linked to variations of its fibrillation propensity, as well as the morphology of the final structures. These findings pave the way towards a better understanding of the forces governing protein self-assembly, allowing the development of strategies to suppress unwanted aggregation and control the growth of tuneable protein-based biomaterials. [ABSTRACT FROM AUTHOR]

Machine learning methods are increasingly used in proteomics research, especially in analyzing and predicting protein structures, functions, subcellular localizations and interactions. However, much research in recent years has focused on protein misfolding problem and the impact of unfolded and defective proteins on cell dysfunction, due to its considerable importance for molecular medicine. These abnormal proteins degradation and deposition often result in the formation of certain plaque cores among them the so-called amyloid fibrils which are responsible for an increasing number of highly debilitating disorders in humans. Yet, a significant challenge remains, especially in understanding the underlying causes and major risk factors of these harmful deposits in vital organs and tissues. This paper explores the potential of string kernel-based support vector machines in the prediction of amyloidogenic regions in proteins by incorporating the most informative features of the protein sequence such as predicted secondary structure and solvent accessibility, with a special focus on α -helical conformations which seem to be primarily concerned with amyloidogenesis. The performances compared with the most popular methods on Pep424 and Reg33 benchmark datasets indicate the robustness of the predictive model. Furthermore, the results showed accurate prediction of regions promoting fibrillogenesis for experimentally determined amyloid proteins and revealed that the five amino acids Leucine, Glycine, Alanine, Valine and Serine are predominantly present in amyloid-prone regions and confirm that the core regions of an amyloid aggregate are not necessarily fully buried. [ABSTRACT FROM AUTHOR]

The giant muscle protein, titin, is the third most abundant protein in muscle (after myosin and actin). It was shown previously that smooth muscle titin (SMT) with a molecular mass of 500 kDa can form in vitro amorphous amyloid aggregates in two conditions: in solution of low ionic strength (0.15 M Glycine-KOH, pH 7.0) (SMT(Gly) aggregates) and in solution with ionic strength in the physiological range (0.2 M KCl, 20 mM imidazole, pH 7.2–7.4) (SMT(KCl) aggregates). Such aggregation in vivo, which may play a pathological or functional role, is not excluded. In view of the fact that some pathological amyloids can activate the classical and alternative pathways of complement system, we investigated the binding of complement component C1q and C3b to smooth muscle titin amyloid aggregates. The binding of С1q and C3b to SMT aggregates was not observed with ELISA assay. Since SMT aggregates do not activate the complement system, they are hardly implicated in the inflammatory process caused by muscle damage in amyloidoses. Abbreviations: SMT: smooth muscle titin; SMT(KCl) aggregates: SMT aggregates in solution containing 0.2 M KCl, 10 mM imidazole, pH 7.0; SMT(Gly) aggregates: SMT aggregates in solution containing 0.15 M glycine-KOH, pH 7.2-7.4; MAC: membrane attack complex; DLS: dynamic light scattering; NHS: Normal Human Serum [ABSTRACT FROM AUTHOR]

Timely detection of amyloid aggregations has a critical role in the treatment of degenerative nervous system disorders such as Alzheimer's, Parkinson's disease and systemic amyloidosis. Thioflavin T (ThT) is a dye considered for the detection of amyloids. However, ThT cannot cross the blood–barrier barrier due to positive charge and low lipophilicity. In the present study, a variety of 2-benzylidene-1,3-indandione derivatives 5a–5j were synthesized as neutral fluorescence probe candidates for identification of amyloid. Among these compounds, only compound 2-(2-hydroxybenzylidene)-1,3-indandione 5a was selected for further examination, because its fluorescence intensity showed the significant increasing upon interaction with amyloid aggregates. The compound 5a was compared to standard and conventional probe such as ThT. The compound 5a was excited at its specified wavelength, and the fluorescence emission signal was recorded in the presence of different concentrations of the protein/peptide (amyloid aggregated and native protein). According to the obtained results, it can be seen that 2-(2-hydroxybenzylidene)-1,3-indandione 5a selectively and specifically bind to amyloid fibrils, such as ThT. Due to the neutrality of electrical charge and high lipophilicity coefficient of the synthesized compound 5a, it is possible for it to cross from the blood–brain barrier. Our results show that this synthetic derivative can be considered as a suitable probe to detect in vitro amyloid aggregations. [ABSTRACT FROM AUTHOR]

Silkworm silk protein fibroin is widely exploited to develop novel silk-based biomaterials due to its stable β-sheet structure, providing high crystallinity and tensile strength. The polymorphic behaviour of silk fibroin provides a window to modulate its structural transitions during self-assembly for different functional outcomes. Most studies are therefore mainly focused on formation of well-developed β-sheet structure and self-assembly of silk fibroin which are regulated by many parameters. Glyoxal, a highly reactive α-oxoaldehyde, reacts with different proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of protein modification by glyoxal-derived AGEs, in the present study the effect of glyoxal (250, 500 and 1000 μM) on the structure of silk fibroin has been investigated. CD and fluorescence studies reveal that higher concentrations of the α-oxoaldehyde induce considerable alterations of secondary and tertiary structure of the protein leading to aggregation following incubation with for 3 weeks. The aggregates exhibit fibrillar morphology with amyloidal nature as evident from SEM, FTIR and XRD experiments. The findings highlight that glycation-induced modification can be a possible approach for modulating the conformation of the silk protein which may be relevant in connection to clinical, biomedical or synthetic biology based applications. [ABSTRACT FROM AUTHOR]

Silkworm silk protein fibroin is widely exploited to develop novel silk-based biomaterials due to its stable β-sheet structure, providing high crystallinity and tensile strength. The polymorphic behaviour of silk fibroin provides a window to modulate its structural transitions during self-assembly for different functional outcomes. Most studies are therefore mainly focused on formation of well-developed β-sheet structure and self-assembly of silk fibroin which are regulated by many parameters. Glyoxal, a highly reactive α-oxoaldehyde, reacts with different proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of protein modification by glyoxal-derived AGEs, in the present study the effect of glyoxal (250, 500 and 1000 μM) on the structure of silk fibroin has been investigated. CD and fluorescence studies reveal that higher concentrations of the α-oxoaldehyde induce considerable alterations of secondary and tertiary structure of the protein leading to aggregation following incubation with for 3 weeks. The aggregates exhibit fibrillar morphology with amyloidal nature as evident from SEM, FTIR and XRD experiments. The findings highlight that glycation-induced modification can be a possible approach for modulating the conformation of the silk protein which may be relevant in connection to clinical, biomedical or synthetic biology based applications. [ABSTRACT FROM AUTHOR]

The production and accumulation of toxic amyloid plaques is one of the hallmarks of Alzheimer’s disease (AD). Amyloid beta (Aβ) peptides undergo self-aggregation to form soluble oligomers, protofibrils and insoluble fibrils. This process is termed as amyloidogenesis and is a major contributor to the observed neuronal damage and memory impairment in the AD brain. Therefore, modulation of Aβ aggregation process is considered to be an effective target to prevent neuronal damage under AD conditions. Modulation of amyloidogenesis involves inhibition of aggregation to form a toxic species or acceleration to drive the aggregation process to form species that are nontoxic by employing well-designed external ligands. In this context, we report a set of 2,3-dihydrophthalazine-1,4-dione (dihydrophthalazinedione, Phz) based small molecules (Phz 1–4) to modulate the Aβ42 aggregation and in cellular toxicity. Our detailed study (thioflavin T fluorescence assay, dot blot and transmission electron microscopy analysis) revealed fluorine containing Phz 4 as the potent modulator of Aβ42 aggregation by accelerating the process to form nontoxic aggregated species through hydrophobic and halogen interactions. Aβ42 aggregates formed in the presence of Phz 4 are mostly nontoxic when compared to the normal amyloid aggregates in the cellular milieu (PC12 cells). This study established that the hydrophobic and halogen interactions can be employed to develop anti-AD drug candidates. The excellent cell viability, effective modulation of Aβ42 aggregation to form nontoxic species and cellular (neuronal) rescue by Phz 4 offer a novel platform to develop therapeutic strategies for AD. [ABSTRACT FROM AUTHOR]

Alzheimer's diseases (AD) and type 2 diabetes (T2D) are two age-related diseases characterized by amyloid fibrillogenesis. Prevention of amyloid aggregation is a promising therapeutic strategy for AD and T2D. Two spermine alkaloids, kukoamines A and B, isolated from Lycii Cortex (LyC) were investigated for their inhibitory effect on amyloid aggregation. Both kukoamines A and B inhibited aggregation of amyloid β (Aβ) and human islet amyloid polypeptide (hIAPP) in a dose-dependent manner. Kukoamine B showed stronger inhibitory activity than kukoamine A. These results on the inhibitory activity of kukoamines A and B on Aβ and hIAPP indicate that the catechol moiety is essential for inhibition of amyloid aggregation. [ABSTRACT FROM AUTHOR]

α-Synuclein (α-syn), a disordered cytoplasmatic protein, plays a fundamental role in the pathogenesis of Parkinson's disease (PD). Here, we have shown, using photophysical measurements, that addition of FKBP12 to α-syn solutions, dramatically accelerates protein aggregation, leading to an explosion of dendritic structures revealed by fluorescence and phase-contrast microscopy. We have further demonstrated that this aberrant α-syn aggregation can be blocked using a recently discovered non-immunosuppressive synthetic inhibitor of FKBP12, ElteN378. The role of FKBP12 and of ElteN378 in the α-syn aggregation mechanism has been elucidated using molecular dynamics simulations based on an effective coarse-grained model. The reported data not only reveal a new potent synthetic drug as a candidate for early stage treatment of α-syn dependent neurodegenerations but also pave the way to a deeper understanding of the mechanism of action of FKBP12 on α-syn oligomeric aggregation, a topic which is still controversial. [ABSTRACT FROM AUTHOR]