Formation of amyloid fibrils by the regulatory 14-3-3ζ protein.

The 14-3-3 proteins are a highly conserved adaptor protein family with multi-layer functions, abundantly expressed in the brain. The 14-3-3 proteins modulate phosphorylation, regulate enzymatic activity and can act as chaperones. Most importantly, they play an important role in various neurodegenerative disorders due to their vast interaction partners. Particularly, the 14-3-3ζ isoform is known to co-localize in aggregation tangles in both Alzheimer's and Parkinson's diseases as a result of protein-protein interactions. These abnormal clumps consist of amyloid fibrils, insoluble aggregates, mainly formed by the amyloid-ß, tau and α-synuclein proteins. However, the molecular basis of if and how 14-3-3ζ can aggregate into amyloid fibrils is unknown. In this study, we describe the formation of amyloid fibrils by 14-3-3ζ using a comprehensive approach that combines bioinformatic tools, amyloid-specific dye binding, secondary structure analysis and atomic force microscopy. The results presented herein characterize the amyloidogenic properties of 14-3-3ζ and imply that the well-folded protein undergoes aggregation to ß-sheet-rich amyloid fibrils.

Investigating lysozyme amyloid fibril formation and structural variability dependence on its initial folding state under different pH conditions.

Protein fibril formation and accumulation are associated with dozens of amyloidoses, including the widespread and yet-incurable Alzheimer's and Parkinson's diseases. Currently, there are still several aspects of amyloid aggregation that are not fully understood, which negatively contributes to the development of disease-altering drugs and treatments. One factor which requires a more in-depth analysis is the effect of the environment on both the initial state of amyloidogenic proteins and their aggregation process and resulting fibril characteristics. In this work, we examine how lysozyme's folding state influences its amyloid formation kinetics and resulting aggregate structural characteristics under several different pH conditions, ranging from acidic to neutral. We demonstrate that both the initial state of the protein and the solution's pH value have a significant combined effect on the variability of the resulting aggregate secondary structures, as well as their stabilities, interactions with amyloid-specific dye molecules, and self-replication properties.

The Major Components of Cerebrospinal Fluid Dictate the Characteristics of Inhibitors against Amyloid-Beta Aggregation.

The main pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-ß into amyloid fibrils, leading to a neurodegeneration cascade. The current medications are far from sufficient to prevent the onset of the disease, hence requiring more research to find new alternative drugs for curing AD. In vitro inhibition experiments are one of the primary tools in testing whether a molecule may be potent to impede the aggregation of amyloid-beta peptide (Aß42). However, kinetic experiments in vitro do not match the mechanism found when aggregating Aß42 in cerebrospinal fluid. The different aggregation mechanisms and the composition of the reaction mixtures may also impact the characteristics of the inhibitor molecules. For this reason, altering the reaction mixture to resemble components found in cerebrospinal fluid (CSF) is critical to partially compensate for the mismatch between the inhibition experiments in vivo and in vitro. In this study, we used an artificial cerebrospinal fluid that contained the major components found in CSF and performed Aß42 aggregation inhibition studies using oxidized epigallocatechin-3-gallate (EGCG) and fluorinated benzenesulfonamide VR16-09. This led to a discovery of a complete turnaround of their inhibitory characteristics, rendering EGCG ineffective while significantly improving the efficacy of VR16-09. HSA was the main contributor in the mixture that significantly increased the anti-amyloid characteristics of VR16-09.

Inhibitory effects of fluorinated benzenesulfonamides on insulin fibrillation.

Amyloid fibrils are protein aggregates formed by protein assembly through cross ß structures. Inhibition of amyloid fibril formation may contribute to therapy against amyloid-related disorders like Parkinson's, Alzheimer's, and type 2 diabetes. Here we report that several fluorinated sulfonamide compounds, previously shown to inhibit human carbonic anhydrase, also inhibit the fibrillation of different proteins. Using a range of spectroscopic, microscopic and chromatographic techniques, we found that the two fluorinated sulfonamide compounds completely inhibit insulin fibrillation over a period of 16 h and moderately suppress α-synuclein and Aß fibrillation. In addition, these compounds decreased cell toxicity of insulin incubated under fibrillation-inducing conditions. We ascribe these effects to their ability to maintain insulin in the native monomeric state. Molecular dynamic simulations suggest that these compounds inhibit insulin self-association by interacting with residues at the dimer interface. This highlights the general anti-aggregative properties of aromatic sulfonamides and suggests that sulfonamide compounds which inhibit carbonic anhydrase activity may have potential as therapeutic agents against amyloid-related disorders.

Rapid restructurization of conformationally-distinct alpha-synuclein amyloid fibrils at an elevated temperature.

Protein aggregation in the form of amyloid fibrils is linked with the onset and progression of more than 30 amyloidoses, including multiple neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Despite countless studies and years of research, the process of such aggregate formation is still not fully understood. One peculiar aspect of amyloids is that they appear to be capable of undergoing structural rearrangements even after the fibrils have already formed. Such a phenomenon was reported to occur in the case of alpha-synuclein and amyloid beta aggregates after a long period of incubation. In this work, we examine whether incubation at an elevated temperature can induce the restructurization of four different conformation alpha-synuclein amyloid fibrils. We show that this structural alteration occurs in a relatively brief time period, when the aggregates are incubated at 60 °C. Additionally, it appears that during this process multiple conformationally-distinct alpha-synuclein fibrils all shift towards an identical secondary structure.

Exploring the Formation of Polymers with Anti-Amyloid Properties within the 2'3'-Dihydroxyflavone Autoxidation Process.

Amyloid-ß and α-synuclein aggregation into amyloid fibrils is linked to the onset and progression of Alzheimer's and Parkinson's diseases. While there are only a few disease-modifying drugs, it is essential to search for new, more effective ways to encounter these neurodegenerative diseases. Multiple research articles have shown that the autoxidation of flavone is a critical factor for activating the inhibitory potential against the protein aggregation. Despite this, the structure of the newly-formed inhibitors is unknown. In this research, we examined the autoxidation products of 2',3'-dihydroxyflavone that were previously shown to possess one of the most prominent inhibitory effects against amyloid-ß aggregation. Their analysis using HPLC suggested the formation of polymeric molecules that were isolated using a 3 kDa cut-off. These polymeric structures were indicated as the most potent inhibitors based on protein aggregation kinetics and AFM studies. This revelation was confirmed using MALDI-TOF and NMR. We also show that active molecules have a tendency to reduce the Amyloid-ß and α-synuclein aggregates toxicity to SH-SY5Y cells.

Superoxide dismutase-1 alters the rate of prion protein aggregation and resulting fibril conformation.

The assembly of amyloidogenic proteins into highly-structured fibrillar aggregates is related to the onset and progression of several amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases. Despite years of research and a general understanding of the process of such aggregate formation, there are currently still very few drugs and treatment modalities available. One of the factors that is relatively insufficiently understood is the cross-interaction between different amyloid-forming proteins. In recent years, it has been shown that several of these proteins or their aggregates can alter each other's fibrillization properties, however, there are still many unknowns in the amyloid interactome. In this work, we examine the interaction between amyloid disease-related prion protein and superoxide dismutase-1. We show that not only does superoxide dismutase-1 increase the lag time of prion protein fibril formation, but it also changes the conformation of the resulting aggregates.

Interplay between epigallocatechin-3-gallate and ionic strength during amyloid aggregation.

The formation and accumulation of protein amyloid aggregates is linked with multiple amyloidoses, including neurodegenerative Alzheimer's or Parkinson's disease. The mechanism of such fibril formation is impacted by various environmental conditions, which greatly complicates the search for potential anti-amyloid compounds. One of these factors is solution ionic strength, which varies between different aggregation protocols during in vitro drug screenings. In this work, we examine the interplay between ionic strength and a well-known protein aggregation inhibitor-epigallocatechin-3-gallate. We show that changes in solution ionic strength have a major impact on the compound's inhibitory effect, reflected in both aggregation times and final fibril structure. We also observe that this effect is unique to different amyloid-forming proteins, such as insulin, alpha-synuclein and amyloid-beta.

Polymorphism of Alpha-Synuclein Amyloid Fibrils Depends on Ionic Strength and Protein Concentration.

Protein aggregate formation is linked with multiple amyloidoses, including Alzheimer's and Parkinson's diseases. Currently, the understanding of such fibrillar structure formation and propagation is still not sufficient, the outcome of which is a lack of potent, anti-amyloid drugs. The environmental conditions used during in vitro protein aggregation assays play an important role in determining both the aggregation kinetic parameters, as well as resulting fibril structure. In the case of alpha-synuclein, ionic strength has been shown as a crucial factor in its amyloid aggregation. In this work, we examine a large sample size of alpha-synuclein aggregation reactions under thirty different ionic strength and protein concentration combinations and determine the resulting fibril structural variations using their dye-binding properties, secondary structure and morphology. We show that both ionic strength and protein concentration determine the structural variability of alpha-synuclein amyloid fibrils and that sometimes even identical conditions can result in up to four distinct types of aggregates.

Autoxidation Enhances Anti-Amyloid Potential of Flavone Derivatives.

The increasing prevalence of amyloid-related disorders, such as Alzheimer's or Parkinson's disease, raises the need for effective anti-amyloid drugs. It has been shown on numerous occasions that flavones, a group of naturally occurring anti-oxidants, can impact the aggregation process of several amyloidogenic proteins and peptides, including amyloid-beta. Due to flavone autoxidation at neutral pH, it is uncertain if the effective inhibitor is the initial molecule or a product of this reaction, as many anti-amyloid assays attempt to mimic physiological conditions. In this work, we examine the aggregation-inhibiting properties of flavones before and after they are oxidized. The oxidation of flavones was monitored by measuring the UV-vis absorbance spectrum change over time. The protein aggregation kinetics were followed by measuring the amyloidophilic dye thioflavin-T (ThT) fluorescence intensity change. Atomic force microscopy was employed to image the aggregates formed with the most prominent inhibitors. We demonstrate that flavones, which undergo autoxidation, have a far greater potency at inhibiting the aggregation of both the disease-related amyloid-beta, as well as a model amyloidogenic protein-insulin. Oxidized 6,2',3'-trihydroxyflavone was the most potent inhibitor affecting both insulin (7-fold inhibition) and amyloid-beta (2-fold inhibition). We also show that this tendency to autoxidize is related to the positions of the flavone hydroxyl groups.

Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils.

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases-a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.

Using lysozyme amyloid fibrils as a means of scavenging aggregation-inhibiting compounds.

The aggregation of amyloidogenic proteins is linked to several amyloidoses, including neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Currently there are very few effective cures or treatments available, despite countless screenings and clinical trials. One of the most challenging aspects of potential anti-amyloid drug discovery is finding which molecules are the actual inhibitors out of mixtures, which may contain hundreds of distinct compounds. Considering that anti-amyloid compounds would interact with the aggregate, this affinity could be used as a means of separating such compounds from ineffective ones. In this work, we attempt to scavenge potential aggregation-inhibiting molecules out of four, different complexity mixtures, ranging from oxidized gallic acid to tea extract, using lysozyme amyloid fibrils. We show that these compounds bind to aggregates with high affinity and can be later separated from them by different methods.

Exploring the occurrence of thioflavin-T-positive insulin amyloid aggregation intermediates.

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.

Lysozyme Fibrils Alter the Mechanism of Insulin Amyloid Aggregation.

Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of how natively non-harmful proteins convert to these highly cytotoxic amyloid aggregates is still not sufficient, with new ideas and hypotheses being presented each year. Recently it has been shown that more than one type of protein aggregates may co-exist in the affected tissue of patients suffering from amyloid-related disorders, sparking the idea that amyloid aggregates formed by one protein may induce another protein's fibrillization. In this work, we examine the effect that lysozyme fibrils have on insulin amyloid aggregation. We show that not only do lysozyme fibrils affect insulin nucleation, but they also alter the mechanism of its aggregation.

Methyl 2-Halo-4-Substituted-5-Sulfamoyl-Benzoates as High Affinity and Selective Inhibitors of Carbonic Anhydrase IX.

Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and selective binding to CAIX over the remaining eleven catalytically active CA isozymes to limit undesired side effects. It has been postulated that such drugs may have anticancer properties and could be used in tumor treatment. Here we have designed a series of compounds, methyl 5-sulfamoyl-benzoates, which bear a primary sulfonamide group, a well-known marker of CA inhibitors, and determined their affinities for all twelve CA isozymes. Variations of substituents on the benzenesulfonamide ring led to compound 4b, which exhibited an extremely high observed binding affinity to CAIX; the Kd was 0.12 nM. The intrinsic dissociation constant, where the binding-linked protonation reactions have been subtracted, reached 0.08 pM. The compound also exhibited more than 100-fold selectivity over the remaining CA isozymes. The X-ray crystallographic structure of compound 3b bound to CAIX showed the structural position, while several structures of compounds bound to other CA isozymes showed structural reasons for compound selectivity towards CAIX. Since this series of compounds possess physicochemical properties suitable for drugs, they may be developed for anticancer therapeutic purposes.

Endogenous motion of liver correlates to the severity of portal hypertension.

BACKGROUND: Degree of portal hypertension (PH) is the most important prognostic factor for the decompensation of liver cirrhosis and death, therefore adequate care for patients with liver cirrhosis requires timely detection and evaluation of the presence of clinically significant PH (CSPH) and severe PH (SPH). As the most accurate method for the assessment of PH is an invasive direct measurement of hepatic venous pressure gradient (HVPG), the search for non-invasive methods to diagnose these conditions is actively ongoing. AIM: To evaluate the feasibility of parameters of endogenously induced displacements and strain of liver to assess degree of PH. METHODS: Of 36 patients with liver cirrhosis and measured HVPG were included in the case-control study. Endogenous motion of the liver was characterized by derived parameters of region average tissue displacement signal (d antero, dr etro, d RMS) and results of endogenous tissue strain imaging using specific radiofrequency signal processing algorithm. Average endogenous strain µ and standard deviation σ of strain were assessed in the regions of interest (ROI) (1 cm × 1 cm and 2 cm × 2 cm in size) and different frequency subbands of endogenous motion (0-10 Hz and 10-20 Hz). RESULTS: Four parameters showed statistically significant (P < 0.05) correlation with HVPG measurement. The strongest correlation was obtained for the standard deviation of strain (estimated at 0-10 Hz and 2 cm × 2 cm ROI size). Three parameters showed statistically significant differences between patient groups with CSPH, but only d retro showed significant results in SPH analysis. According to ROC analysis area under the curve (AUC) of the σ ROI[0…10Hz, 2 cm × 2 cm] parameter reached 0.71 (P = 0.036) for the diagnosis of CSPH; with a cut-off value of 1.28 µm/cm providing 73% sensitivity and 70% specificity. AUC for the diagnosis of CSPH for µ ROI[0…10Hz, 1 cm × 1 cm] was 0.78 (P = 0.0024); with a cut-off value of 3.92 µm/cm providing 73% sensitivity and 80% specificity. D retro parameter had an AUC of 0.86 (P = 0.0001) for the diagnosis of CSPH and 0.84 (P = 0.0001) for the diagnosis of SPH. A cut-off value of -132.34 µm yielded 100% sensitivity for both conditions, whereas specificity was 80% and 72% for CSPH and SPH respectively. CONCLUSION: The parameters of endogenously induced displacements and strain of the liver correlated with HVPG and might be used for non-invasive diagnosis of PH.

Identifying Insulin Fibril Conformational Differences by Thioflavin-T Binding Characteristics.

Amyloidogenic protein aggregation into highly structured fibrils is linked to more than 30 amyloidoses, including several neurodegenerative disorders. Despite significant progress in trying to understand the process of amyloid formation, there is still no cure or effective treatment available. A number of studies involving potential anti-amyloid compounds rely on the use of a fluorescent probe-thioflavin-T-to track the appearance, growth, or disassembly of these cytotoxic aggregates. Despite the wide application of this dye molecule, its interaction with amyloid fibrils is still poorly understood. Recent reports have shown it may possess distinct binding modes and fluorescence intensities based on the conformation of the examined fibrils. In this work, we generate insulin fibrils under four different conditions and attempt to identify distinct conformations using both classic methods, such as atomic force microscopy and Fourier-transform infrared spectroscopy, as well as their ThT binding ability and fluorescence quantum yield. We show that there is a significant variance of ThT fluorescence quantum yields, excitation/emission maxima positions, and binding modes between distinct insulin fibril conformations.

Gallic acid oxidation products alter the formation pathway of insulin amyloid fibrils.

Amyloidogenic protein assembly into insoluble fibrillar aggregates is linked with several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease, affecting millions of people worldwide. The search for a potential anti-amyloid drug has led to the discovery of hundreds of compounds, none of which have passed all clinical trials. Gallic acid has been shown to both modulate factors leading to the onset of neurodegenerative disorders, as well as directly inhibit amyloid formation. However, the conditions under which this effect is seen could lead to oxidation of this polyphenol, likely changing its properties. Here we examine the effect of gallic acid and its oxidised form on the aggregation of a model amyloidogenic protein-insulin at low pH conditions. We show a vastly higher inhibitory potential of the oxidised form, as well as an alteration in the aggregation pathway, leading to the formation of a specific fibril conformation.

Quantification of Endogenous Brain Tissue Displacement Imaging by Radiofrequency Ultrasound.

The purpose of this paper is a quantification of displacement parameters used in the imaging of brain tissue endogenous motion using ultrasonic radiofrequency (RF) signals. In a preclinical study, an ultrasonic diagnostic system with RF output was equipped with dedicated signal processing software and subject head-ultrasonic transducer stabilization. This allowed the use of RF scanning frames for the calculation of micrometer-range displacements, excluding sonographer-induced motions. Analysis of quantitative displacement estimates in dynamical phantom experiments showed that displacements of 55 µm down to 2 µm were quantified as confident according to Pearson correlation between signal fragments (minimum p ≤ 0.001). The same algorithm and scanning hardware were used in experiments and clinical imaging which allows translating phantom results to Alzheimer's disease patients and healthy elderly subjects as examples. The confident quantitative displacement waveforms of six in vivo heart-cycle episodes ranged from 8 µm up to 263 µm (Pearson correlation p ≤ 0.01). Displacement time sequences showed promising possibilities to evaluate the morphology of endogenous displacement signals at each point of the scanning plane, while displacement maps-regional distribution of displacement parameters-were essential for tissue characterization.

The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG.

Millions of people around the world suffer from amyloid-related disorders, including Alzheimer's and Parkinson's diseases. Despite significant and sustained efforts, there are still no disease-modifying drugs available for the majority of amyloid-related disorders, and the overall failure rate in clinical trials is very high, even for compounds that show promising anti-amyloid activity in vitro. In this study, we demonstrate that even small changes in the chemical environment can strongly modulate the inhibitory effects of anti-amyloid compounds. Using one of the best-established amyloid inhibitory compounds, epigallocatechin-3-gallate (EGCG), as an example, and two amyloid-forming proteins, insulin and Parkinson's disease-related α -synuclein, we shed light on the previously unexplored sensitivity to solution conditions of the action of this compound on amyloid fibril formation. In the case of insulin, we show that the classification of EGCG as an amyloid inhibitor depends on the experimental conditions select, on the method used for the evaluation of the efficacy, and on whether or not EGCG is allowed to oxidise before the experiment. For α -synuclein, we show that a small change in pH value, from 7 to 6, transforms EGCG from an efficient inhibitor to completely ineffective, and we were able to explain this behaviour by the increased stability of EGCG against oxidation at pH 6.