ABSTRACT
Amyloid-beta aggregation is considered one of the factors influencing the onset of the Alzheimer's disease. Early prevention of such aggregation should alleviate disease condition by applying small molecule compounds that shift the aggregation equilibrium toward the soluble form of the peptide or slow down the process. We have discovered that fluorinated benzenesulfonamides of particular structure slowed the amyloid-beta peptide aggregation process by more than three-fold. We synthesized a series of ortho-para and meta-para double-substituted fluorinated benzenesulfonamides that inhibited the aggregation process to a variable extent yielding a detailed picture of the structure-activity relationship. Analysis of compound chemical structure effect on aggregation in artificial cerebrospinal fluid showed the necessity to arrange the benzenesulfonamide, hydrophobic substituent, and benzoic acid in a particular way. The amyloid beta peptide aggregate fibril structures varied in cross-sectional height depending on the applied inhibitor indicating the formation of a complex with the compound. Application of selected inhibitors increased the survivability of cells affected by the amyloid beta peptide. Such compounds may be developed as drugs against Alzheimer's disease.
ABSTRACT
Protein liquid-liquid phase separation (LLPS) is a rapidly emerging field of study on biomolecular condensate formation. In recent years, this phenomenon has been implicated in the process of amyloid fibril formation, serving as an intermediate step between the native protein transition into their aggregated state. The formation of fibrils via LLPS has been demonstrated for a number of proteins related to neurodegenerative disorders, as well as other amyloidoses. Despite the surge in amyloid-related LLPS studies, the influence of protein condensate formation on the end-point fibril characteristics is still far from fully understood. In this work, we compare alpha-synuclein aggregation under different conditions, which promote or negate its LLPS and examine the differences between the formed aggregates. We show that alpha-synuclein phase separation generates a wide variety of assemblies with distinct secondary structures and morphologies. The LLPS-induced structures also possess higher levels of toxicity to cells, indicating that biomolecular condensate formation may be a critical step in the appearance of disease-related fibril variants.
ABSTRACT
Protein aggregation in the form of amyloid fibrils has long been associated with the onset and development of various amyloidoses, including Alzheimer's, Parkinson's or prion diseases. Recent studies of their fibril formation process have revealed that amyloidogenic protein cross-interactions may impact aggregation pathways and kinetic parameters, as well as the structure of the resulting aggregates. Despite a growing number of reports exploring this type of interaction, they only cover just a small number of possible amyloidogenic protein pairings. One such pair is between two neurodegeneration-associated proteins: the pro-inflammatory S100A9 and prion protein, which are known to co-localize in vivo. In this study, we examined their cross-interaction in vitro and discovered that the fibrillar form of S100A9 modulated the aggregation pathway of mouse prion protein 89-230 fragment, while non-aggregated S100A9 also significantly inhibited its primary nucleation process. These results complement previous observations of the pro-inflammatory protein's role in amyloid aggregation and highlight its potential role against neurodegenerative disorders.
Subject(s)
Amyloid , Calgranulin B , Prion Proteins , Protein Aggregates , Calgranulin B/metabolism , Calgranulin B/chemistry , Animals , Mice , Prion Proteins/chemistry , Prion Proteins/metabolism , Amyloid/metabolism , Amyloid/chemistry , Peptide Fragments/metabolism , Peptide Fragments/chemistry , KineticsABSTRACT
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.
Subject(s)
Neurodegenerative Diseases , Parkinson Disease , Humans , Amyloid/chemistry , 14-3-3 Proteins/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism , Parkinson Disease/metabolism , Amyloid beta-Peptides/metabolism , Neurodegenerative Diseases/metabolismABSTRACT
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.
Subject(s)
Amyloid , Protein Folding , Amyloid/chemistry , Muramidase/chemistry , Protein Structure, Secondary , Hydrogen-Ion ConcentrationABSTRACT
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.
Subject(s)
Alzheimer Disease , Catechin , Humans , Peptide Fragments/chemistry , Amyloid beta-Peptides/chemistry , Alzheimer Disease/pathology , Amyloid , Catechin/chemistryABSTRACT
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.
Subject(s)
Carbonic Anhydrases , Diabetes Mellitus, Type 2 , Humans , Insulin/chemistry , Amyloid/chemistry , Sulfonamides/pharmacology , Carbonic Anhydrase Inhibitors/pharmacologyABSTRACT
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.
Subject(s)
Parkinson Disease , alpha-Synuclein , Humans , alpha-Synuclein/chemistry , Amyloid/chemistry , Amyloid beta-Peptides/chemistry , Temperature , Parkinson Disease/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Peptide Fragments/metabolism , Prion Proteins/metabolism , Protein Aggregates/drug effects , Superoxide Dismutase-1/metabolism , Animals , Hydrogen Bonding , Mice , Peptide Fragments/chemistry , Prion Proteins/chemistry , Protein Conformation, beta-Strand/drug effects , Protein Multimerization/drug effectsABSTRACT
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.
Subject(s)
Amyloid/chemistry , Protein Aggregates , Protein Aggregation, Pathological , alpha-Synuclein/chemistry , Amyloid/metabolism , In Vitro Techniques/methods , Kinetics , Osmolar Concentration , Parkinson Disease/metabolism , Protein Binding , Protein Structure, Secondary , alpha-Synuclein/metabolismABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Amyloid/chemistry , Prion Proteins/chemistry , Protein Multimerization , Temperature , Protein ConformationABSTRACT
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.
Subject(s)
Amyloid , Neurodegenerative Diseases , Humans , MuramidaseABSTRACT
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.
Subject(s)
Amyloid/metabolism , Insulin/metabolism , Muramidase/metabolism , Protein Aggregation, Pathological/metabolism , Amyloid/ultrastructure , Animals , Chickens , Humans , Protein Aggregates , Recombinant Proteins/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Benzoates/pharmacology , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrase Inhibitors/pharmacology , Sulfonamides/pharmacology , Catalytic Domain/drug effects , Crystallography, X-Ray/methods , Humans , Isoenzymes/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Protein Binding/physiology , Structure-Activity Relationship , Thermodynamics , Tumor Microenvironment/drug effects , BenzenesulfonamidesABSTRACT
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.
Subject(s)
Benzothiazoles , Insulin , Amyloid/metabolism , Fluorescent Dyes , Insulin/chemistry , Microscopy, Atomic Force , Protein BindingABSTRACT
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.