amyloid fibrils
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2022 ◽  
Vol 8 ◽  
Tatsuhito Matsuo ◽  
Alessio De Francesco ◽  
Judith Peters

Lysozyme amyloidosis is a hereditary disease, which is characterized by the deposition of lysozyme amyloid fibrils in various internal organs. It is known that lysozyme fibrils show polymorphism and that polymorphs formed at near-neutral pH have the ability to promote more monomer binding than those formed at acidic pH, indicating that only specific polymorphs become dominant species in a given environment. This is likely due to the polymorph-specific configurational diffusion. Understanding the possible differences in dynamical behavior between the polymorphs is thus crucial to deepen our knowledge of amyloid polymorphism and eventually elucidate the molecular mechanism of lysozyme amyloidosis. In this study, molecular dynamics at sub-nanosecond timescale of two kinds of polymorphic fibrils of hen egg white lysozyme, which has long been used as a model of human lysozyme, formed at pH 2.7 (LP27) and pH 6.0 (LP60) was investigated using elastic incoherent neutron scattering (EINS) and quasi-elastic neutron scattering (QENS). Analysis of the EINS data showed that whereas the mean square displacement of atomic motions is similar for both LP27 and LP60, LP60 contains a larger fraction of atoms moving with larger amplitudes than LP27, indicating that the dynamical difference between the two polymorphs lies not in the averaged amplitude, but in the distribution of the amplitudes. Furthermore, analysis of the QENS data showed that the jump diffusion coefficient of atoms is larger for LP60, suggesting that the atoms of LP60 undergo faster diffusive motions than those of LP27. This study thus characterizes the dynamics of the two lysozyme polymorphs and reveals that the molecular dynamics of LP60 is enhanced compared with that of LP27. The higher molecular flexibility of the polymorph would permit to adjust its conformation more quickly than its counterpart, facilitating monomer binding.

2022 ◽  
Vol 23 (2) ◽  
pp. 783
Diana Fedunova ◽  
Andrea Antosova ◽  
Jozef Marek ◽  
Vladimir Vanik ◽  
Erna Demjen ◽  

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.

2022 ◽  
Benedikt Kolbrink ◽  
Theresa Riebeling ◽  
Nikolas K. Teiwes ◽  
Claudia Steinem ◽  
Hubert Kalbacher ◽  

Murine cytomegalovirus protein M45 contains a RIP homotypic interaction motif (RHIM) that is sufficient to confer protection of infected cells against necroptotic cell death. Mechanistically, the N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils, and interacts with the endogenous RHIM domains of receptor-interacting protein kinases (RIPK) 1, RIPK3, Z-DNA binding protein 1, and TIR domain-containing adaptor-inducing interferon-β. Remarkably, all four mammalian proteins harbouring such a RHIM domain are key components of inflammatory signalling and regulated cell death processes. Immunogenic cell death by regulated necrosis causes extensive tissue damage in a wide range of diseases, including ischemia reperfusion injury, myocardial infarction, sepsis, stroke and organ transplantation. To harness the cell death suppression properties of M45 protein in a therapeutically usable manner, we developed a synthetic peptide encompassing only the RHIM domain of M45. To trigger delivery of RHIM into target cells, we fused the transactivator protein transduction domain of human immunodeficiency virus 1 to the N-terminus of the peptide. The fused peptide could efficiently penetrate eukaryotic cells, but unexpectedly it killed all tested cancer cell lines and primary cells irrespective of species without further stimulus through a necrosis-like cell death. Typical inhibitors of different forms of regulated cell death cannot impede this process, which appears to involve a direct disruption of biomembranes. Nevertheless, our finding has potential clinical relevance; reliable induction of a necrotic form of cell death distinct from all known forms of regulated cell death may offer a novel therapeutic approach to combat resistant tumour cells.

2022 ◽  
Vol 13 (1) ◽  
Thomas Heerde ◽  
Matthies Rennegarbe ◽  
Alexander Biedermann ◽  
Dilan Savran ◽  
Peter B. Pfeiffer ◽  

AbstractSeveral studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 418
Sara Linse

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein “as is” with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.

2022 ◽  
Robert Bücker ◽  
Carolin Seuring ◽  
Cornelia Cazey ◽  
Katharina Veith ◽  
Maria García-Alai ◽  

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs) across kingdoms of life. Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-β fibrils of mated β-sheets at atomic resolution. Uperin 3.5 displayed substantial polymorphism with a protofilament of two mated β-sheets. The determined structure was a polymorph showing a 3-blade symmetrical propeller of nine peptides per fibril layer including tight β-sheet interfaces. This cross-β cryo-EM structure complements the cross-α fibril conformation previously determined by a crystal structure, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked β-sheets allowing a rounded compactness of the fibril. The kinked β-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. The amyloidal properties of antimicrobial peptides shed light on a mechanism of regulation of animicrobial activity involving self-assembly and fibril morphological variations. Moreover, the known endurance of amyloid structures can provide a template for the design of sturdy antimicrobials.

2022 ◽  
Gunasekhar Burra ◽  
Mahmoud Bukar Maina ◽  
Louise C. Serpell ◽  
Ashwani Thakur

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size ~7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

Natalya Ryabova ◽  
Liliia Fakhranurova ◽  
Vitaly Balobanov ◽  
Victor Marchenkov ◽  
Anatoly Glukhov ◽  

И.Е. Стрелкова

Амилоидоз – это группа заболеваний, характеризующихся накоплением в интерстиции различных органов и тканей белка специфической фибриллярной структуры. Понятие «амилоидоз» объединяет более 30 различных по своей патофизиологии состояний, в основе каждого из которых лежит нарушение синтеза 30 различных белков-предшественников. Однако 95% амилоидных кардиомиопатий связаны всего с двумя белками: белком, образованным из легких цепей иммуноглобулинов, и белком транстиретином. Определение белка-предшественника является краеугольным камнем ведения пациента с амилоидной кардиомиопатией. Транстиретин – это белок-переносчик тироксина, ретинола и других веществ, выполняющий жизненно важные функции. По наследственным или возрастным причинам происходит нарушение синтеза транстиретина в печени, и образующиеся мономеры, попадая в кровь, образуют токсичные промежуточные продукты и амилоидные фибриллы. Амилоидоз сердца (или амилоидная кардиомиопатия) до недавнего времени считался редким заболеванием. В недалеком прошлом возможности терапии амилоидоза сердца ограничивались назначением диуретиков, антагонистов минералокортикоидных рецепторов и антикоагулянтов, так как другие средства не переносятся пациентами или переносятся в минимальных дозах. С появлением в России первого средства специфического лечения транстиретиновой амилоидной кардиомиопатии резко возросла необходимость в повышении настороженности в отношении транстиретинового амилоидоза среди врачей-терапевтов и кардиологов и во внедрении современных алгоритмов диагностики данного заболевания. Своевременное выявление транстиретинового амилоидоза и грамотная дифференциальная диагностика от других видов амилоидной кардиомиопатии могут сыграть решающую роль в прогнозе заболевания. Препарат тафамидис доказанно снижает частоту госпитализаций и летальность у пациентов с транстиретиновым амилоидозом. Amyloidosis is a group of diseases characterized by accumulation of a protein of a specific fibrillar structure in the interstitium of various organs and tissues. The concept of ≪amyloidosis≫ unites more than 30 different pathophysiological conditions, each of which is based on abnormal synthesis of 30 different precursor proteins. However, 95% of amyloid cardiomyopathies are associated with just two proteins: a protein derived from light chains of immunoglobulins and a protein called transthyretin. Determination of the precursor protein is a cornerstone of management of patients with amyloid cardiomyopathy. Transthyretin is a carrier protein of thyroxine, retinol and other substances, that performs vital functions. For hereditary or age-related reasons, TTR misfolding occurs in the liver. The resulting monomers, entering blood, form toxic intermediate products and amyloid fibrils. Cardiac amyloidosis (or amyloid cardiomyopathy) used to be considered a rare disease. In the recent past, possibilities of therapy for cardiac amyloidosis were limited by prescription of diuretics, mineralocorticoid receptor antagonists and anticoagulants, since other drugs are not tolerated well by patients or are tolerated in minimal doses. Advent of the first drug specific for treatment of transthyretin amyloid cardiomyopathy in Russia increased a need of awareness of ATTR-CM among general practitioners and cardiologists, and introduction of modern diagnostic algorithms for this disease. Timely detection and competent differential diagnosis of ATTR-CM from other types of amyloid cardiomyopathy can play a decisive role in the prognosis of this disease. Tafamidis is a treatment that was shown to reduce mortality and CV-related hospitalization in ATTR-CM patients.

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