Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies—amyloidoses—are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented.

Structural development of amyloid precursors in insulin B chain and the inhibition effect by fibrinogen

Amyloid fibrils are abnormal protein aggregates that relate to a large number of amyloidoses and neurodegenerative diseases. The oligomeric precursors, or prefibrillar intermediates, which emerge prior to the amyloid fibril formation, have been known to play a crucial role for the formation. Therefore, it is essential to elucidate the mechanisms of the structural development of the prefibrillar intermediates and ways to prevent its fibril formation. An insulin-derived peptide, insulin B chain, has been known for its stable accumulation of the prefibrillar intermediates. In this study, structural development of B chain prefibrillar intermediates was monitored by transmission electron microscopy and small-angle X-ray scattering combined with size exclusion chromatography and solid-state NMR spectroscopy to elucidate the stability and secondary structure. We further tracked its inhibition process by fibrinogen (Fg), which has been known to effectively prevent the amyloid fibril formation of B chain. We demonstrated that prefibrillar intermediates are wavy structures with low β-sheet content, growing in a multistep manner toward the nucleation for the amyloid fibril formation. In the presence of Fg, the formation of the prefibrillar intermediates slowed down by forming specific complexes. These observations suggest that the prefibrillar intermediates serve as reaction fields for the nucleation and its propagation for the amyloid fibril formation, whereas the inhibition of prefibrillar intermediate elongation by Fg is the significant factor to suppress the fibril formation. We propose that the obtained molecular picture could be a general inhibition mechanism of the amyloid fibril formation by the inhibitors.

Proteins in Wonderland: The Magical World of Pressure

Admitting the “Native”, “Unfolded” and “Fibril” states as the three basic generic states of proteins in nature, each of which is characterized with its partial molar volume, here we predict that the interconversion among these generic states N, U, F may be performed simply by making a temporal excursion into the so called “the high-pressure regime”, created artificially by putting the system under sufficiently high hydrostatic pressure, where we convert N to U and F to U, and then back to “the low-pressure regime” (the “Anfinsen regime”), where we convert U back to N (U→N). Provided that the solution conditions (temperature, pH, etc.) remain largely the same, the idea provides a general method for choosing N, U, or F of a protein, to a great extent at will, assisted by the proper use of the external perturbation pressure. A successful experiment is demonstrated for the case of hen lysozyme, for which the amyloid fibril state F prepared at 1 bar is turned almost fully back into its original native state N at 1 bar by going through the “the high-pressure regime”. The outstanding simplicity and effectiveness of pressure in controlling the conformational state of a protein are expected to have a wide variety of applications both in basic and applied bioscience in the future.

Amyloidogenesis of SARS-CoV-2 Spike Protein

SARS-CoV-2 infection is associated with a surprising number of morbidities. Uncanny similarities with amyloid-disease associated blood coagulation and fibrinolytic disturbances together with neurologic and cardiac problems led us to investigate the amyloidogenicity of the SARS-CoV-2 Spike protein (S-protein). Amyloid fibril assays of peptide library mixtures and theoretical predictions identified seven amyloidogenic sequences within the S-protein. All seven peptides in isolation formed aggregates during incubation at 37°C. Three 20-amino acid long synthetic Spike peptides (sequence 191-210, 599-618, 1165-1184) fulfilled three amyloid fibril criteria: nucleation dependent polymerization kinetics by ThT, Congo red positivity and ultrastructural fibrillar morphology. Full-length folded S-protein did not form amyloid fibrils, but amyloid-like fibrils with evident branching were formed during 24 hours of S-protein co-incubation with the protease neutrophil elastase (NE) in vitro. NE efficiently cleaved S-protein rendering exposure of amyloidogenic segments and accumulation of the peptide 193-202, part of the most amyloidogenic synthetic Spike peptide. NE is overexpressed at inflamed sites of viral infection and at vaccine injection sites. Our data propose a molecular mechanism for amyloidogenesis of SARS-CoV-2 S-protein in humans facilitated by endoproteolysis. The potential implications of S-protein amyloidogenesis in COVID-19 disease associated pathogenesis and consequences following S-protein based vaccines should be addressed in understanding the disease, long COVID-19, and vaccine side effects.

Acceleration of amyloid fibril formation by multichannel sonochemical reactor

Formation of amyloid fibrils of various amyloidogenic proteins is dramatically enhanced by ultrasound irradiation. For applying this phenomenon to the study of protein aggregation science and diagnosis of neurodegenerative diseases, a multichannel ultrasound irradiation system with individually adjustable ultrasound-irradiation conditions is necessary. Here, we develop a sonochemical reaction system, where an ultrasonic transducer is placed in each well of a 96-well microplate to perform ultrasonic irradiation of sample solutions under various conditions with high reproducibility, and applied it for studying amyloid-fibril formation of amyloid $\beta$, $\alpha$-synuclein, $\beta$2-microglobulin, and lysozyme. The results clearly show that our instrument is superior to conventional shaking method in terms of degree of acceleration and reproducibility of fibril formation reaction. The acceleration degree is controllable by controlling the driving voltage applied to each transducer. We have thus succeeded in developing a useful tool for the study of amyloid fibril formation in various proteins.

325 Dealing with cardiac amyloidosis diagnosis: keep calm and use the magnifying glasses!

Methods and results Case report— male, 71 years old. Past medical history—arterial hypertension, dyslipidemia, tobacco abuse. COPD on nocturnal CPAP therapy. Rheumatic polymyalgia on steroid therapy. Previous unprovoked deep vein thrombosis on anticoagulation with rivaroxaban. Bilateral carpal tunnel surgeries 8 years ago. Spontaneous left biceps tendon rupture 4 year ago. IgA kappa monoclonal gammopathy of undetermined significance (MGUS). Mild interventricular septum (IVS) hypertrophy on echocardiography since 2018. In 2019 IVS was 18 mm with granular sparkling appearance. In February 2020 he was hospitalized for initial heart failure and COPD exacerbation. In 2021 he developed worsening dyspnoea. He underwent cardiological evaluation in a spoke hospital and a cardiac magnetic resonance (CMR) suggested infiltrative cardiomyopathy. Bone scintigraphy showed moderate cardiac uptake (Perugini Score 2). Following haematological evaluation, fat pad biopsy was performed, and amyloid was detected on Congo red staining. Classification of the amyloid fibril protein was not performed. Bone marrow biopsy, even though of suboptimal quality, was negative for amyloid and for plasma cellular infiltration. Bone marrow aspirate showed 11% of plasma cells and multiple myeloma was therefore hypothesized. Recent medical history—he was evaluated in our Cardiac Amyloid Outpatient Clinic in May 2021. He was symptomatic for dyspnoea (NYHA class III) and exercise intolerance, diffuse osteo-muscolar pain, and extremities paresthesia. His blood pressure was on the low side of normality with necessity of anti-hypertensive therapy downgrading. Signs and symptoms of hematological disease were not present. We required to analyse the fat pad specimen in order to perform amyloid fibril protein typing; with immunoelectron microscopy, transthyretin (TTR) was identified as the amyloid fibrils precursor (no light chains could be identified). We considered performing endomyocardial biopsy to exclude the coexistence of ATTR amyloidosis and light chains (AL) amyloidosis in the heart but, given the history, clinical picture, and fat pad biopsy results, we felt that cardiac ATTR was the most probable diagnosis and we decided to proceed with a close cardiological and haematological follow-up. TTR genetic testing is ongoing. Conclusions ATTR cardiac amyloidosis is an emerging cause of heart failure, especially with preserved ejection fraction, in the older population. However, these patients frequently present with dysproteinemias and bone marrow abnormalities, up to multiple myeloma, raising the issue of differential diagnosis between ATTR and AL amyloidosis. According to the latest European Consensus Document, in the presence of cardiac uptake at bone scintigraphy (Grades 1–3) and positive haematologic tests, histological confirmation (usually cardiac) is necessary to subtype amyloid infiltration. In our case, the patient had positive Congo Red-stained fat pad biopsy, but the typing of the amyloid deposition was not performed. After referral to a Center with a Cardiac Amyloid Outpatient Clinic with a specialized Pathology Unit, we could further proceed with diagnostic workup and identify the amyloid deposition as ATTR; of note, fat pad biopsy is positive in just 15–25% of ATTR amyloidosis. Moreover, close collaboration with Hematology was necessary to assess the risk of AL amyloidosis and to provide a close and targeted follow-up. Endomyocardial biopsy was not performed after consideration of the various elements suggestive for ATTR cardiac amyloidosis, but the patient will be evaluated periodically and closely to potentially reassess this decision.