Mechano-enzymatic cleavage of transthyretin in systemic amyloidosis: elucidation of mechanism and characterization of putative proteases

Lead Research Organisation: University College London
Department Name: Medicine

Abstract

Amyloidosis is a group of rare, serious and usually fatal diseases, responsible for the deaths of about one per thousand people who die in developed countries. It is caused by deposition of abnormal, insoluble, protein fibres, known as amyloid, in the tissues and organs of the body including the heart, kidneys, bowel, nerves and skin. Amyloid is derived by transformation of the body's own normal proteins into a mass of solid amyloid fibres; this leads to disruption and eventually organ failure. Transthyretin (TTR) is one of these "amyloidogenic" proteins and can result in a familial (hereditary) form of the disease. TTR amyloid also occurs in the elderly, making TTR amyloidosis a disease of ageing with profound clinical and socio-economic consequences. We have recently discovered that plasmin, an important enzyme in the normal blood clotting process is important in the development of TTR amyloid fibrils. Under conditions which are found in the heart, this enzyme breaks down TTR and leads directly to the formation of amyloid fibres. We will look at all the steps in the formation of TTR amyloid, particularly looking at the effects of both currently available and newly developed drugs. We will examine the relationship between the physiological (normal) pathway of blood clotting and the pathogenic (abnormal) pathway of TTR fibrillogenesis. Plasmin not only breaks down blood clots but may also activate the formation of amyloid by our newly discovered route. Our new understanding of how these two natural systems interact will allow us to evaluate the effect of anti-plasmin drugs on TTR amyloid formation. We believe that this work will improve our understanding of the causes of TTR amyloidosis, but will also be of major interest to the mechanism of other protein misfolding diseases.

Technical Summary

We have identified a new pathway for TTR amyloidogenesis which is primed by proteolytic cleavage followed by TTR dissociation and fibril formation. We have compelling evidence that plasmin, the key proteolytic enzyme of the physiological fibrinolytic pathway, is central to TTR amyloid formation. In WP1 we will determine the rate constants for the 5 central reactions involved in the transformation of native tetrameric TTR into amyloid fibrils: 1. Selective cleavage of native TTR at the peptide bond Lys48-Thr49 (k1). 2. Proteolytic degradation of truncated 49-127 TTR fragment (k2). 3. Proteolytic degradation of full-length 1-127 TTR protomer (k3). 4. Oligomers formation (k4). 5. Aggregation of full-length and/or truncated protomer into amyloid fibrils (k5). In WP2 we plan to establish the intensity of shear stress required for the activation of the mechano-enzymatic cleavage for the most common amyloidogenic variants and investigate the relationship between the shear stress required for each individual variant and their corresponding thermodynamic stability. In WP3 we will test the effect of drugs, including those currently in clinical use, on the model of fibrillogenesis designed in WP1. We will carry out a comparative analysis of the efficacy of monovalent and bivalent TTR ligands in inhibiting the fibril formation. We will examine how TTR ligands can affect the composition of amyloid fibrils and whether they may reduce the amount of truncated TTR species which is converted into fibrils. In WP4 we will characterize how the physiological activation of plasminogen to plasmin can affect cleavage and amyloidogenesis of TTR. We aim to understand how modulation of this enzyme by its activators and inhibitors can affect the kinetics of the fibrillogenic pathway, the final yield of amyloid fibrils and their composition.

Planned Impact

This research, studying the new mechano-enzymatic mechanism of transthyretin (TTR) amyloidosis identified in our laboratory, will have a number of impacts going beyond the academic sector into clinical treatment. Systemic amyloidosis, with extracellular deposition of amyloid fibrils in the viscera and connective tissues producing organ failure, causes about one per thousand of all deaths in developed countries. In humans both wild type TTR and several variants can cause various forms of systemic amyloidosis in which cardiac involvement, described in over half of the reported variants, can represent the most negative prognostic factor. Cardiac amyloidosis is a predominant clinical feature in senile systemic amyloidosis (SSA), and is caused by deposition of wild type TTR affecting around 20% of people over 75. The heart is also the main target of patients heterozygous for the mutation Val122Ile whose allele frequency is 3.9% of African American population. This amyloidosis represents an excellent example in which a circulating protein is targeted by innovative therapies designed to prevent misfolding and fibrillar conversion. This research will offer new insights into the development and progression of the disease, and, should lead to the development of new drugs. Of particular importance is the ability to re-evaluate current treatments in light of this new pathogenic mechanism. The low pH-mediated fibrillogenesis methods currently used in vitro for studying TTR amyloid conversion and for testing the effects of drugs are very far from being biocompatible. Novel therapies, such as tafamidis, were designed based on these non-physiological assays, and this probably explains their limited efficacy in clinical trials.
Our discovery of a method of fibrillogenesis implying a selective proteolytic cleavage which is fully compatible with the physiological environment opens a completely new approach to study the pathogenesis of the disease, and also for developing new effective drugs. Some TTR ligands, in particular the bivalent compounds, capable of completely stabilizing the folding state and reducing the susceptibility to cleavage should be reconsidered for therapeutic exploitation based on this new mechano-enzymatic mechanism.
We believe that our approach will lead to a paradigm shift in the understanding of TTR amyloidosis whilst at the same time, allowing us to re-examine some of the huge amount of work undertaken on TTR ligands designed purely to stabilize the tetramer against acid denaturation. As we move on from the identification of this new molecular mechanism of TTR amyloidosis into the development of new effective therapies, our work will result in a better understanding of the factors affecting the development of the disease, and will also offer hope to a growing patient cohort facing severe illness.
The discovery that plasmin may be the enzyme causing the cleavage and fibrillogenesis of TTR can deeply transform our knowledge of the pathogenesis and natural history of the disease, thus promoting new research on the genetic basis of the susceptibility to the disease as well on the role of the environment and lifestyle. In fact, plasmin is essential for maintaining a physiological equilibrium between clotting and fibrinolysis. The cleavage of TTR by plasmin may represent the dark side of the lifesaving complex process that, at the same time guarantees, throughout life, integrity and patency of the blood vessels and remodelling of the extracellular matrix.

Publications

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Mangione PP (2018) Plasminogen activation triggers transthyretin amyloidogenesis in vitro. in The Journal of biological chemistry

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Moura A (2020) Lysozyme amyloid: evidence for the W64R variant by proteomics in the absence of the wild type protein. in Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis

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Governing Council Of The Italian Society Of Biochemistry And Molecular Biology (2020) Basic and applied science at the time of COVID-19. in FEBS letters

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Canetti D (2020) Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre. in Clinical chemistry and laboratory medicine

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Cantarutti C (2022) Calcium Binds to Transthyretin with Low Affinity. in Biomolecules

 
Description ATTR stabiliser Licensing Project
Amount £1,500,000 (GBP)
Funding ID UTF 17-009 
Organisation University College London 
Department Technology Fund
Sector Academic/University
Country United Kingdom
Start 03/2018 
End 12/2021
 
Title Physiologically relevant model of TTR fibrillogenesis in vitro 
Description Identification of plasmin as one of the proteases which may be physiologically relevant in TTR proteolysis and amyloid formation (Mangione et al. J Biol Chem, 293:14192-99, 2018) allowed us to establish an even more biocompatible and physiologically relevant model of TTR fibrillogenesis in vitro. In fact, trypsin worked extremely well for the proof of concept of the mechano-enzymatic mechanism underlying TTR amyloidogenesis (Marcoux et al. EMBO Mol Med, 7:1337-49, 2015). However, it is unlikely to be involved in the pathogenesis of systemic TTR amyloidosis in patients since it is synthesized only by the exocrine pancreas and secreted exclusively into the small bowel lumen. After a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, plasmin was selected and its role for selective cleavage of human TTR under physiological conditions was confirmed in vitro. A comparative structural and thermodynamic analysis demonstrated that the mechano-enzymatic product obtained with plasmin was notably more similar to the ex vivo amyloid fibrils than the acidic-mediated in vitro aggregates (Raimondi et al. J Biol Chem 295:11379-87, 2020). 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Establishment of our model of fibrillogenesis, based on the combination of plasmin and biomechanical forces, will be the most suitable tool for therapeutically relevant studies of TTR amyloidosis. This important finding gave the basis for the generation of the first murine model of cardiac amyloidosis associated to the amyloidogenic Ser52Pro variant transthyretin. The animal model was developed in the transgenic Unit at the UCL Royal Free Campus in collaboration with Dr Paul Simons. 
 
Description Amyloidosis and drug development (CAAPP and NAC) 
Organisation University College London
Department Centre for Amyloidosis and Acute Phase Proteins
Country United Kingdom 
Sector Academic/University 
PI Contribution Bellotti has had a long-standing and successful collaboration with the Centre for Amyloidosis and Acute Phase Proteins. As a result, he was appointed as Professor of Medical Biochemistry at UCL in October 2011, and now has a team working within the Centre. Apart from his independent work on ß2-microglobulin, transthyretin, a-synuclein and Apolipoprotein CIII, he is also central to a number of the Centre's drug development programmes targeting amyloidosis and other diseases. These include a novel therapy based on antibody-mediated depletion of serum amyloid P component, the stabilisation of transthyretin to reduce protein deposition in vivo and a small molecule inhibitor of C-reactive protein to limit CRP-mediated tissue damage following myocardial infarction. The identification of the so-called mechano-enzymatic mechanism of transthyretin amyloidogenesis has provided novel drug discoveries targets and further insight into the mode of action of current drugs which stabilize the native state of transthyretin.
Collaborator Contribution The UCL Centre for Amyloidosis and Acute Phase Proteins comprises the Wolfson Drug Discovery Unit directed by Prof Sir Mark Pepys FRS FMedSci and the NHS National Amyloidosis Centre headed by Prof Philip Hawkins FMedSci. The clinical and non-clinical groups are closely linked, and allow both groups to share and build upon findings from laboratory-based and clinical (patient based) studies. The UCL transgenic mouse unit (headed by Dr Paul Simons) is central to a number of our projects including the production of transgenic mice expressing pathogenic protein variants such as human D76N ß2-microglobulin (created with the support of Bellotti's MRC Strategic Award) and S52P human transthyretin.
Impact 26 Publications (PMID: 34876572, 35237660, 32069225, 32067519, 31393717, 30740936, 30018138, 29016222, 28647518, 28429761, 28298647, 26790392, 26400472, 26646718, 26286619, 24474780, 24014031, 22693999, 21059958, 20962779, 19372378, 16642000, 9039909, 8915605, 26286619, 26400472)
Start Year 2006
 
Description Atomic force microscopy in amyloid (Annalisa Relini) 
Organisation University of Genoa
Department Department of Physics
Country Italy 
Sector Academic/University 
PI Contribution We have always tried to identify new techniques to better understand the mechanisms of amyloid formation. In this case, we initiated acollaboration with Annalisa Relini and colleagues, using atomic force microscopy to examine the structure of amyloid fibrils during their growth, from a range of precursor proteins which we have intensively studied over the past years; these include Apolipoprotein A-I, abeta peptide, transthyretin, a -synuclein, apolipoprotein C-III, wild type and D76N ß2-microglobulin.
Collaborator Contribution The application of atomic force microscopy for the analysis of amyloid fibrils from wild type and D76N ß2-microglobulin, Leu174Ser ApolipoproteinA-I, abeta peptide, a -synuclein, apolipoprotein C-III, all prepared by the Bellotti group.
Impact 16 Publications (PMID: 30018138, 27806283, 26921323, 26790392, 25505181, 24474780, 24014031, 22271711, 21296086, 21068391, 21067308, 19626482, 18056266, 17242436, 16601119, 15337168)
Start Year 2006
 
Description NMR analysis of alpha-synuclein, apolipoprotein C-III and transthyretin (John Christodoulou) 
Organisation University College London
Department Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Bellotti and his group are investigating the mechansim of aggregation of a pathogenic variant of alpha synuclein as part of a major project in collaboration within UCL. Collaboration with Professor John Christodoulou's group has been invaluable for the identification of structural features underlying the peculiar aggregation propensity of this variant. At the same time, our collaboration with Professor Christodoulou has been extended to another project related to a novel variant of apoliprotein C-III which is associated to a new type of hereditary amyloidosis. Following Bellotti's seminal discovery of a novel mechano-enzymatic mechanism underlying transthyretin (TTR) amyloidogenesis in vivo (See recent publication in EMBO Mol Med, 2015) a new project has started to characterize this novel molecular mechanism by NMR using the highly amyloidogenic recombinant Ser52Pro TTR. The project will be very challenging and ultimately NMR will be used to study the binding of TTR by ligands and their potential inhibitory effect on proteolysis and fibrillogenesis.
Collaborator Contribution Christodoulou and colleagues have carried out NMR study of alpha synuclein and apoliprotein-CIII isoforms. The new project related to TTR will also involved the full training of Bellotti's PhD student (supported by the recent M427 Rosetrees Trust award) to NMR spectroscopy, now currently post-doc funded by his MRC MR/R016984/1 grant. Meanwhile another expert in NMR has joined the team, Prof Alessandra Corazza (University of Udine, Italy) who has been visting Professor at UCL since 2016. She has been an invaluable addition to the team conducting a very challenging experimental part of the NMR study of TTR in complex with ligands as well as helping out with the training of the staff.
Impact The first manuscript related to the alpha-synuclein project has been already published (PMID 25505181). The second manuscript on the apolipoprotein C-III has been published on a high impact journal (PMID 26790392). A seminal paper on the long distance effect induced by bivalent ligands bound to TTR has been published on a high impact journal (PMID 31393717). The NMR based study will be beneficial to any further development on TTR binding potential drugs.
Start Year 2013