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

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.

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.

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.