Plasminogen activation triggers transthyretin amyloidogenesis in vitro

Abstract

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation.

Carpal tunnel syndrome in cardiac amyloidosis: implications for early diagnosis and prognostic role across the spectrum of aetiologies

“…While the epidemiological association between CTS and CA is well established, the underlying mechanisms remain unclear. The reason for these two tissues being exclusively involved, as well as the observed temporal delay between the two remain unexplained, but it has been suggested that a repeated mechanical stimulus on tendons and myocardium could favour fibrillogenesis and/or tissue infiltration . Our retrospective study was not designed to explore the mechanistic relationship between CTS and CA and did not include a histological analysis of the tenosinovial tissue of patients who underwent CTS surgery.…”

“…The reason for these two tissues being exclusively involved, as well as the observed temporal delay between the two remain unexplained, but it has been suggested that a repeated mechanical stimulus on tendons and myocardium could favour fibrillogenesis and/or tissue infiltration. 28 Our retrospective study was not designed to explore the mechanistic relationship between CTS and CA and did not include a histological analysis of the tenosinovial tissue of patients who underwent CTS surgery. The presence of TTR amyloid in the tenosinovium of patients with CTS has been previously demonstrated (online supplementary Table S8), [10][11][12][13]29 but the co-existence of CA has been rarely investigated, and when it has, the prevalence was found to be particularly low.…”

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

“…The advantages of this method are that it is easy to apply in highly aggregated systems, it has a low-cost, and it has high reproducibility. As relevant examples, we can mention actin polymerization kinetics, hemoglobin fibrillation [ 74 ] and liquid phase separation of TDP-43 C-terminal domain [ 75 ], alpha-synuclein aggregation and its effects on Tau protein [ 76 ], TTR amyloid aggregation in the presence of plasminogen [ 77 ], and the β-amyloid peptide [ 78 ]. From a pharmaceutical point of view, it is employed to evaluate the effect of stirring on the aggregation processes of an antibody, in the presence or absence of polysorbate 80 by using turbidity measurements (at 350 and 550 nm) [ 79 ].…”

Targeting Amyloid Aggregation: An Overview of Strategies and Mechanisms

“…Bellotti’s group has recently discovered that disassembly of TTR and fibrillogenesis can be achieved under physiologic conditions by mechano-enzymatic mechanism consisting of a proteolytic cleavage permitted by the perturbation of the folded state in the presence of physiologic biomechanical forces [ 149 , 150 ]. Fibrils formed through this procedure display chemical and structural properties extremely similar to those extracted from natural deposits and highlight the role of a truncated form of TTR [ 149 ] in priming the amyloidogenesis.…”