Comparative study of the stabilities of synthetic in vitro and natural ex vivo transthyretin amyloid fibrils

Abstract

Systemic amyloidosis caused by extracellular deposition of insoluble fibrils derived from the pathological aggregation of circulating proteins, such as transthyretin, is a severe and usually fatal condition. Elucidation of the molecular pathogenic mechanism of the disease and discovery of effective therapies still represents a challenging medical issue. The in vitro preparation of amyloid fibrils that exhibit structural and biochemical properties closely similar to those of natural fibrils is central to improving our understanding of the biophysical basis of amyloid formation in vivo and may offer an important tool for drug discovery. Here, we compared the morphology and thermodynamic stability of natural transthyretin fibrils with those of fibrils generated in vitro using either the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were −12.36 kcal mol-1, −8.10 kcal mol-1 and −10.61 kcal mol-1, respectively. The fibrils generated via plasmin cleavage were more stable than those prepared at low pH and were thermodynamically and morphologically similar to natural fibrils extracted from human amyloidotic tissue. Determination of thermodynamic stability is an important tool that is complementary to other methods for structural comparison between ex vivo fibrils and fibrils generated in vitro. Our finding that fibrils created via an in vitro amyloidogenic pathway are structurally similar to ex vivo human amyloid fibrils does not necessarily establish that the fibrillogenic pathway is the same for both, but it narrows the current knowledge gap between in vitro models and in vivo pathophysiology.

AA amyloid fibrils from diseased tissue are structurally different from in vitro formed SAA fibrils

“…3a ). These differences might be assumed to result in a higher thermodynamic stability of pathogenic fibrils, which would be in accord with a recent study demonstrating that ex vivo fibrils from transthyretin protein are more resistant to guanidine denaturation (and more thermodynamically stable) than the in vitro fibrils from this protein 29 .…”

“…7 ). In the case of the ex vivo fibrils from transthyretin it was found that even guanidine isothiocyanate had to be used to fully denature these assemblies 29 . We conclude that properties other than simple thermodynamic stability must be more relevant when rationalizing the pathological involvement of the observed ex vivo amyloid fibrils.…”

Transthyretin: From Structural Stability to Osteoarticular and Cardiovascular Diseases

“…In the familial type of the disease, TTR depositions are localized predominantly extracellularly in the brain and heart tissues, and SSA depositions associated with age are detected in many organs, mainly in the heart, lungs, and blood vessels [ 51 , 66 , 67 , 68 ]. Amyloid depositions in SSA (and in some FAP) patients are extensive and diffused, and contain tightly packed, short, and randomly oriented fibrils (amyloid A) [ 66 , 67 , 69 , 70 ]. Amyloid A contains truncated TTR devoid of the N -terminal fragment, starting at approximately amino acid 49 [ 66 , 67 , 69 , 70 , 71 ].…”

“…Amyloid depositions in SSA (and in some FAP) patients are extensive and diffused, and contain tightly packed, short, and randomly oriented fibrils (amyloid A) [ 66 , 67 , 69 , 70 ]. Amyloid A contains truncated TTR devoid of the N -terminal fragment, starting at approximately amino acid 49 [ 66 , 67 , 69 , 70 , 71 ]. Amyloid depositions of various morphologies, which were observed only in FAP patients, are small and contain long fibrils formed from mutated full-length TTR.…”

“…Recently, plasmin was shown to specifically cleave TTR, which results in the formation of TTR fibrils containing truncated TTR molecules (49–127) [ 190 ]. Plasmin-derived TTR fibrils resemble the fibrils isolated from TTR amyloid formed in vivo [ 70 ]. These observations indicate that physiological fibrinolysis in vivo is interconnected with the formation of TTR amyloid [ 190 ].…”