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 either using the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were -12.36, -8.10, 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 of 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.
Highlights
Understanding the mechanisms of diseases is vital to efficiently tackling unmet medical needs, and this is especially true for diseases of high complexity, such as systemic amyloidosis
Most of our knowledge of the pathogenesis of TTR amyloidosis derives from the experimental model of in vitro fibrillogenesis at low pH in which protein aggregation is primed by tetramer disassembly [3]; this method has been the key tool for identifying drugs, such as tafamidis, currently used to treat the disease [4,5,6,7]
Based on the observation that truncated forms of TTR are present in natural fibrils [8], we have established a new, more physiological, system of fibrillogenesis in which biomechanical forces combined with specific proteolytic enzymes play a central role [9,10,11]
Summary
The aggregation of recombinant V122I variant TTR was carried out in vitro using the mechano-enzymatic mechanism we have described recently [11] and using the widely used low-pH procedure established by Colon and Kelly [3]. MS analysis was carried out to identify the main species in the mechano-enzymatic and ex vivo fibrils, in contrast to the material treated at low pH, which is homogenously composed of full-length recombinant V122I TTR only. Data were fitted with the linear polymerization model using Equation 4 as described under “Experimental procedures” [21, 22] (Fig. 2B) to yield the main thermodynamic parameters, including the midpoint denaturant concentration and the change in the Gibbs free energy of elongation in the absence of denaturant, DG0el. All the species identified in the natural fibrils contained both WT and V122I variant TTR, as demonstrated after further digestion of each band with trypsin or AspN proteases (data not shown)
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