Abstract
Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1–3% older adults. Fragmented hTTR is the standard composition of amyloid deposits in SSA, but the protease(s) responsible for amyloidogenic fragments generation in vivo is(are) still elusive. Here, we show that subtilisin secreted from Bacillus subtilis, a gut microbiota commensal bacterium, translocates across a simulated intestinal epithelium and cleaves hTTR both in solution and human plasma, generating the amyloidogenic fragment hTTR(59–127), which is also found in SSA amyloids in vivo. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart.
Highlights
Aggregation of human wild-type transthyretin, a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1–3% older adults
Identification of subtilisin as a human wild-type transthyretin (hTTR)-cleaving protease. hTTR was first purified to homogeneity from human plasma by the phenol precipitation method[29], followed by ion-exchange chromatography and size-exclusion chromatography (Supplementary Fig. 1), allowing to obtain in good yields (>35%) highly pure (>98%) hTTR preparations, as obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDSPAGE), where hTTR predominantly migrates as a monomer at ~13 kDa while minute amounts of hTTR dimer are still present in the denaturing sample loading buffer of the SDS-PAGE (Supplementary Fig. 1C), as already reported[30]
Circular dichroism and fluorescence spectra, along with analytical size-exclusion chromatography and dynamic light scattering (DLS) measurements indicate that our purified hTTR has conformational properties identical to those of other plasma hTTR preparations[32] and that it exists as a monodispersed tetramer, with an apparent molecular weight of 57 ± 3 kDa and a hydrodynamic radius (Rh) of 3.8 ± 1.0 nm, very close to that determined earlier[33] (Supplementary Fig. 2 and Table 1)
Summary
Aggregation of human wild-type transthyretin (hTTR), a homo-tetrameric plasma protein, leads to acquired senile systemic amyloidosis (SSA), recently recognised as a major cause of cardiomyopathies in 1–3% older adults. To the best of our knowledge, these findings highlight a novel pathogenic mechanism for SSA whereby increased permeability of the gut mucosa, as often occurs in elderly people, allows subtilisin (and perhaps other yet unidentified bacterial proteases) to reach the bloodstream and trigger generation of hTTR fragments, acting as seeding nuclei for preferential amyloid fibrils deposition in the heart. At variance with ATTR, SSA involves wild type hTTR, more often affects elderly people (usually >75 years), it is generally associated to cardiomyopathic complications, and leads to progressive heart failure[10,11]. HTTR amyloid fibrils were found in 25% of post-mortem hearts from patients >80 years of age[11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
