Calcium Binds to Transthyretin with Low Affinity

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop

C. elegans expressing human β2-microglobulin: a novel model for studying the relationship between the molecular assembly and the toxic phenotype.

Availability of living organisms to mimic key step of amyloidogenesis of human protein has become an indispensable tool for our translation approach aiming at filling the deep gap existing between the biophysical and biochemical data obtained in vitro and the pathological features observed in patients. Human β(2)-microglobulin (β(2)-m) causes systemic amyloidosis in haemodialysed patients. The structure, misfolding propensity, kinetics of fibrillogenesis and cytotoxicity of this protein, in vitro, have been studied more extensively than for any other globular protein. However, no suitable animal model for β(2)-m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β(2)-m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β(2)-m affects the larval growth of C. elegans and the severity of the damage correlates with the intrinsic propensity to self-aggregate that has been reported in previous in vitro studies. We have no evidence of the formation of amyloid deposits in the body-wall muscles of worms. However, we discovered a strict correlation between the pathological phenotype and the presence of oligomeric species recognized by the A11 antibody. The strains expressing human β(2)-m exhibit a locomotory defect quantified with the body bends assay. Here we show that tetracyclines can correct this abnormality confirming that these compounds are able to protect a living organism from the proteotoxicity of human β(2)-m

Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

C-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system, but their chaperone activities remain poorly understood. Here, we examined their effects on the amyloid fibril formation from Alzheimer’s amyloid β (Aβ) (1-40) and on that from D76N β2-microglobulin (β2-m) which is related to hereditary systemic amyloidosis. CRP and SAP dose-dependently and substoichiometrically inhibited both Aβ(1-40) and D76N β2-m fibril formation in a Ca2+-independent manner. CRP and SAP interacted with fresh and aggregated Aβ(1-40) and D76N β2-m on the fibril-forming pathway. Interestingly, in the presence of Ca2+, SAP first inhibited, then significantly accelerated D76N β2-m fibril formation. Electron microscopically, the surface of the D76N β2-m fibril was coated with pentameric SAP. These data suggest that SAP first exhibits anti-amyloidogenic activity possibly via A face, followed by pro-amyloidogenic activity via B face, proposing a model that the pro- and anti-amyloidogenic activities of SAP are not mutually exclusive, but reflect two sides of the same coin, i.e., the B and A faces, respectively. Finally, SAP inhibits the heat-induced amorphous aggregation of human glutathione S-transferase. A possible role of pentraxins to maintain extracellular proteostasis is discussed

Diagnostic amyloid proteomics: experience of the UK National Amyloidosis Centre

Systemic amyloidosis is a serious disease which is caused when normal circulating proteins misfold and aggregate extracellularly as insoluble fibrillary deposits throughout the body. This commonly results in cardiac, renal and neurological damage. The tissue target, progression and outcome of the disease depends on the type of protein forming the fibril deposit, and its correct identification is central to determining therapy. Proteomics is now used routinely in our centre to type amyloid; over the past 7 years we have examined over 2000 clinical samples. Proteomics results are linked directly to our patient database using a simple algorithm to automatically highlight the most likely amyloidogenic protein. Whilst the approach has proved very successful, we have encountered a number of challenges, including poor sample recovery, limited enzymatic digestion, the presence of multiple amyloidogenic proteins and the identification of pathogenic variants. Our proteomics procedures and approaches to resolving difficult issues are outlined

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

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

C. elegans expressing D76N β_{2}-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β_{2} -m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β_{2} -m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β_{2} -m expressing worms. We also demonstrated the specificity of the β_{2} -m variant in determining the pathological phenotype by rescuing the wild type phenotype when β_{2} -m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

Human wild-type and D76N β_{2}-microglobulin variants are significant proteotoxic and metabolic stressors for transgenic C. elegans

β2-microglobulin (β2-m) is a plasma protein derived from physiological shedding of the class I major histocompatibility complex (MHCI), causing human systemic amyloidosis either due to persistently high concentrations of the wild-type (WT) protein in hemodialyzed patients, or in presence of mutations, such as D76N β2-m, which favor protein deposition in the adulthood, despite normal plasma levels. Here we describe a new transgenic Caenorhabditis elegans (C. elegans) strain expressing human WT β2-m at high concentrations, mimicking the condition that underlies dialysis-related amyloidosis (DRA) and we compare it to a previously established strain expressing the highly amyloidogenic D76N β2-m at lower concentrations. Both strains exhibit behavioral defects, the severity of which correlates with β2-m levels rather than with the presence of mutations, being more pronounced in WT β2-m worms. β2-m expression also has a deep impact on the nematodes' proteomic and metabolic profiles. Most significantly affected processes include protein degradation and stress response, amino acids metabolism, and bioenergetics. Molecular alterations are more pronounced in worms expressing WT β2-m at high concentration compared to D76N β2-m worms. Altogether, these data show that β2-m is a proteotoxic protein in vivo also in its wild-type form, and that concentration plays a key role in modulating pathogenicity. Our transgenic nematodes recapitulate the distinctive features subtending DRA compared to hereditary β2-m amyloidosis (high levels of non-mutated β2-m vs. normal levels of variant β2-m) and provide important clues on the molecular bases of these human diseases

Clinical ApoA-IV amyloid is associated with fibrillogenic signal sequence

Apolipoprotein A-IV amyloidosis is an uncommon form of the disease normally resulting in renal and cardiac dysfunction. ApoA-IV amyloidosis was identified in sixteen patients attending the National Amyloidosis Centre and in eight clinical samples received for histology review. Unexpectedly, proteomics identified the presence of ApoA-IV signal sequence residues (p.18–43 to p.20–43) in 16/24 trypsin-digested amyloid deposits, but in only 1/266 non-ApoA-IV amyloid samples examined. These additional signal residues were also detected in the cardiac sample from the Swedish patient in which ApoA-IV amyloid was first described, and in plasma from a single cardiac ApoA-IV amyloidosis patient. The most common signal-containing peptide observed in ApoA-IV amyloid, p.20–43 and to far lesser extent the N-terminal peptide, p.21–43 were fibrillogenic in vitro at physiological pH generating Congo red positive fibrils. The addition of a single signal-derived alanine residue to the N-terminus has resulted in markedly increased fibrillogenesis. If this effect translates to the mature circulating protein in vivo, then the presence of signal may result in preferential deposition as amyloid, perhaps acting as seed for the main circulating native form of the protein; it may also influence other ApoA-IV associated pathologies

Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis

Cardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments