Amyloidogenic Domain Research Articles

Responses to Amyloids of Microbial and Host Origin Are Mediated through Toll-like Receptor 2

Curli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like beta-amyloid 1-42, which is associated with brain inflammation and Alzheimer's disease, curli fibrils have been implicated in the induction of host inflammatory responses. However, the underlying mechanisms of amyloid-induced inflammation are not fully understood. In a mouse sepsis model, we show that curli fibrils contributed to Nos2 expression, a hallmark of inflammation, by stimulating Toll-like receptor (TLR) 2. The TLR2 agonist activity was reduced by an amyloidogenicity-lowering amino acid substitution (N122A) in CsgA. Amyloid-forming synthetic peptides corresponding to beta-amyloid 1-42 or CsgA 111-151 stimulated Nos2 production in macrophages and microglia cells through a TLR2-dependent mechanism. This activity was abrogated when an N122A substitution was introduced into the synthetic CsgA peptide. The induction of TLR2-mediated responses by bacterial and eukaryotic amyloids may explain the inflammation associated with amyloids and the resulting pathologies.

The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization

Curli are functional amyloid fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In E. coli, the polymerization of the major curli fiber subunit protein CsgA into an amyloid fiber depends on the minor curli subunit protein, CsgB. The outer membrane-localized CsgB protein shares approximately 30% sequence identity with the amyloid-forming protein CsgA, suggesting that CsgB might also have amyloidogenic properties. Here, we characterized the biochemical properties of CsgB and the molecular basis for CsgB-mediated nucleation of CsgA. Deletion analysis revealed that a CsgB molecule missing 19 amino acids from its C terminus (CsgB(trunc)) was not outer membrane-associated, but secreted away from the cell. CsgB(trunc) was overexpressed and purified from the extracellular milieu of cells as an SDS-soluble, nonaggregated protein. Soluble CsgB(trunc) assembled into fibers that bound to the amyloid-specific dyes Congo red and thioflavin-T. CsgB(trunc) fibers were able to seed soluble CsgA polymerization in vitro. CsgB(trunc) displayed modest nucleator activity in vivo, as demonstrated by its ability to convert extracellular CsgA into an amyloid fiber. Unlike WT CsgB, CsgB(trunc) was only able to act as a nucleator when cells were genetically manipulated to secrete higher concentrations of CsgA. This work represents a unique demonstration of functional amyloid nucleation and it suggests an elegant model for how E. coli guides efficient amyloid fiber formation on the cell surface.

Recombinant amyloidogenic domain of ApoA-I: Analysis of its fibrillogenic potential

A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1–93 fragment. Similarly to the polypeptide isolated ex vivo, a pH switch from 7 to 4 induces a fast and reversible conformational transition to a helical state and leads to the identification of a key intermediate in the fibrillogenesis process. Limited proteolysis experiments suggested that the C-terminal region is involved in helix formation. The recombinant polypeptide generates fibrils at pH 4 on a time scale comparable with that of the native fragment. These findings open the way to studies on structural, thermodynamic, and kinetic aspects of ApoA-I fibrillogenesis.

Destabilization of Human IAPP Amyloid Fibrils by Proline Mutations Outside of the Putative Amyloidogenic Domain: Is There a Critical Amyloidogenic Domain in Human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.

A model for amyloid fibril formation in immunoglobulin light chains based on comparison of amyloidogenic and benign proteins and specific antibody binding

In an attempt to understand the mechanism of amyloid fibril formation in light chain amyloidosis, the properties of amyloidogenic (SMA) and benign (LEN) immunoglobulin light chain variable domains (VL) were compared. The conformations of LEN and SMA were measured using secondary and tertiary structural probes over the pH range from 2 and 8. At all pH values, LEN was more stable than SMA. The CD spectra of LEN at pH 2 were comparable to those of SMA atpH 7.5, indicating that the low pH conformation of LEN closely resembles that of SMA at physiological pH. At low pH, a relatively unfolded intermediate conformation is populated for SMA and rapidly leads to amyloid fibrils. The luck of such an intermediate with LEN, is attributed to sequence differences and accounts for the lack of LEN fibrils in the absence of agitation. A κIV-specific monoclonal antibody that recognizes the N-terminal of SMA caused unraveling of the fibrils to the protofilaments and was observed to bind to one end of SMA protofilaments by atomic force microscopy. The antibody result indicates that each protofilament is asymmetric with different ends. A model for the formation of fibrils by SMA is proposed.

Identification of a novel human islet amyloid polypeptide β-sheet domain and factors influencing fibrillogenesis

Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent β-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to β-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state.

Intracellular production of beta A4 amyloid of Alzheimer's disease: modulation by phosphoramidon and lack of coupling to the secretion of the amyloid precursor protein.

The amyloid precursor protein (APP) undergoes abnormal metabolism in Alzheimer's disease, resulting in the accumulation of beta A4 amyloid in the brain. Normal APP metabolism includes the release of a truncated form (sAPP) which has been cleaved at the alpha-secretase site within the beta A4 amyloidogenic domain. However, intact forms of beta A4 protein may also be generated by the beta- and gamma-secretases. Soluble forms of beta A4 have been detected in various cell lines and in cerebrospinal fluid. Previous studies of protein kinase C activation have suggested a reciprocal relationship between sAPP secretion and beta A4 production and release. We find that phorbol ester activation of protein kinase C in untransfected SH-SY5Y neuroblastoma cells increases the release of sAPP without affecting beta A4 secretion. We provide further evidence for intracellular beta A4 production. Treatment of SY5Y cells with the protease inhibitor phosphoramidon results in a 2-fold increase in beta A4 secretion and an increase in the amount of beta A4 recovered from cell lysates, yet it does not affect sAPP secretion. The protease inhibitors thiorphan and N-[(RS)-2-carboxy-3-phenylpropanoyl]-L-leucine had no effect on beta A4 or sAPP secretion. The lysosomotropic agents chloroquine and NH4Cl decreased beta A4 secretion, providing additional evidence for the involvement of intracellular acidic compartments in the production of beta A4. Our results therefore demonstrate a double dissociation between the secretion of sAPP and beta A4 in the SH-SY5Y cell line. The effect of phosphoramidon supports previous studies which show that metalloproteases are involved in the biogenesis of beta A4.