12 research outputs found
Influence of the Carboxy Terminus of Serum Amyloid A on Protein Oligomerization, Misfolding, and Fibril Formation
The fibrillar deposition of serum amyloid A (SAA) has
been linked
to the disease amyloid A (AA) amyloidosis. We have used the SAA isoform,
SAA2.2, from the CE/J mouse strain, as a model system to explore the
inherent structural and biophysical properties of SAA. Despite its
nonpathogenic nature in vivo, SAA2.2 spontaneously forms fibrils in
vitro, suggesting that SAA proteins are inherently amyloidogenic.
However, whereas the importance of the amino terminus of SAA for fibril
formation has been well documented, the influence of the proline-rich
and presumably disordered carboxy terminus remains poorly understood.
To clarify the inherent role of the carboxy terminus in the oligomerization
and fibrillation of SAA, we truncated the proline-rich final 13 residues
of SAA2.2. We found that unlike full-length SAA2.2, the carboxy-terminal
truncated SAA2.2 (SAA2.2ÎC) did not oligomerize to a hexamer
or octamer, but formed a high molecular weight soluble aggregate.
Moreover, SAA2.2ÎC also exhibited a pronounced decrease in the
rate of fibril formation. Intriguingly, when equimolar amounts of
denatured SAA2.2 and SAA2.2ÎC were mixed and allowed to refold
together, the mixture formed an octamer and exhibited rapid fibrillation
kinetics, similar to those for full-length SAA2.2. These results suggest
that the carboxy terminus of SAA, which is highly conserved among
SAA sequences in all vertebrates, might play important structural
roles, including modulating the folding, oligomerization, misfolding,
and fibrillation of SAA
Characterization of hSAA1.1 and MetSAA1.1 by SDS-PAGE, SEC, far UV-CD, tryptophan fluorescence, and thermal denaturation studies.
<p>(A) SDS-PAGE gel (lanes: 1, protein ladder; 2, hSAA1.1; 3, MetSAA1.1 (B) SEC elution profiles of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line); (C) far UV-CD spectra of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line); (D) Thermal denaturation profiles of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line) (E) Tryptophan emission spectra of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line); (F) Tryptophan fluorescence-based thermal denaturation profiles of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line). The concentration of protein used in all the experiments was 20 ”M. All experiments were performed at 4°C.</p
Biophysical characterization of aggregates formed by MetSAA1.1 and hSAA1.1.
<p>AFM analysis of (A) MetSAA1.1, 3 h, 37°C; (B) MetSAA1.1, 72 h, 37°C; (C) hSAA1.1, 3 h, 37°C; (D) hSAA1.1, 200 h, 37°C; Immunoblot analysis of aggregates formed by MetSAA1.1 using (E) A11 antibody and (F) OC antibody; Immunoblot analysis of aggregates formed by hSAA1.1 using (G) A11 antibody and (H) OC antibody. All scale bars for AFM images represent 1 ”m.</p
Characterization of âseedingâ properties of MetSAA1.1 and hSAA1.1 by ThT fluorescence assay.
<p>(A) ThT fluorescence intensity profile for freshly refolded MetSAA1.1 only (black bars) and MetSAA1.1+ MetSAA1.1 âseedâ (gray bars); (B) ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ hSAA1.1 âseedâ (gray bars). The concentration of protein was 20 ”M. ThT fluorescence intensities were recorded by incubating the samples at 37°C.</p
Characterization of aggregation of MetSAA1.1 and hSAA1.1 by the ThT Fluorescence assay, Congo red binding assay, far UV CD, and solubility assay.
<p>(A) ThT fluorescence intensity profile for MetSAA1.1 (black bars) and hSAA1.1 (red bars); (B) Congo red absorbance spectra for Congo red only (blue solid line); Congo red plus MetSAA1.1 sample (red solid line); MetSAA1.1 difference spectra (red dash line); Congo red plus hSAA1.1 sample (black solid line); hSAA1.1 difference spectra (black dash line); (C) Far UV CD spectra of MetSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (D) Far UV CD spectra of hSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (E) solubility profile for MetSAA1.1 (black solid line) and hSAA1.1 (red solid line). The starting concentration of protein was 20 ”M. All assays were performed after the proteins were allowed to aggregate at 37°C.</p
Cartoon representing the proposed pathway for oligomerization and fibrillation of MetSAA1.1 and hSAA1.1.
<p>Figures are not drawn to scale.</p
Biomineralized Anisotropic Gold MicroplateâMacrophage Interactions Reveal Frustrated Phagocytosis-like Phenomenon: A Novel Paclitaxel Drug Delivery Vehicle
This study reports a facile biomineralization
route for gold microplates (GMPs) synthesis using bovine serum albumin
(BSA) as a reductant and stabilizing agent. Adding BSA to HAuCl<sub>4</sub> solution yields spontaneous versatile anisotropic and partially
hollow GMPs upon aging. We hypothesize that the instantaneous protein
denaturation at low pH enabled access to serine and threonine hydroxyl,
and sulfhydryl groups of BSA, which act as a reductant and stabilizer,
respectively. This reaction could be hastened by increasing the temperature
well beyond 65 °C. Transmission electron microscopy/X-ray diffraction
studies revealed highly crystalline and anisotropic structures (triangle,
pentagon, and rectangle). Atomic force microscopy/scanning electron
microscopy analyses demonstrated unique morphology of microplates
with a partially void core and BSA mineralized edge structure. RAW
264.7 mice peritoneal macrophageâmicroplate interaction studies
using live cell confocal imaging reveal that cells are capable of
selectively internalizing smaller GMPs. Large GMPs are preferentially
picked with sharp vertices but cannot be internalized and exhibit
frustrated phagocytosis-like phenomenon. We explored particle phagocytosis
as an actin mediated process that recruits phagosome-like acidic organelles,
shown by a lysosensor probe technique. The biocompatible GMPs exhibited
âŒ70% paclitaxel (PCL) loading and sustained release of PCL,
showing antitumor activity with the MCF-7 cell line, and could be
a novel drug carrier for breast cancer therapy