Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

<div><p>Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.</p></div

Extracellular Chaperones are overrepresented in DLT individuals—SDS-PAGE analysis of plasma proteins from four individuals after orthotic liver transplantation (OLT) and four individuals after domino transplantation (DLT) and Western blot to detect TTR, Fibrinogen, Alpha 1 anti-trypsin (ITI), Vitamin D binding protein (VBP), alpha 2 microglobolin and C-reactive protein (CRP).

<p>Molecular weight markers are represented on the left hand of the figure and included in the western blots as blue markers.</p

Proteome analysis of plasma from ATTR individuals.

<p>A– 2D-PAGE analysis of plasma proteins. Labeled spots show a statistically significant variation (p<0.05) and a minimal fold variation of 1.5. These spots were excised, tryptic digested and proteins identified by MS/MS analysis. Average normalized volumes and protein identifications are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125392#pone.0125392.t001" target="_blank">Table 1</a>. B–Principle component analysis (PCA) of the 2D results. Each data point in the PCA represents the global expression values for all spots with a significant ANOVA value (p<0.05). A separation between the control and the ATTR individuals is clearly observed. C- 2D image analysis of four protein spots and normalized volumes, shown as examples. D-Over expression of western blot analysis of plasma from four control and four FAP individuals to detect TTR. E—Western blot analysis of a 2DE of serum from four control and four FAP individuals to detect TTR with super imposition of spots identified as TTR in 2DE.</p

Plasma from ATTR individuals presents a higher proteolytic activity.

<p>A- Proteolytic activity of control and ATTR plasma measured by fluorescent protease assay kit. B–Sequence coverage obtained for three spots identified as albumin with decreased molecular weight.</p

Functional annotation enrichment analysis of the identified proteins using the Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7.

<p>Annotation terms are representative of a particular cluster. FDR – False discovery rate.</p

Detailed expression profiles for all the identified differentially expressed proteins, according to its functional categories: (A) cell metabolism; (B) unknown function; (C) Cell redox homeostasis; (D) protein folding and degradation; (E) translation.

<p>Grey bars represent fold change in protein expression in BTTR-wt versus the control while black bars represent fold change in protein expression in BTTR-L55P versus the control. The vertical axis indicates the identified protein while the horizontal axis represents the fold variation in protein expression. Additional information for each protein, including full name, can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050123#pone-0050123-t001" target="_blank">Table 1</a>. For proteins identified in different spots (with slightly different fold variations) the average is represented in the graph.</p

Differentially expressed proteins identified by MALDI-TOF-TOF MSMS.

<p>All the listed proteins showed a statistical difference of spot volume ratio between the control/BTTR-wt and control/BTTR-L55P with an ANOVA p<0.05.</p>a)<p>Gene code as in the yeast genome database (<a href="http://www.yeastgenome.org" target="_blank">www.yeastgenome.org</a>).</p>b)<p>CM – carbohydrate metabolism; AM – amino acid metabolism; NM – nucleotide metabolism; EM – energy metabolism; LM – lipid metabolism; U – unknown; CRH – cell redox homeostasis; T – translation; TP – transport; PFD – protein folding and degradation.</p>c)<p>Accession code of the uniprot database (<a href="http://www.uniprot.org" target="_blank">www.uniprot.org</a>).</p>d)<p>Spot number on the master gel (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050123#pone-0050123-g001" target="_blank">fig. 1A</a>).</p>e)<p>Summary of the protein identification results. The protein and the peptide score as given by the GPS Explorer software (Applied Biosystems). The number of peptides with MSMS data is also given.</p

TTR misfolding and protein quality control mechanisms.

<p>Differentially expressed proteins are highlighted, those induced framed in purple and those repressed are framed in green. Protein full names are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050123#pone-0050123-t001" target="_blank">Table 1</a>.</p