Amyloidogenic and Associated Proteins in Systemic Amyloidosis Proteome of Adipose Tissue

Abstract

In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of ␣B-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration. Molecular & Cellular Proteomics 7:1570 -1583, 2008.

Role of Glycosaminoglycan Sulfation in the Formation of Immunoglobulin Light Chain Amyloid Oligomers and Fibrils

“…The present study indicates that the specific posttranslational modifications present within full-length LC dramatically influence the effects of HS on fibril formation. The importance of the presence of both the VL and the CL regions for aggregation and stability has been recently proposed (34,35) and suggests that there may be a difference in the binding of sulfated GAGs. The latter reports support the demonstrated differences in pI detected within LCs, specifically with the VL region tending to be more acidic (18).…”

Biochemical markers in early diagnosis and management of systemic amyloidoses

“…In the differential proteomics approaches [51,76,80], such as in the MudPIT-based method developed by investigators of the Pavia Amyloid Research and Treatment Center in collaboration with colleagues at CNR in Milan [51,66,76], identification of the deposited protein is instead achieved on the whole tissue, without prior selection of the amyloid-positive areas. Typing is obtained through comparison of the diseased sample protein profile with a corresponding reference profile from control tissue (obtained from non-affected individuals).…”

Evidence for a Functional Role of the Molecular Chaperone Clusterin in Amyloidotic Cardiomyopathy

“…The complexity of amyloid deposits in fat and other tissues has been demonstrated in proteomic studies. 1 Two serum proteins that can misfold, aggregate, and form amyloid deposits in the heart and other organs are transthyretin (TTR) and immunoglobulin light chain (LC). Familial TTR-associated amyloidosis (ATTR) is caused by point mutations in the TTR gene that give rise to destabilized mutant proteins.…”