Hydrogen exchange mass spectrometry (HXMS) for studying human serum amyloid A fibril formation

Research project objectives/ Research hypothesis

Human serum amyloid A (hSAA) is a small apolipoprotein (12 kDa) produced by hepatocytes under regulation by interleukin (IL)- 1, IL- 6, and tumor necrosis factor. Many chronic inflammatory diseases such as rheumatic diseases, chronic infections (e.g. tuberculosis), and non-infectious diseases, such as Hodgkin’s disease, lead to elevation of SAA level and may result in deposition of normally soluble serum amyloid A in the form of insoluble fibrils, impairing tissue structure and function. A life-threatening complication of chronic inflammatory is a secondary type amyloidosis called amyloid A protein (AA) amyloidosis. It has been clearly established that SAA is the serum precursor of amyloid A protein. The N-terminal 76-amino acid fragment of SAA has been found as the main component of amyloid A deposits accumulating predominately in the kidneys, liver, and spleen. A number of diseases (e.g., Alzheimer’s disease, type II diabetes, and secondary amyloidosis) associated with the formation of amyloid deposits is known, and they are all serious social problem worldwide. The deposits are the cause of death of thousands individuals per annum in the world.

Since AA amyloidosis is a conformational disease, any change in the secondary structure of SAA may induce the assembly of protein molecules into aggregates and amyloid fibrils. Despite three decades of research, the mechanism by which amyloid fibrils are formed from the precursor protein is still unclear.

The aims of our project are to understand the mechanism of human serum amyloid A (SAA) aggregation, and to find compounds able to prevent this pathological process. The first part of the work will be focused on application of mass spectrometric analysis combined with the hydrogen/deuterium exchange for structural determination of amyloid fibrils derived from recombinant human serum amyloid A protein and its 76-amino acid N-terminal fragment. The main objective of this task is to probe differences in H/D exchange patterns between the monomer and oligomers, and reveal regions interacting with each other in the course of oligomerization. In the second part we will focus on search of short peptides and peptidomimetics able to diminish or prevent aggregation of the studied protein and its aggregation-prone fragments. In this task we are going to test the hypothesi s that short peptidic sequences derived from the parent aggregating protein are capable to suppress formation of fibrillar deposits. The concept of our project is based on the hypothesis that short synthetic peptides would interact with the analogous region in the full-length serum amyloid A molecule and block its assembly into oligomers and amyloid fibrils. To identify the appropriate binding region, we will synthesize a library of short peptides with overlapping sequences, spanning the N-terminal SAA primary structure. Producing native SAA monomers is complicated by their apparent toxicity for bacterial system. Also isolation of SAA from serum is complicated by i s low concentration and limited solubility. For these reasons this protein is very expensive. To test the tendency of short peptides for inhibition of amyloid fibril formation we will use synthetic peptides corresponding to the first amino acids (e.g. 1-12) from the sequence of SAA. The results of already performed studies, including ours, indicate that the N-terminal 10-15 amino acids of SAA may correspond to the amyloidogenic core of the protein. Peptidic inhibitors capable to arrest aggregation in short amyloidogenic fragments of SAA will be subsequently tested for their ability to inhibit aggregation of ful l-length and 1-76 fragment of SAA.

Research project methodology

Hydrogen/deuterium exchange followed by mass spectrometry (HXMS) has emerged as a new powerful and versatile technique to study proteins in solution. HXMS can be used to study the local and global changes in protein’s structure and dynamics due to a ligand binding, protein activation-inactivation by its modification, as well as protein-protein interactions. In addition, the technique can also be used to monitor protein folding or to determine the stability of proteins under various conditions. A combination of isotopic exchange with mass spectrometry will give insight into the SAA oligomers/aggregates structure. The HXMS studies of fibrils created by both the  full-length and 1-76 fragment of SAA will allow monitoring of subtle structural changes during fibril formation and will provide global information on the extent of the β-sheet network and protection against the isotopic exchange within the fibrils. Studies with the shortened and full-length protein would explain the role of the central and C-terminal region of SAA in AA amyloid formation. HXMS experiments allow to determine the number of backbone amide groups involved in the stable H-bonded structure. Utilizing HXMS combined with enzymatic digestion we will be able to determine which parts of aggregated SAA molecules are protected from the solvent. In this work we will also focus our attention on usage of short peptides derived from the sequence of the parent aggregating protein as an important research tool for investigation of the molecular recognition and self-assembly mechanisms that promote the formation of amyloid fibril deposits. It is also believed that utilizing a short peptides provide information about importance of aromatic interactions in amyloid formation process. Moreover, studies concerning the short peptides may be crucial for developing small molecular mass inhibitors of the amyloidogenic process.

Expected impact of the research project on the development of science, civilization and society

Diseases associated with the systemic amyloidosis are a serious social problem, both in Poland and abroad. There are currently no effective medical treatment of such diseases. This study may help to explain the causes and mechanism of the protein aggregation. The results of our studies could have

broad application in research concerning the aging-related diseases such as Alzheimer disease, type II diabetes and rheumatoid arthritis. We believe that results of the presented project open up new possibilities in designing compounds that are able to prevent formation of amyloid deposits. The new peptidic inhibitors will provide a useful tool for the diagnostic and therapeutic approach to amyloid diseases. Peptides with the proved capability of aggregation inhibition could be a starting point for the design of peptidomimetic molecules more suitable as potential drugs.