Serpin Acceleration of Amyloid Fibril Formation: A Role for Accessory Proteins

Abstract

Protein aggregation underlies an increasing number of human diseases. Recent experiments have shown that the aggregation reaction is exquisitely specific involving particular interactions between non-native proteins. However, aggregation of certain proteins, for example β-amyloid, in vivo leads to the recruitment of other proteins into the aggregate. Antichymotrypsin, a non-fibril forming protein, is always observed to be associated with β-amyloid plaques in Alzheimer's sufferers. The role of antichymotrypsin is controversial with studies showing it can either accelerate or inhibit the aggregation reaction. To investigate the role of antichymotrypsin in fibrillogenesis we have studied its interaction with apolipoprotein C-II, a well characterized model system for the study of fibrillogenesis. Our data demonstrate that sub-stoichiometric amounts of antichymotrypsin and its alternate structural forms can dramatically accelerate the aggregation of apolipoprotein C-II, whereas the presence of α₁-antitrypsin, a structural homologue of antichymotrypsin, cannot. Sedimentation velocity experiments show more apolipoprotein C-II fibrils were formed in the presence of antichymotrypsin. Using pull-down assays and immuno-gold labeling we demonstrate an interaction between antichymotrypsin and apolipoprotein C-II fibrils that specifically occurs during fibrillogenesis. Taken together these data demonstrate an interaction between antichymotrypsin and apolipoprotein C-II that accelerates fibrillogenesis and indicates a specific role for accessory proteins in protein aggregation.

Introduction

Protein misfolding and self-association into insoluble aggregates forms the basis of numerous diseases such as, Alzheimer's disease (AD), Parkinson's disease, and Creutzfeldt-Jakob disease.1., 2., 3. These diseases and others occur when a specific protein aggregates, resulting in either intra or extra-cellular deposition. Aggregating proteins and peptides often form elongated rope-like structures commonly termed fibrils. Fibril formation is a complex multi-step mechanism, involving the formation of intermediate species and small protofibrils, followed by the rapid elongation of the fibril through the recruitment of proteins.⁴ These can then form higher order structures by the ordered incorporation of multiple fibrils to produce rope-like structures leading to the formation of insoluble tangles.

Mature amyloid plaques were originally thought to be the causative agents in AD, however, the correlation between plaque load and disease manifestation does not support the theory that mature plaques are solely responsible for AD.5., 6. Most recently the focus has been turned to the impact of the early oligomers and protofibrillar species. Recent evidence suggests that the neurotoxicity associated with aggregation is linked predominantly to small aggregates such as dimers and trimers of the β-amyloid peptide as well as protofibrils.7., 8.

There is an inherent specificity to the aggregation process, such that aggregates consist of only one fibrillar protein. This was clearly shown when two distinct, aggregation prone proteins were co-expressed and allowed to aggregate in the cell, resulting in the formation of discrete aggregates.⁹ There are, however, a number of other non-aggregated proteins often found localized to disease related aggregates. These non-fibrillar components include the serine protease inhibitor (serpin) antichymotrypsin (ACT),10., 11., 12. serum amyloid P component,¹³ and apolipoprotein E.¹⁴ Previous studies have indicated that ACT co-localizes with all β-amyloid plaques and also has elevated expression levels in the brain of AD sufferers.10., 11., 12., 15. These studies all suggest that ACT plays an important, but poorly understood role in amyloid formation. The role of ACT in fibril formation is contentious, with some studies indicating that ACT can form a complex with amyloid β-peptide accelerating amyloid fibril formation and the resultant deposition in brain tissues.16., 17., 18. Other studies suggest that ACT is able to inhibit fibril formation or disaggregate mature fibrils.19., 20. The molecular mechanisms involved in these interactions are as yet undetermined.

ACT is an acute phase plasma protein and a member of the serpin superfamily consisting of three β-sheets, nine α-helices and a flexible reactive centre loop (RCL) (Figure 1(a)).²¹ Flexibility in the RCL of ACT is essential to its inhibitory properties, as it is this solvent exposed loop that interacts with target proteases.²² RCL cleavage by a protease drives insertion of the RCL into the central A β-sheet of the molecule resulting in the cleaved conformation (Figure 1(b)). The mobility of the RCL is also associated with a tendency to insert into the A β-sheet without cleavage by a protease, forming the inactive latent conformation (Figure 1(c)). ACT is also able to aggregate, forming long-chain polymers (Figure 1(d)).23., 24. In vivo these polymers are formed by changes in environmental factors, or the presence of mutations, which destabilize the native state. The polymeric form of ACT has been found in the sera of patients with probable late onset AD²⁵ increasing the need to investigate alternate conformations. The role of each of the alternate conformations in fibrillogenesis is unclear.

The current literature presents a contrasting picture about the role of ACT in fibrillogenesis. Here we perform a detailed study of the molecular mechanisms and specificity involved in the interaction between ACT and the fibril forming protein apolipoprotein C-II (apoC-II). ApoC-II fibrils have been implicated in the activation of macrophages during the development of atherosclerosis.²⁶ ApoC-II forms classical thioflavin T and Congo Red reactive fibrils²⁷ at room temperature, providing a consistent and reproducible fibrillogenic system to investigate the properties of fibril formation. Our research investigates the role of ACT and its alternate conformers in apoC-II fibril formation and demonstrates a specific and co-operative interaction between these proteins during the formation of fibrils.