Molecular Aspects of Transthyretin Amyloid Disease

This thesis was made to get a deeper understanding of how chaperones interact with unstable, aggregation prone, misfolded proteins involved in human disease. Over the last two decades, there has been much focus on misfolding diseases within the fields of biochemistry and molecular biotechnology research. It has become obvious that proteins that misfold (as a consequence of a mutation or outer factors), are the cause of many diseases. Molecular chaperones are proteins that have been defined as agents that help other proteins to fold correctly and to prevent aggregation. Their role in the misfolding disease process has been the subject for this thesis. Transthyretin (TTR) is a protein found in human plasma and in cerebrospinal fluid. It works as a transport protein, transporting thyroxin and holo-retinol binding protein. The structure of TTR consists of four identical subunits connected through hydrogen bonds and hydrophobic interactions. Over 100 point mutations in the TTR gene are associated with amyloidosis often involving peripheral neurodegeneration (familial amyloidotic polyneuropathy (FAP)). Amyloidosis represents a group of diseases leading to extra cellular deposition of fibrillar protein known as amyloid. We used human SH-SY5Y neuroblastoma cells as a model for neurodegeneration. Various conformers of TTR were incubated with the cells for different amounts of time. The experiments showed that early prefibrillar oligomers of TTR induced apoptosis when neuroblastoma cells were exposed to these species whereas mature fibrils were not cytotoxic. We also found increased expression of the molecular chaperone BiP in cells challenged with TTR oligomers. Point mutations destabilize TTR and result in monomers that are unstable and prone to aggregate. TTR D18G is naturally occurring and the most destabilized TTR mutant found to date. It leads to central nervous system (CNS) amyloidosis. The CNS phenotype is rare for TTR amyloid disease. Most proteins associated with amyloid disease are secreted proteins and secreted proteins must pass the quality control check within the endoplasmic reticulum (ER). BiP is a Hsp70 molecular chaperone situated in the ER. BiP is one of the most important components of the quality control system in the cell. We have used TTR D18G as a model for understanding how an extremely aggregation prone protein is handled by BiP. We have shown that BiP can selectively capture TTR D18G during co-expression in both E. coli and during over expression in human 293T cells and collects the mutant in oligomeric states. We have also shown that degradation of TTR D18G in human 293T cells occurs slower in presence of BiP, that BiP is present in amyloid deposition in human brain and mitigates cytotoxicity of TTR D18G oligomers.