The Role of proteases and chaperones in extracellular proteostasis

Endogenous and exogenous stresses (e.g. mutations, reduction and oxidation, extremes of pH or temperature) can cause the unfolding or misfolding of proteins. Failure to refold or degrade misfolded/unfolded proteins can result in their aggregation to form either structured fibrils or unstructured amorphous aggregates. These can accumulate and deposit within intraor extracellular environments to cause pathologies by physically disrupting tissue function (deposits) or exerting cytotoxicity (soluble protein oligomers). To deal with this, a complex network of protein quality control mechanisms have evolved to maintain proteins at their correct levels and in their native structures. As a consequence of aging, however, protein quality control systems lose efficacy and the ability of the body to defend itself against a variety of serious protein deposition diseases is decreased. Intracellularly, chaperones and proteases cooperatively maintain protein homeostasis (proteostasis) by ensuring correct protein folding, maintaining protein solubility, and degrading non-native or aggregated proteins. Relatively little is known, however, about the extracellular counterpart(s) of these processes even though many protein misfolding diseases have pathologies associated with extracellular protein aggregation and deposition. Recently discovered extracellular chaperones (such as alpha-2 macroglobulin and clusterin) are believed to function similarly to small heat shock proteins, by binding to and solubilising non-native proteins and protein oligomers, and facilitating their clearance by receptor-mediated endocytosis. The tissue plasminogen activation system also appears to play a role in extracellular proteostasis via its ability to activate plasminogen to plasmin in response to fibrillar protein aggregates, and the ability of plasmin to subsequently degrade these species. This thesis reports that the chaperone activity of alpha-2 macroglobulin can be enhanced in response to a variety of conditions related to diseases, such as reductive/oxidative stress, and increased protease secretion. The ability of alpha-2 macroglobulin dimers and monomers formed by oxidation and reduction, respectively, and protease activated alpha-2 macroglobulin to inhibit protein aggregation was found to be more potent than that of the native alpha-2 macroglobulin tetramer. In addition, the level of chaperone activity of alpha-2 macroglobulin correlated with the level of surface-exposed hydrophobicity, with alpha-2 macroglobulin dimers/monomers and alpha-2 macroglobulin complexed with proteases having significantly higher surface-exposed hydrophobicity than native alpha-2 macroglobulin. Pre-formed alpha-2 macroglobulin:protease complexes were able to partially degrade some aggregating chaperone client proteins, however, these complexes are rapidly cleared in vivo and thus the limited degradation of client proteins is unlikely to be physiologically relevant. However, protease activation of alpha-2 macroglobulin following its binding to misfolded proteins would assist in clearance of the complexes by cell-surface receptors. Further findings presented in this thesis suggest the existence of a novel system in which proteases and circulating extracellular chaperones act synergistically as key agents in extracellular proteostasis, together mediating the progressive degradation and safe clearance of large insoluble protein deposits. Tissue plasminogen activator and plasminogen were shown to co-localise on the surface of amorphously aggregated proteins via their binding to lysine residues, enhancing the formation of plasmin. It was also shown that when bound to insoluble protein aggregates, tissue plasminogen activator was partially shielded from inhibition by plasminogen activator inhibitor type-2 and active plm was shielded from inhibition by alpha-2 antiplasmin. The action of plasmin on amorphous protein aggregates was shown to release smaller soluble fragments of protein. The plasmin-generated protein fragments were bound and internalised (via different mechanisms) to both endothelial and microglial cells, and were subsequently trafficked to lysosomes in both cell types. When incubated with cells, protein fragments generated from different types of protein aggregates all elicited the formation of reactive oxygen species and ensuing cytotoxicity. Extracellular chaperones were able to bind to these toxic protein fragments, significantly ameliorating their negative effects on cells. These results indicate that extracellular proteases and chaperones may act together in extracellular proteostasis to inhibit the development of age-related protein deposition diseases. The results reported in this thesis are a promising first step towards understanding how extracellular proteostasis is maintained. Continued advances in this field may lead to confirmation of the operation of these processes in vivo and, ultimately, to the development of effective therapeutic strategies to combat serious degenerative protein deposition diseases.