Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal neurodegenerative disorders caused by a change in conformation of the prion protein from the normal cellular form (PrPC) to a misfolded form (PrPSc). Prion protein has long been known as a copper binding protein. Although the functional implication of copper binding to PrP is not yet clear, it is believed that copper is an important cofactor in prion disease. Therefore, the aim of this work is to determine the potential role of copper in prion conversion. Copper can effectively bind to PrPC via histidine residues in the octapeptide repeats (OR) and the non-OR region located in the disordered N-terminal of the protein. Our hypothesis is that if copper binding plays a role in prion disease, removal of histidine residues in N-terminal domain may affect the prion conversion process. To examine this, we created a series of mutant murine PrP (MoPrP) molecules by replacing histidine residue at OR and non-OR region by tyrosine. These constructs were transfected into ScN2a cells and the efficiencies of prion conversion were evaluated. The results showed that replacing histidine by tyrosine at non-OR region led to an increasing of PrPSc conversion. When copper was removed by cuprizone, the construct with tyrosine at non-OR site (MoPrP H95Y) did not alter the level of PrPSc which meanwhile increased in case of the MoPrP wild-type (WT). To test these mutants in vitro, we produced recombinant protein, did the fibrilization assay and compared the lag phase. The result clearly showed that these constructs with tyrosine (H95Y, H110Y, H95Y/H110Y) need a shorter time to aggregate making fibrils faster than WT MoPrP. In particular, N2aPrP-/- cells expressing non-OR mutations PrP spontaneously cause prion formation that can be detected by ASA or PMCA. Moreover, transgenic mice overexpressing MoPrP H95Y showed clinical signs and died at 100 days with PK-resistant PrP in their brain. Based on these data, we can concluded that the substitution of histidine by tyrosine at non-OR region can enhance PrPC-PrPSc conversion process, and the non-OR copper binding site may have a critical role in this process.
|Autori interni:||Tran, Thanh Hoa|
|Autori:||Tran, Thanh Hoa|
|Titolo:||The non-octarepeat copper-binding site of the prion protein and its potential role in prion conversion|
|Data di pubblicazione:||9-feb-2016|
|Appare nelle tipologie:||8.1 PhD thesis|