Other alterations are less well defined in terms of their contribution to the final neoplastic phenotype, and include activation of Src, which has been shown in up to 300% of breast cancers by our lab and others

ions and cofactors affecting the susceptibility of a murine PrPC substrate to seeded PrPres formation, we report here that PrPres formation is significantly and specifically inhibited by the degradation of endogenous nucleic acids or heparan sulphate. We further show that treatment to modify the degree of GAG sulphation has a differential effect on the ability of wild-type PrP and PrP encoding a mutation associated with familial prion disease to act as a substrate for conversion to PrPres. This may be attributed to the differing ability of wild-type and mutant PrPC to bind to GAGs, suggesting that cellular cofactors differentially 881681-00-1 modulate sporadic and familial forms of prion disease and implicates subtle changes in the GAG repertoire in the pathogenesis of prion disease. Results Heparan sulphate and electrostatic involvement in cell free PrPres formation The Conversion Activity Assay generates PrPres from a PrPC substrate derived from an uninfected brain homogenate seeded with a prion infected brain homogenate. Using the M1000 mouse adapted prion strain as the IBH seed, PrPres formation occurs in a time and PrPC dependent manner with PrPres generated from the balb/c but 18325633 not Prnp2/2 mouse brain homogenates. While the PrPC 25581517 contained within the WT UBH was efficiently converted, there is evidence of further limiting, non-PrP factors in the process as only a small proportion of the available PrPC substrate is converted in the reaction using UBH derived from PrPC over expressing Tga20 mice. That an increase in PrPC does not significantly increase conversion efficiency suggests that factors other than PrPC in the UBH may limit the output of the assay. Electrostatic forces mediate many biological interactions and have been reported to affect the folding and stability of PrP. To investigate whether electrostatic forces play a role in the cell-free formation of PrPres the CAA was performed in buffers of increasing ionic strength. Using a similar assay, the ability of IBH derived PrPSc to drive the amplification of PrPres has been shown to decrease in the absence of NaCl. However, the interaction was also significantly reduced in high ionic strength buffers, consistent with a 2 August 2010 | Volume 5 | Issue 8 | e12351 Prion Protein Misfolding physiologically relevant interaction and implicating electrostatic interactions in the seeded formation of PrPres. Electrostatic interactions may exist between polyanionic molecules, such as sulphated GAG species and the polybasic regions of PrP. The contribution of sGAG to PrPres formation using the CAA described here was investigated by specific depletion of the endogenous sGAG content of the UBH used as the PrPC substrate in the CAA. Following optimisation of the conditions required for efficient sGAG digestion, the presence of sulphated species in the UBH was decreased and a reduction of polysaccharide chains shown by decreased absorbance of purified GAGs separated using an anion exchange column. The capacity of the UBH to act as a conversion substrate in the CAA was specifically and significantly reduced following heparinase III treatment to preferentially degrade heparan sulphate but not other sulphated GAG species, including heparin and chondroitin sulphate species. Treatment to deplete GAGs from the substrate did not reduce the amount of available PrPC substrate. It has been previously reported that the conversion activity of 263K, a hamster adapted sheep scrapie strain, is decreased by enzymatic tr