The Development of a System to Study the Prion Protein at the Membrane Surface

The Cellular Prion Protein (PrPC), discovered by Nobel Laureate Dr. Stanley Prusiner, represents the causative agent of a class of transmissible neurodegenerative diseases known as prion diseases. Despite knowledge that PrPC initiates early events of toxicity at the cell surface, limited research efforts and therapeutics have targeted the membrane environment. To meet this end, we are designing fully new and important ways of studying prion-induced toxicity at the membrane surface. Given that membrane-anchored PrPC purified from mammalian cells have heterogeneous post-translational modifications that are not amenable to biophysical assays, it is essential to instead chemically modify recombinantly expressed PrPC with a synthetic anchor. It is also important to prepare a simple representative membrane system that creates a controlled environment for studying the protein’s molecular and structural mechanisms. We first determine that liposomes are an ideal membrane system given their similar morphology to the native membrane, their ability to incorporate peripheral membrane proteins and their amenability to various biophysical assays. We then test the expression of mPrP S230pAcPhe and subsequent modification with a representative membrane anchor. We produce a protein construct that can be expressed using a modified expression system that incorporates the unnatural amino acid para-acetyl phenylalanine. We identify a two-step scheme that will selectively modify the protein construct with a synthetic membrane anchor. We successfully modify the protein with a dPEG linker that has aminooxy functionality, but struggled to further modify the construct with lipid tails. Alternatively, we successfully demonstrate that mutation of two residues near the extreme C-terminus of PrPC to cysteines allows for selective modification with a synthetic maleimide-functionalized membrane anchor. The development of this system enables us to investigate PrPC in the context of a membrane environment using biophysical approaches. Given that toxicity occurs at the membrane surface, such information will help identify interactions to target in the progression of prion diseases and other neurodegenerative diseases with similar pathology