PrP (122-139) is a covert mitochondrial targeting signal of prion protein and it specifically triggers the perinuclear clustering of mitochondria in neuronal culture cells.

In many neurodegenerative diseases, mitochondria are actively involved in the onset and/or progression of diseases because the energy depletion of the neuronal cells directly leads to the dysfunction and degeneration of cells. In the case of prion diseases, mitochondrial involvement has been reported recently and evidence that prion protein (PrP) is localized in mitochondria is increasing. Despite these findings, the precise molecular mechanism by which PrP targets mitochondria remains unclear. PrP is a secretory protein and does not have a pre-sequence that targets the mitochondria, therefore, we thought that there was a covert signal in the amino acid sequence of PrP. To find the sequence, we constructed various GFP-fused PrP-truncations and colocalization with mitochondria was verified by live-cell imaging. Consequently, we found that 18 amino acids, PrP (122-139), are indispensable for the mitochondrial targeting of PrP. In addition, fluorescent microscopy observation revealed that PrP-localized mitochondria were accumulated at the perinuclear region in neuronal cells such as mouse neuroblastoma Neuro2a (N2a) and prion persistent infection N2a strain (ScN2a), anterograde movement of the mitochondria toward the cell membrane was completely inhibited because of the stacking of PrP on the outer membrane. The cristae formation of perinuclear accumulated mitochondria was disappeared indicating the reduced mitochondrial activity. Surprisingly, PrP-dependent mitochondrial perinuclear accumulation was specifically occurred on neuronal cells, whereas in epithelial HeLa cells and fibroblast COS-7 cells, no perinuclear accumulation observed even after the mitochondrial targeting of PrP.

Structures and reaction mechanisms of the two related enzymes, PurN and PurU.

The crystal structures of glycinamide ribonucleotide transformylases (PurNs) from Aquifex aeolicus (Aa), Geobacillus kaustophilus (Gk) and Symbiobacterium toebii (St), and of formyltetrahydrofolate hydrolase (PurU) from Thermus thermophilus (Tt) were determined. The monomer structures of the determined PurN and PurU were very similar to the known structure of PurN, but oligomeric states were different; AaPurN and StPurN formed dimers, GkPurN formed monomer and PurU formed tetramer in the crystals. PurU had a regulatory ACT domain in its N-terminal side. So far several structures of PurUs have been determined, yet, the mechanisms of the catalysis and the regulation of PurU have not been elucidated. We, therefore, modelled ligand-bound structures of PurN and PurU, and performed molecular dynamics simulations to elucidate the reaction mechanisms. The evolutionary relationship of the two enzymes is discussed based on the comparisons of the structures and the catalytic mechanisms.