Lysine residues are not required for proteasome-mediated proteolysis of cellular prion protein

• Published in: Biochemical and biophysical research communications Vol. 735; p. 150807
• Main Authors: Nishinakagawa, Takuya, Homma, Takujiro, Ikeda, Aoi, Hazekawa, Mai, Morita, Yurie, Nakagaki, Takehiro, Atarashi, Ryuichiro, Nishida, Noriyuki, Ishibashi, Daisuke
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.10.2024
• Subjects: Degradation; Lysine residue; Prion protein; Proteasome; Ubiquitination; Degradation; Ubiquitination; Prion protein; Lysine residue; Proteasome
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2024.150807

Cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. The mature cell-surface PrPC is internalized and subsequently degraded by lysosomes. Although, proteasomes are proposed to be involved, the precise mechanism of PrPC degradation remains uncertain. Given that proteins are ubiquitinated primarily on lysine residues, we sought to determine whether lysine residues within PrPC are involved in the ubiquitination and subsequent degradation of PrPC. We generated a plasmid vector expressing a mutant PrPC (called lysine-null PrPC) in which all lysine residues were replaced with arginine residues. Subsequently, we established stably transformed cell lines (designated HpL2-1 PrP-WT and HpL2-1 PrP-K/R, respectively) using the mouse PrPC-deficient neuronal cell line (HpL2-1) and plasmids expressing wild-type (WT) or lysine-null PrPC (PrP-K/R). We found that HpL2-1 PrP-WT and HpL2-1 PrP-K/R cells correctly expressed their respective PrPC which translocated efficiently to the plasma membrane. Subsequently, using immunoblotting and confocal microscopy, we found that treatment with cycloheximide (CHX; a protein synthesis inhibitor) significantly reduced PrPC expression in both these transformed cell lines, indicating that WT and lysine-null PrPC are degraded similarly. Taken together, these results indicate that the lysine residues of PrPC do not regulate its degradation. •Lysine residues in cellular prion protein (PrPC) are highly conserved across various species.•Lysine residues in PrPC are not affect its transportation to the plasma membrane in secretory pathway.•Lysine residues of PrPC are not critical for regulating its degradation.