Figure 5.

PCK1 is neddylated in three lysines after caloric restriction and fasting

(A) Pie chart showing the percentage of neddylated proteins from the total hepatic proteins identified in bionedd8 mice, as determined by LC-MS/MS proteomics (n = 4 or 5).

(B–D) (B) PANTHER functional enrichment analysis of neddylated proteins in liver. Neddylation of PCK1 during a 4-day caloric restriction (of 60%) (C) or 24-h fasting (D), as shown by immunoblot after immunoprecipitation of PCK1.

(E and F) PCK1 and NAE1 protein levels after the immunoprecipitation of PCK1, and NAE1 protein levels relativized to immunoprecipitated PCK1, from the livers of mice fed ad libitum or subjected to a 4-day caloric restriction (of 60%) (E) or 24-h fasting (F); n = 2–4.

(G) Immunoprecipitated PCK1 was subjected to mass spectrometry analysis. Possible modified sites are shown. K^∗, modified with GG.

(H) Ribbon representation of M. musculus PCK1 structure with identified neddylation sites colored in dark blue. Loops 95–118 and 460–470 are highlighted in cyan, and the Mn²⁺ ion, in light green.

(I) Average structures of PCK1 (left, olive) and PCK1-K387-NEDD8 (right, red) after molecular dynamics simulation. Neddylation of PCK1 alters the dynamics and conformation of loops 95–118 and 460–470 (cyan dashed lines). In-depth view of the opening the protein’s crevice upon PCK1 neddylation that makes the Mn²⁺ ion accessible to the substrate.